Cell and Tissue Biology

Tiago Fernandes

email: tfernandes@tecnico.ulisboa.pt
phone: 210 407 056
office: room 13.25 (Taguspark)
 Todays’ Summary

General methods to study cell and tissue organization:

- Overview of the cell organization and function.

- Classic and high-resolution microscopy techniques.
- Cellular labelling techniques (Molecular probes; Optogenetics).
 How many cells do we have?

body=3.72*1013 (Ann Hum Biol 40, 463-471, 2013)

milli 10-3
brain=1.7x 1011
micro 10-6
nano 10-9
pico 10-12
What is the size of a “normal” cell? Diameter and volume and mass? femto 10-15

Sphere volume (4/3)pr3; cell density: 1.1g/cm3 atto 10-18

10 µm, 523 µm3 (or fL); 576 pg

20 µm, 4187 µm3 (or fL); 4605 pg

Component Amount per HeLa cell
(polyploid)
Total dry weight 400 pg

How much DNA has a human cell? Total DNA 15 pg*

Total RNA 30 pg
genome size=3x109 bp; 1bp=660Da (or gmol-1); NA=6x1023 mol-1 Total protein 300 pg

Cytoplasmic ribosomes 4x106

6.6pg (somatic cells) Cytoplasmic tRNAs 6x107

Cytoplasmic mRNAs 7x105

Cell structure and physiology

Lodish et al., Molecular Cell Biology, W.H. Freeman

The cell architecture
(structure/function) is based on
several types of microscopy. Because
there is no one “correct” view of a
cell, it is essential to understand the
characteristics of the key cell-viewing
techniques, the types of images they
produce, and their limitations.

Lodish et al., Molecular Cell Biology, W.H. Freeman

 Methods to study subcellular organization of eukaryotic cells
and tissue organization of multicellular organisms

R. Hooke, A. von Leeuwenhoek, M. Schleiden, T. Schwann, R. Virchow and others after them led to the
development of the Cell theory:

”Cell is basic unit of structure and function of all living organisms”

Hooke’s compound
microscope (20-30X),
Leeuwenhoek’s microscope
1665
(<270X), 1673

Cork in Micrographia Hooke’s book,

1665

- All organisms are composed of one or more cells (Schleiden & Schwann) (1838)

- The cell is the basic unit of life in all living things (Schleiden & Schwann) (1838)

- All cells are produced by the division of preexisting cells (Virchow) (1858)
 Cytochemistry/histochemistry
Cell & tissue microscopic observations: in vivo, ex vivo, Staining, Fixation, Sectioning

Green unicellular algae Arabidopsis petals Mouse

Fixation and Sectioning of samples for microscopy

Tissues should be cut (using a microtome) in slices thin enough to avoid

complete absorbance of the incident light or electrons (e.g. the sections for electron
microscopy must have only 50-100 nm, which is about 0.2% of the thickness of a single cell)

Fixation aims to denature and cross-links groups on adjacent molecules of

proteins and nucleic acids, rendering them insoluble and stable for subsequent
procedures and observation
Groups of fixatives: Aldehydes: (formaldehyde, glutaraldehyde), Alcohols (methanol, ethanol) or
Oxidizing agents (potassium permanganate, potassium dichromate and osmium tetroxide)
 Staining

Most cellular constituents (e.g. nuclei, mitochondria) are not colored and absorb about the same degree of
visible light, so that they are hard to distinguish under a light microscope and staining is needed.

Many chemical stains bind to molecules that have specific features, e.g.:
eosin binds to basic amino acids (lysine and arginine) on many different proteins, and hematoxylin binds to
acidic molecules (such as DNA, and aspartate and glutamate side chains)

Schematic view of cross section of intestine

 Staining
- Modifying a substrate into a colored product or into a precipitate by an endogenous
enzyme

Cytochemical staining. Light micrograph of cross section of

human skeletal muscle stained for succinate
dehydrogenase, an enzyme found only in mitochondria.
From Burkitt & Daniels “Functional Histology”

- In situ hybridization allows localization and detecting of specific mRNA (also DNA) sequences in tissues by the
hybridization of the complementary strand of a nucleotide probe
A B C
Differential expression of von Willebrand factor mRNA in
Endothelial cells from different tissues. The Relative level
of expression of von Willebrand factor mRNA in murine
tissues compared by FISH (fluorescence in situ
hybridization. (A) lung, (B) brain, (C) liver; magnification of
× 250.
(Blood 1998 92:2791-2801)
 Immunocytochemistry/immunohistochemistry
Dyes (colorimetric, electron-dense or fluorescent) have a low and nonspecific affinity for biological molecules, but they can
be chemically coupled to antibodies specific for almost any desired protein (and other macromolecules).

Localization of catalase by immunocytochemistry and TEM Localization of actin by immunocytochemistry and fluorescence microscopy

Fluorophore

(actin)

The antibody is allowed to interact with a specific antigen (catalase) and then
incubated with electron-dense 5-7 nm diameter gold particles. The gold
nanoparticles are located exclusively in peroxisomes. Lodish et al., Molecular Cell Biology, W.H. Freeman
By staining a specimen with two or three dyes that fluoresce at different wavelengths, multiple
proteins can be localized within a cell
Fluorescent techniques for high resolution imaging in living cells

Main advantage of fluorescence microscopy: live cells! But a low resolution if compared with EM
Current research is focused on improving spatial resolution up to 1-5 nm in live cells

Comparison of the spatial and temporal resolutions of biological imaging techniques

The size scale is logarithmic. Average sizes of biological

features are given; specific sizes vary widely among
different species and cell lines. The spatial and temporal
resolutions are estimates of current practices, and some
were taken from REF. 120. The spatial resolution is given
for the focal plane. The temporal resolution is not
applicable (NA) for electron microscopy (EM) or near-field
scanning optical microscopy (NSOM) because they image
static samples. Ground-state depletion (GSD) and
saturated structured-illumination microscopy (SSIM) have
not been shown on biological samples, and thus their
temporal resolutions are not determined (ND). ER,
endoplasmic reticulum; MRI, magnetic resonance
imaging; OCT, optical coherence tomography; PALM,
photoactivated localization microscopy; PET, positron-
emission tomography; STED, stimulated emission
depletion; STORM, stochastic optical reconstruction
microscopy; TIRF, total internal reflection fluorescence;
US, ultrasound; WF, wide-field microscopy.

Nat Rev. Mol. Cell Biol.9, 929-943 (2008)

 Fluorescence microscopy methods
Conventional

Confocal

Sea urchin fertilized egg

White et al., J Cell Biol 104:41 (1987)

Living HEK293 cells

Pellett et al., Biomed Optics Exp 2:2364 (2011)
 Probes for fluorescence super-resolution imaging:

1- Labelling by Fusion with reporter Fluorescent proteins, genetically encoded probes:

- XFPs “1994 old-fashioned” fluorescent proteins

Mice expressing GFP Brainbow mice (hippocampal
under UV light compared dentate gyrus)
Nat Rev Neurosci 9: 417–422 (2008)
to normal mouse.
BMC Cancer 2012, 12:21

- Photoactivatable-FPs, those that convert from a dark state to a bright fluorescent state
- Photoshiftable-FPs, those that change fluorescence wavelength on irradiation;
(PA and PS FPs change their spectral properties on irradiation with light of a specific wavelength)

2- Organic small-molecule fluorophores, non-genetically encoded probes

FRET (Förster resonance energy transfer) is a mechanism
describing energy transfer between two chromophores.

de:Sven Jähnichen
 Synthetic fluorophores and fluorescent proteins for microscopy

Synthetic dyes have relatively poor targeting efficiency and generally generate large background signal and
cannot be genetically encoded like fluorescent proteins. But synthetic fluorophores can be advantageous over
fluorescent proteins for super-resolution imaging due to their high intrinsic brightness, excellent photostability,
good contrast, and greater fatigue resistance.
http://zeiss-campus.magnet.fsu.edu/articles/superresolution/palm/introduction.html
 Optogenetics
- Involves the use of light to control neurons that have been genetically modified to express light-sensitive ion channels.
Cell type-specific depolarization or silencing can be optically induced by heterologous expression of light-sensitive
microbial membrane proteins.

The optogenetic principle: changing the membrane voltage potential of

excitable cells. Activating tools-channelrhodopsins: channelrhodopsin-2
from Chlamydomonas reinhardtii (ChR2) and channelrhodopsin-1 Volvox
carteri (VChR1) from nonselective cation channels leading to
depolarization of target cells.
Silencing tools-ion pumps: archaerhodopsin-3 (Arch) from Halorubrum
sodomense works as a proton pump and leads to hyperpolarization of
the target cell such as the chloride pump NpHR (NpHR) from
Natronomonas pharaonis.

Mol Genet Genomics 287: 95-109 (2012)

Optogenetics video (https://www.nature.com/collections/tqxhytcpwh/video)
 A live cell captured by Lattice Light Sheet Microscopy developed by Nobel prizewinner Eric Betzig Nature 526, S50 (2015)

Lattice Light Sheet Microscopy video 1 (https://www.youtube.com/watch?v=hBWhIDuq1Xw)

Lattice Light Sheet Microscopy video 2 (https://www.youtube.com/watch?v=UmxKxpKua2M)

In addition to microscopy (and optogenetics) the biochemical methods are very useful tools
to study structure/function of cells and tissues
- Measurement of biomolecule activity and concentration (e.g. enzymatic assays, ELISA, Western blotting,
sequencing (DNA, RNA), PCR, DNA microarrays (indirectly measuring mRNAs), flow cytometry, etc, etc),
Lattice Light Sheet Microscopy video 1 (https://www.youtube.com/watch?v=hBWhIDuq1Xw)
Lattice Light Sheet Microscopy video 2 (https://www.youtube.com/watch?v=UmxKxpKua2M)
FURTHER READING
 Resolution and magnification

Microscope Resolution Magnification

Optical 200 nm 1,500X
TEM ~0.5 nm 500,000X
SEM ~2 nm 200,000X
TEM, Transmission Electron Microscopy; SEM, Scanning Electron Microscopy

Limit of resolution (r) is defined as the shortest distance between two

points that can still be distinguished as separate entities

where l is the imaging wavelength, n the refractive index of

medium and NA the numerical aperture

Ɵ
 Fluorescent sensors for monitoring cellular signals

(a) Sensors based on single fluorescent proteins (FPs). Single FP-based

voltage-sensitive fluorescent proteins (VSFPs, voltage probes) are derived
from a combination of a membrane-integrated voltage sensor domain (gray
and purple transmembrane domains) and cpXFPs (cpVSFPs) or XFPs (VSFP3s).
Single FP calcium indicators include scaffolds based on GFP and the
calmodulin (such as Pericam, GCaMPs, and Case) and troponin-based
scaffolds (Camgaroo). SynaptopHluorins are indicators of vesicle release and
recycling, consisting of a pH-sensitive form of GFP (pHluorin) fused to the
luminal side of a vesicle-associated membrane protein (VAMP). Sinphos are
detectors of protein phosphorylation (kinase activity) made of a fusion
between a cpXFP, a phosphorylable substrate peptide, and a phosphoamino
acid binding domain.

(b) FRET sensors. These sensors are traditionally based on a FRET pair of FPs
such as CFP and YFP. VSFP2s are FRET-based voltage sensors. FRET calcium
indicators include cameleons (based on the calmodulin) and the TN sensor
family (based on troponin). Chloride sensors (Clomeleon and Cl-sensors) take
advantage of the fact that chloride can efficiently quench YFP fluorescence,
thus reducing the FRET signal of a CFP-YFP pair. In BioSensor-GlyR, Cl-sensor
proteins are grafted to the subunits of a glycine receptor (GlyR) in order to
sense chloride ions flowing through the receptor. FRET kinase activity sensors
(Phocuses and XKARs) have been developed using the same rationale as for
Sinphos (see a). Finally, glutamate can be detected using FRET sensors based
on bacterial periplasmic binding proteins (PBPs) or on a metabotropic
glutamate receptor (mGluR1).

Membranes are represented with the cytoplasmic side toward the bottom.

Progress in Brain Research, 196: 1-28 (2012)

Nat Rev. Mol. Cell Biol.9, 929-943 (2008)
 Small-molecule fluorophores

* Photoactivation of these fluorophores is strongly facilitated by the presence of an activator fluorophore, such as Cy2, Cy3 or Alexa Fluor 405, to induce photoswitching

Nat Rev. Mol. Cell Biol.9, 929-943 (2008)

 Next Class…

• Module:

A. Cell and Tissue Biology

 Todays’ Summary

Integration of cells into tissues:

- Extracellular matrix.
- Cell-cell and cell-matrix adhesion and communication.
- Extracellular matrix molecules and their ligands.
- Principles of cell signaling.
All biomembranes contain a phospholipid bilayer with proteins, glycoproteins, cholesterol
and other steroids, and glycolipids. The presence of specific sets of proteins and lipids
permits each type of membrane to carry out distinctive functions.

Lodish et al., Molecular Cell Biology, W.H. Freeman

The protein and also the phospholipid composition differs in two membrane leaflets
Lipid (and protein) composition in each leaflet is not homogenous: lipid rafts

Lodish et al., Molecular Cell Biology, W.H. Freeman

 The cytoskeleton provides structural support and shape for the cell, organizes cytoplasm (polarity) and
permits directed movement of organelles, chromosomes, and the cell itself. Also, through association with
extracellular matrix and other cells it stabilizes tissues.

The polymerization/depolymerization (assembling/disassembling) of the cytoskeleton elements is precisely and tightly regulated

Composition:
Microfilaments (f=7nm) G-actin which polymerizes to F-actin
Main functions: cell motility, cell contractility
actin

Microtubules (f=24nm) a and b tubulin,

Main functions: vesicular transport, mitosis, cilia and flagella
tubulin

Intermediate filaments (f=9-11nm) filamentous proteins (keratin, vimentin, lamin,

desmin, nestin, GFAP, etc)
vimentin
Main functions: essentially as structural components Lodish et al., Molecular Cell Biology, W.H. Freeman
 Integrating Cells into Tissues

Direct interactions between cells, as well as between cells and the extracellular
matrix, are critical to the development and function of multicellular organisms

Lodish et al., Molecular Cell Biology, W.H. Freeman

The extracellular matrix, ECM, is a complex structural entity surrounding and supporting
cells that are found within mammalian tissues.

Functions: mechanical strength (rigidity and compressibility), adhesion,

migration, chemical selectivity, proliferation, differentiation, apoptosis and cell
shape. All of them depend on ECM composition and density.

Composition:
- Structural proteins: e.g. collagens and non-collagenous proteins (e.g. elastin)
- Specialized proteins: e.g. fibronectin and laminin
- Glycosaminoglycans (GAGs): polysaccharides
- Proteoglycans: a protein core attached to GAGs
Cell-cell and cell-ECM adhesion and communication
is dependent on specialized structures (Gap junctions, Tight junctions, Adherens junctions,
Adhesion plaques, Desmosomes, Hemidesmosomes) and molecules (Cell adhesion molecules)
associated with microfilaments and intermediate filaments
 Gap junctions ("junções comunicantes")
- consist of assemblies of six (x2) connexins (>20 types), which form open channels through the
plasma membranes of adjacent cells where some ions and small molecules pass through
(movement of molecules smaller than 1 kDa or <2nm (e.g. peptide < 8 aa))

Lodish et al., Molecular Cell Biology, W.H. Freeman

 Tight junctions (“junções apertadas")

- ribbon-like bands connecting adjacent cells that prevent leakage of fluid across the cell layer
- are formed by interactions between strands of transmembrane proteins (occludin and claudins)
on adjacent cells.
Tight junctions in epithelial cells of small intestine and glucose transport
from intestine lumen and blood

Lodish et al., Molecular Cell Biology, W.H. Freeman

 Desmosomes (D), Hemidesmosomes (HD),
Adherens Junctions (AJ), Adhesion Plaques (AP)
- are dense protein plaques (D, HD) or belts (AJ, AP) that mediate
adhesion between cells (D and AJ) or between cells and ECM (HD
and AP). Desmosomes and Hemidesmosomes bind to intermediate
filaments and Adherens Junctions and Adhesion Plaques attach to
microfilaments.

Lodish et al., Molecular Cell Biology, W.H. Freeman

 Stable cell-cell junctions mediated by the cadherins

Interactions between cadherins mediate two types of stable cell-cell adhesions:

- In adherens junctions, the cadherins are linked to bundles of actin filaments via the catenins

- In desmosomes, desmoplakin links members of the cadherin superfamily (desmogleins and

desmocollins) to intermediate filaments

Adherens Junction Desmosome

ECM exerts control over many cellular fate processes through binding to a class of receptors, integrins.

Lodish et al., Molecular Cell Biology, W.H. Freeman

- The extracellular matrix is critically important for many cellular processes including growth,
differentiation, survival, and morphogenesis.
- Cells remodel and reshape the ECM by degrading and reassembling it, playing an active role in sculpting
their surrounding environment and directing their own phenotypes.
- Both mechanical and biochemical molecules influence ECM dynamics in multiple ways; by releasing small
bioactive signaling molecules, releasing growth factors stored within the ECM, eliciting structural changes to
matrix proteins which expose cryptic sites and by degrading matrix proteins directly.
- The dynamic reciprocal communication between cells and the ECM plays a fundamental role in tissue
development, homeostasis, and wound healing.
Current Opinion in Biotechnology 24: 830-833 (2013)
Understanding and controlling cell physiology requires a deep knowledge of cell-cell
and cell-ECM interactions and also ECM architecture (components and organization).

Each ECM is comprised of unique compositional and topographical features*. The stiffness
and elasticity of the ECM has important implications in cell migration, gene expression, and
differentiation.

*Topographies. The three-dimensional qualities of surfaces or structures, including contours and relief. In the context of
the ECM this includes features such as peaks and valleys, changes in roughness and geometric features.
Nat Rev Mol Cell Biol 15, 771–785 (2014)
Inner live of a cell (video)
(http://www.xvivo.net/animation/the-inner-life-of-the-cell/)
 Cell-cell signaling
(how cells communicate with one another)

- The overall process of converting signals into cellular responses, as well as the individual steps in this process, is termed
signal transduction

- Extracellular signaling molecules or ligands (hormones, neurotransmitters, cytokines, etc) are used in cell-to-cell
communication

- Signaling molecules are synthesized and released by signaling cells to produce a specific response only in target cells that
have receptors for the signaling molecules

Synthesis
↓
Release from the signaling cell
↓
Transport to the target cell
↓
Binding to receptor
↓
Cellular response
↓
Removal of signal
 Signaling molecules operate at different distances

e.g. epinephrine acts as a neurotransmitter (paracrine) and as a systemic hormone (endocrine)

Lodish et al., Molecular Cell Biology, W.H. Freeman

 Lipophilic signaling molecules

- Retinoic acid acts through Hox genes, which ultimately control

embryonic anterior/posterior patterning in early developmental stages.

- Thyroxine is involved in controlling the rate of metabolic processes in the

body and influencing physical development.

- Cortisol is involved in response to stress and anxiety. It increases blood

pressure and blood sugar, and reduces immune responses.

- Progesterone is involved in the female menstrual cycle, pregnancy and embryogenesis of

humans and other species

- Estradiol has not only a critical impact on reproductive and sexual functioning in females, but
also affects other organs including the bones

- Testosterone is the principal male sex hormone and an anabolic steroid (males and females).
 Hydrophilic signaling molecules
- Insulin causes cells in the liver, muscle, and fat tissue to take up glucose from the blood, storing it as glycogen in the liver and muscle,
and stopping use of fat as an energy source
- Glucagon is released when blood glucose levels start to fall too low, causing the liver to convert stored glycogen into glucose and release
it into the bloodstream
- Growth factors are a diverse group of molecules capable of stimulating cellular growth
- Epinephrine is released into the bloodstream in response to physical or mental stress, as from fear or injury. It initiates many bodily
responses, including the stimulation of heart action and an increase in blood pressure, metabolic rate, and blood glucose concentration.
- Histamine triggers the inflammatory response by increasing the permeability of the capillaries to white
blood cells and proteins
- Serotonin ('wonder drug‘) is a neurotransmitter involved in the control of appetite, sleep, memory and
learning, temperature regulation, mood, behaviour, cardiovascular function, muscle contraction, endocrine
regulation and depression. Is also found in wasp stings and scorpion venom where its function is of an irritant,
since intravenous injection of serotonin in humans leads to pain, gasping, coughing, a tingling and prickling
sensation, nausea, cramps and other unpleasant symptoms

- Acetylcholine is a neurotransmitter that has functions both in the peripheral nervous system where activates muscles and in the central
nervous system in association with neurons form a neurotransmitter system, the cholinergic system, which tends to cause excitatory
actions.

- Erythropoietin controls erythropoiesis, or red blood cell production

- Interferons are proteins which trigger the protective defenses of the immune system that eradicate pathogens (such as viruses, bacteria,
or parasites) or tumors.
 In many, but not all, signaling pathways, ligand binding to a receptor leads to activation of
transcription factors in the cytosol, permitting them to translocate into the nucleus and
stimulate (or occasionally repress) transcription of their target genes.

Alternatively, receptor stimulation

may lead to activation of cytosolic
protein kinases that then
translocate into the nucleus and
regulate the activity of nuclear
transcription factors.

Lodish et al., Molecular Cell Biology, W.H. Freeman

Lodish et al., Molecular Cell Biology, W.H. Freeman
 G protein-coupled receptors (GPCRs)
- are a class of cell-surface receptors that activate G-proteins

The human genome encodes several thousand GPCRs; e.g. receptors in the visual, olfactory, and gustatory systems,
many neurotransmitter receptors, and most of the receptors for hormones that control carbohydrate, amino acid,
and fat metabolism, and light activated receptors (rhodopsins) in the eye.

About 40% of all commercialized pharmaceutical drugs interacts with GPCRs!...

Lodish et al., Molecular Cell Biology, W.H. Freeman

Operational model for ligand-induced activation of effector proteins associated with GPCRs

Sign
al tr
ansd
uctio
n

Stop
sign
alin
g

Lodish et al., Molecular Cell Biology, W.H. Freeman

G protein regulate the activity of effector proteins like adenylyl cyclase, phospholipase C, PDE
or ion channels that produce/degrade 2nd messengers or alter membrane voltage

Lodish et al., Molecular Cell Biology, W.H. Freeman

 Intracellular second messengers
- are intracellular signaling molecules that greatly amplify the original first messenger signal

- may be coupled downstream to kinase /phosphatase cascades

ATP GTP PIP2

(Phosphatidylinositol 4,5-bisphosphate)

Ca2+

Activates PKC and

complexes with calmodulin

Lodish et al., Molecular Cell Biology, W.H. Freeman

 Protein kinases and phosphatases

- are involved directly or indirectly in the signal transduction from cell surface receptors
- can be regulated by second messengers (e.g. cAMP)

The cyclic phosphorylation and dephosphorylation of a protein is a common cellular mechanism for
regulating protein activity. In this example, the target protein R is inactive (light orange) when
phosphorylated and active (dark orange) when dephosphorylated; some proteins have the opposite
pattern.
Lodish et al., Molecular Cell Biology, W.H. Freeman
FURTHER READING
 just to recall!... molecules, cells and organism sizes

Gap junction

0.18 kDa
16 kDa
 Bundle of actin
Microfilaments
actin
The actin cytoskeleton is organized in bundles and filament
networks of filaments which are held together by
actin cross-linking (or branching) proteins protein
cross-link
Actin also interacts with motor proteins (myosins)

Fibroblast
Sperm-egg fusion depends on actin polymerization

SEM image

Rhodamine-phalloidin staining

Lodish et al., Molecular Cell Biology, W.H. Freeman

 Microfilaments
Actin contributes for the shape (biconcave disk) Changes in shape of platelets during blood clotting
of the erythrocyte by interacting with other are due to actin rearrangements
proteins (cytoskeletal and integral proteins)

Lodish et al., Molecular Cell Biology, W.H. Freeman

Resting platelets are smooth and disc
shaped, but after activation become
irregular with many protruding pseudopodia
Microtubules

Intracellular membrane vesicles travel along microtubules

- Kinesin is (+) end-directed microtubule motor protein (anterograde transport)

- Dynein is (-) end-directed microtubule motor protein (retrograde transport)

Lodish et al., Molecular Cell Biology, W.H. Freeman

 Microtubules

The mitotic apparatus is a microtubule machine for separating chromosomes (motor proteins
are also involved)

Taxol and vinblastine blocks spindle formation (inhibits depolymerization) being used as anticancer drugs

Lodish et al., Molecular Cell Biology, W.H. Freeman

Although Intermediate filaments are dynamic polymers they are more
stable than microfilaments and microtubules

Lodish et al., Molecular Cell Biology, W.H. Freeman

Nat Rev Mol Cell Biol 15, 771–785 (2014)
Nat Rev Mol Cell Biol 15, 771–785 (2014)
 Collagens (28 types)
- Types I, II and III are the most abundant and form fibrils of similar structure
- Type IV forms a two-dimensional reticulum and is a major component of the basal lamina
- Collagens are mainly synthesized by fibroblasts but epithelial cells also synthesize them
- “Collagens” is the major protein comprising the ECM (and also from the animal kingdom)

Most of collagens (rich in glycine and proline) are long (300nm) and thin (1.5nm) diameter rod-like proteins consisting of 3
coiled subunits composed in a characteristic right-handed triple helix

Lateral interactions of triple helices of collagens result in the

formation of collagen fibrils roughly 50-200 nm diameter. The
packing of collagen is such that adjacent molecules are displaced
~1/4 of their length (67nm).
 Laminin and fibronectin form bridges between structural ECM molecules, and
connect the ECM to cells and to soluble molecules within the extracellular space

Fibronectins (dimers) bind many cells (via RGD-integrins) to fibrous collagens and other ECM molecules

Laminin and type IV collagen form the 2-D network of basal lamina

Lodish et al., Molecular Cell Biology, W.H. Freeman

Glycosaminoglycans (GAGs): hyaluronic acid, dermatan sulfate, chondroitin sulfate, heparin, heparan
sulfate, and keratan sulfate;

-are long unbranched polysaccharides, highly negatively charged, containing a repeating disaccharide unit: N-
acetylgalactosamine (GalNAc) or N-acetylglucosamine (GlcNAc), and a uronic acid such as glucuronate or
iduronate
GAGs confer high viscosity to the solution and low compressibility, which makes these molecules ideal for a
lubricating fluid in the joints. At the same time, their rigidity provides structural integrity to cells and provides
passageways between cells, allowing for cell migration.

Hyaluronic acid is unique among the GAGs in that it does not contain any sulfate and is not found covalently attached
to proteins as a proteoglycan. It has very large molecular weight (100,000–10,000,000) and can displace a large volume
of water.

GAG Localization Comments

large polymers, shock

Hyaluronate synovial fluid, vitreous humor, ECM of loose connective tissue
absorbing

Chondroitin sulfate cartilage, bone, heart valves most abundant GAG

contains higher acetylated

Heparan sulfate basement membranes, components of cell surfaces
glucosamine than heparin

component of intracellular granules of mast cells more sulfated than

Heparin
lining the arteries of the lungs, liver and skin heparan sulfates

Dermatan sulfate skin, blood vessels, heart valves

Keratan sulfate cornea, bone, cartilage aggregated with chondroitin sulfates

Proteoglycans are proteins linked to GAGs (also called mucopolysaccharides).
- GAGs extend perpendicularly from the core in a brush-like structure. The linkage of GAGs
to the protein core involves a specific trisaccharide which is linked to the protein core
through an O-glycosidic bond to a S or T residue in the protein.

Lodish et al., Molecular Cell Biology, W.H. Freeman

 G-proteins (trimeric and monomeric GTPase switch proteins)

- in their “on” state bind and regulate the activity of effector proteins

Conversion of the active into the inactive form by hydrolysis of the bound GTP is
accelerated by GAPs (GTPase-accelerating proteins) and RGSs (regulators of G-protein-
signaling) and inhibited by GDIs (guanine nucleotide dissociation inhibitors).
Reactivation is promoted by GEFs (guanine nucleotide–exchange factors).
Lodish et al., Molecular Cell Biology, W.H. Freeman
 Next Class…

• Module:

A. Cell and Tissue Biology

 Todays’ Summary

Cell signaling pathways:

- GPCRs (Cont.)
- Intracellular receptors
- Receptors that are ion channels
- Receptors with intrinsic or associated enzymatic activity
- Receptors involving proteolysis
Operational model for ligand-induced activation of effector proteins associated with GPCRs

Sign
al tr
ansd
uctio
n

Stop
sign
alin
g

Lodish et al., Molecular Cell Biology, W.H. Freeman

Lodish et al., Molecular Cell Biology, W.H. Freeman
 GPCRs that regulate adenylyl cyclase
In the liver, glucagon and epinephrine bind to different receptors, but both receptors interact with and
activate the same Gsa, which activates adenylyl cyclase, thereby triggering the same metabolic responses.

Positive and negative regulation of adenylyl cyclase activity occurs in some cell types, providing fine-tuned
control of the cAMP level. For example, stimulation of adipose cells by epinephrine, glucagon, or ACTH
activates adenylyl cyclase, whereas prostaglandin* PGE1 or adenosine inhibits the enzyme.

*Prostaglandins are lipophilic molecules (9 classes: PGA to PGI) that bind to plasma membrane receptors. Usually act in paracrine and
autocrine signaling modulating the response of other signaling molecules.
Lodish et al., Molecular Cell Biology, W.H. Freeman
 Sign
al tr
ansd
uctio
n

cAMP-activated protein kinase A (PKA) mediates various responses in different cells - alters the transcription
of specific genes or the activity of specific proteins- by phosphorylation of serine and threonine residues in the
target proteins (X-R-(R/K)-X-(S/T)-F)

Activation of a
transcription factor

Plasma
membrane
PKA

Nuclear
envelope

At low concentrations of cAMP, the PKA is an inactive tetramer. Binding

of cAMP to the regulatory (R) subunits causes a conformational change
in these subunits that permits release of the active, monomeric catalytic
(C) subunits. cAMP binding is cooperative.

Lodish et al., Molecular Cell Biology, W.H. Freeman

GPCRs that activate gene transcription by a kinase cascade

Kinase cascade that transmits signals downstream

from mating factor receptors in S. cerevisiae.

The receptors for yeast a and a mating factors are

coupled to the same trimeric G protein. Ligand binding
leads to activation and dissociation of the G protein. In
the yeast mating pathway, the dissociated Gbg activates
a protein kinase cascade analogous to the cascade
downstream of Ras that leads to activation of MAP
kinase. The final component, Fus3, is functionally
equivalent to MAP kinase (MAPK) in higher eukaryotes.
Association of several kinases with the Ste5 scaffold
contributes to specificity of the signaling pathway by
preventing phosphorylation of other substrates.

Lodish et al., Molecular Cell Biology, W.H. Freeman

 GPCRs that activate phospholipase C (PLC) via Ca2+ and PKC

Synthesis of DAG and IP3 (diffusible) from membrane-bound phosphatidylinositol (PI)

The level of PIs are regulated by extracellular signals, especially those that
bind to receptor tyrosine kinases or cytokine receptors
Lodish et al., Molecular Cell Biology, W.H. Freeman
IP3/DAG pathway and the elevation of cytosolic Ca2+

Lodish et al., Molecular Cell Biology, W.H. Freeman

 GPCRs that activate phospholipase C (PLC) via Ca2+ and calmodulin

The Ca2+/calmodulin complex regulates the activity of many different proteins (e.g. cAMP-phosphodiesterase,
nitric oxide synthase) and kinases (e.g. GPK that hydrolyses glycogen) and phosphatases that control the
activity of various proteins including transcription factors

Regulation of contractility of arterial smooth muscle by nitric oxide and cGMP

Nitric oxide (NO) is synthesized in endothelial

cells in response to acetylcholine and the
subsequent elevation in cytosolic Ca2+. NO
diffuses to nearby smooth muscle cells where
activates an intracellular NO receptor with
guanylyl cyclase activity. The resulting rise in
cGMP leads to activation of protein kinase G
(PKG) (which indirectly induces
dephosphorylation of myosin), relaxation of
the muscle, and thus vasodilation.
(Nitroglycerin (is converted to NO by
mitochondrial aldehyde dehydrogenase) and
“Viagra” inhibit a cGMP-phosphodiesterase)

Lodish et al., Molecular Cell Biology, W.H. Freeman

GPCRs that activate a PDE (phosphodiesterase)

http://webvision.umh.es/webvision/sretina.html

https://www.sas.upenn.edu/visual-studies/

Lodish et al., Molecular Cell Biology, W.H. Freeman

 Rhodopsin consists of a
protein (opsin) covalently
bound to a light absorbing
pigment 11-cis-retinal

The high level of cGMP present in the dark acts to keep cGMP-gated cation channels open; the light-induced
decrease in cGMP leads to channel closing, membrane hyperpolarization (cell interior more negative), and
reduced neurotransmitter release
Lodish et al., Molecular Cell Biology, W.H. Freeman
 GPCRs that regulate ion channels

Operational model of muscarinic acetylcholine receptor in the heart muscle plasma membrane

The exit of K+ results in hyperpolarization of the cell membrane that slows the rate of muscle contraction
Lodish et al., Molecular Cell Biology, W.H. Freeman
 Stop
sign
alin
g

Mechanisms of GPCR signal termination (3 pathways)

- hydrolysis of GTP to GDP, which is accelerated by GAPs and RGSs, causes Gα to dissociate from effector and bind with Gßɣ

- upon phosphorylation of the GPCR by protein kinases (PKs) or GPCR kinases (GRKs) arrestin binding may occur preventing
G-protein coupling as well triggering the process of receptor internalization through clathrin-mediated endocytosis

(e) GPCR responsive elements such as PKs or GRKs phosphorylate the

intracellular side of the receptor and decouple the G protein by steric exclusion

(f) b-arrestins can recognize the phosphorylated GPCR and trigger the
internalization process

(g) Modifications on the b-arrestin molecule such as dephosphorylation or

ubiquitination define the fate of the internalized molecule either to recycling or
degradation respectively

Trends Biotechnol 30, 566-574 (2012)

Lodish et al., Molecular Cell Biology, W.H. Freeman
 Intracellular receptors

Small lipophilic molecules like steroids (cortisol, progesterone, estradiol, testosterone), thyroxine
and retinoic acid diffuse across plasma membrane and interact with intracellular receptors altering
gene expression at transcription or post-transcription level. Long term stimulus (hours to days).
Lodish et al., Molecular Cell Biology, W.H. Freeman
Sequential activation of gated ion channels at a neuromuscular junction

Arrival of an action potential at the terminus of a

presynaptic motor neuron induces opening of voltage-
gated Ca2+ channels (step 1) and subsequent release of
acetylcholine, which triggers opening of the ligand-gated
nicotinic receptors that are ion channels in the
muscle plasma membrane (step 2). The resulting influx of
Na+ produces a localized depolarization of the
membrane, leading to opening of voltage-gated Na+
channels and generation of an action potential (step 3).
When the spreading depolarization reaches T tubules, it
triggers opening of voltage-gated Ca2+-release channels
and release of Ca2+ from the sarcoplasmic reticulum into
the cytosol (step 4). The rise in cytosolic Ca2+ causes
muscle contraction
Lodish et al., Molecular Cell Biology, W.H. Freeman
 TGFb Receptors and the Direct Activation of Smads

■ Stimulation by TGFb leads to activation of the intrinsic

serine/threonine kinase activity in the cytosolic domain of the type I
(RI) receptor, which then phosphorylates an R-Smad, exposing a
nuclear-localization signal.

■ After phosphorylated R-Smad binds a co-Smad, the resulting

complex translocates into the nucleus, where it interacts with various
transcription factors to induce expression of target genes.

■ TGFb signaling generally inhibits cell proliferation. Loss of various

components of the signaling pathway contributes to abnormal cell
proliferation and malignancy.

Lodish et al., Molecular Cell Biology, W.H. Freeman

 TGFß* receptors
Receptor Serine Kinases That Activate Smads
- have serine/threonine kinase activity

- directly activate Smads and then the transcription of PAI-1

TGFb-Smad signaling pathway.

Step 1a: In some cells, TGFb binds to the type III TGFb receptor (RIII), which presents it to the
type II receptor (RII). Step 1b: In other cells, TGFb binds directly to RII, a constitutively
phosphorylated and active kinase. Step 2: Ligand-bound RII recruits and phosphorylates the
juxtamembrane segment of the type I receptor (RI), which does not directly bind TGFb. This
releases the inhibition of RI kinase activity that otherwise is imposed by the segment of RI
between the membrane and kinase domain. Step 3: Activated RI then phosphorylates Smad3
(shown here) or another R-Smad, causing a conformational change that unmasks its nuclear-
localization signal (NLS). Step 4: Two phosphorylated molecules of Smad3 interact with a co-
Smad (Smad4), which is not phosphorylated, and with importin b (Imp-b), forming a large
cytosolic complex. Steps 5 and 6: After the entire complex translocates into the nucleus,
RanGTP causes dissociation of Imp-b. Step 7: A nuclear transcription factor (e.g., TFE3) then
associates with the Smad3/Smad4 complex, forming an activation complex that cooperatively
binds in a precise geometry to regulatory sequences of a target gene. Shown at the bottom is
the activation complex for the gene encoding plasminogen activator inhibitor (PAI-1).

*Transforming growth factor beta (TGF-β) is a protein that controls proliferation, cellular differentiation, and
other functions in most cells Lodish et al., Molecular Cell Biology, W.H. Freeman
Receptor Tyrosine Kinases and Activation of Ras

Receptor tyrosine kinases (RTKs), which bind to peptide and protein hormones, may
exist as preformed dimers or dimerize during binding to ligands.

Ligand binding leads to activation of the intrinsic protein tyrosine kinase activity of the
receptor and phosphorylation of tyrosine residues in its cytosolic domain. The activated
receptor also can phosphorylate other protein substrates.

Ras is an intracellular GTPase switch protein that acts downstream from most RTKs. Like
G protein, Ras cycles between an inactive GDP-bound form and an active GTP-bound
form.

Ras cycling requires the assistance of two proteins, a guanine nucleotide–exchange

factor (GEF) and a GTPase-activating protein (GAP).

Lodish et al., Molecular Cell Biology, W.H. Freeman

 Cytokine receptors and receptor tyrosine kinases

- share many signaling features

- the phosphotyrosines of the receptors are docking sites for proteins (with SH2 domains)

General structure and ligand-induced activation of

receptor tyrosine kinases (RTKs) and cytokine receptors.

The cytosolic domain of RTKs contains a protein tyrosine kinase

catalytic site, whereas the cytosolic domain of cytokine receptors
associates with a separate JAK kinase (step 1). In both types of
receptor, ligand binding causes a conformational change that
promotes formation of a functional dimeric receptor, bringing
together two intrinsic or associated kinases, which then
phosphorylate each other on a tyrosine residue in the activation
lip (step 2). Phosphorylation causes the lip to move out of the
kinase catalytic site, thus allowing ATP or a protein substrate to
bind. The activated kinase then phosphorylates other tyrosine
residues in the receptor’s cytosolic domain (step 3). The resulting
phosphotyrosines function as docking sites for various signal-
transduction proteins
Lodish et al., Molecular Cell Biology, W.H. Freeman
Activation of Ras following ligand binding to receptor tyrosine kinases (RTKs). The receptors for epidermal
growth factor (EGF) and many other growth factors are RTKs. The cytosolic adapter protein GRB2 binds to a specific
phosphotyrosine on an activated, ligand-bound receptor and to the cytosolic Sos protein, bringing it near its substrate,
the inactive RasGDP. The guanine nucleotide–exchange factor (GEF) activity of Sos then promotes formation of active
RasGTP. Note that Ras is tethered to the membrane by a hydrophobic farnesyl anchor

Lodish et al., Molecular Cell Biology, W.H. Freeman

Kinase cascade that transmits signals downstream from activated Ras protein to
MAP kinase.
In unstimulated cells, most Ras is in the inactive
form with bound GDP; binding of a ligand to its RTK or cytokine receptor leads to formation of
the active RasGTP complex (step 1).
Activated Ras triggers the downstream kinase cascade depicted in steps 2–6, culminating in
activation of MAP kinase (MAPK). In unstimulated cells, binding of the 14-3-3 protein to Raf
stabilizes it in an inactive conformation. Interaction of the Raf N-terminal regulatory domain
with RasGTP relieves this inhibition, results in dephosphorylation of one of the serines that bind
Raf to 14-3-3, and leads to activation of Raf kinase activity (steps 2 and 3). Note that in contrast
to many other protein kinases, activation of Raf does not depend on phosphorylation of the
activation lip. After inactive RasGDP dissociates from Raf, it presumably can be reactivated by
signals from activated receptors, thereby recruiting additional Raf molecules to the membrane. Lodish et al., Molecular Cell Biology, W.H. Freeman
 Cytokine Receptors and the JAK-STAT Pathway

■ Erythropoietin, a cytokine secreted by kidney cells, prevents apoptosis and promotes

proliferation and differentiation of erythroid progenitor cells in the bone marrow. An excess of
erythropoietin or mutations in its receptor that prevent down-regulation result in production
of elevated numbers of red blood cells.

■ All cytokine receptors are closely associated with a JAK protein tyrosine kinase, which can
activate several downstream signaling pathways leading to changes in transcription of target
genes or in the activity of proteins that do not regulate transcription.
and several SOCS proteins

■ The JAK-STAT pathway operates

downstream of all cytokine receptors.
STAT monomers bound to receptors are
phosphorylated by receptor-associated
JAKs, then dimerize and move to the
nucleus, where they activate
transcription.

Lodish et al., Molecular Cell Biology, W.H. Freeman

JAK-STAT signaling pathway.

Following ligand binding to a cytokine receptor and activation of an associated JAK kinase, JAK phosphorylates several
tyrosine residues on the receptor’s cytosolic domain. After an inactive monomeric STAT transcription factor binds to a
phosphotyrosine in the receptor, it is phosphorylated by active JAK. Phosphorylated STATs spontaneously dissociate from
the receptor and spontaneously dimerize. Because the STAT homodimer has two phosphotyrosine–SH2 domain interactions,
whereas the receptor-STAT complex is stabilized by only one such interaction, phosphorylated STATs tend not to rebind to the
receptor. The STAT dimer, which has two exposed nuclear-localization signals (NLS), moves into the nucleus, where it can
bind to promoter sequences and activate transcription of target genes. Lodish et al., Molecular Cell Biology, W.H. Freeman
Lodish et al., Molecular Cell Biology, W.H. Freeman
 Pathways That Involve Signal-Induced Protein Cleavage

■ Wnt pathway is involved in carcinogenesis and in embryonic development. The canonical

pathway avoids β-catenin degradation that can be translocated into the nucleus promoting
transcription of target genes.

■ Upon binding to its ligand Delta on the surface of an adjacent cell, the Notch receptor
protein undergoes two proteolytic cleavages. The released Notch cytosolic segment then
translocates into the nucleus and modulates gene transcription.

■ The NF-kB transcription factor regulates many genes that permit cells to respond to
infection and inflammation.

■ In unstimulated cells, NF-kB is localized to the cytosol, bound to an inhibitor protein, I-kB.
In response to extracellular signals, phosphorylation-dependent ubiquitination and
degradation of I-kB in proteasomes releases active NF-kB, which translocates to the
nucleus.

Lodish et al., Molecular Cell Biology, W.H. Freeman

 Wnt pathway

The canonical Wnt pathway leads to regulation of gene transcription, the noncanonical planar cell polarity
pathway regulates the cytoskeleton that is responsible for the shape of the cell, and the noncanonical
Wnt/calcium pathway regulates calcium inside the cell. Wnt signaling is involved in carcinogenesis and in
embryonic development.
(a) Canonical pathway, signaling through the Frizzled (Fz)
and LRP5/6 receptor complex induces the stabilization of
β-catenin via the DIX and PDZ domains of Dishevelled (Dsh)
and a number of factors including Axin, glycogen synthase
kinase 3 (GSK3) and casein kinase 1 (CK1). β-catenin
translocates into the nucleus where it complexes with
members of the LEF/TCF family of transcription factors to
mediate transcriptional induction of target genes. β-
catenin is then exported from the nucleus and degraded
via the proteosomal machinery. (b) Non-canonical or
planar cell polarity signaling is transduced via Frizzled
independent of LPR5/6. Utilizing the PDZ and DEP domains
of Dsh, this pathway mediates cytoskeletal changes
through activation of the small GTPases Rho and Rac. (c)
Wnt-Ca2+ pathway: Frizzled mediates activation of
heterotrimeric G-proteins, which engage Dsh,
phospholipase C (not shown), calcium-calmodulin kinase 2
(CamK2) and protein kinase C (PKC).
Journal of Biology 2005, 4:2 doi:10.1186/jbiol22
Notch/Delta signaling pathway

The extracellular subunit of Notch on the

responding cell is non covalently associated with
its transmembrane-cytosolic subunit. Binding of
Notch to its ligand Delta on an adjacent signaling
cell (step 1) first triggers cleavage of Notch by
the membrane-bound metalloprotease TACE
(tumor necrosis factor alpha converting enzyme),
releasing the extracellular segment (step 2).
Presenilin 1, an integral membrane protein, then
catalyzes na intramembrane cleavage that
releases the cytosolic segment of Notch (step 3).
Following translocation to the nucleus, this
Notch segment interacts with several
transcription factors that act to affect expression
of genes that in turn influence the determination
of cell fate during development (step 4).
Lodish et al., Molecular Cell Biology, W.H. Freeman
NF-kB signaling pathway

In resting cells, the dimeric transcription factor NF-kB,

composed of p50 and p65, is sequestered in the cytosol,
bound to the inhibitor I-kB. Stimulation by TNF-a or IL-1
induces activation of TAK1 kinase (step 1), leading to
activation of the trimeric I-kB kinase (step 2a). Ionizing
radiation and other stresses can directly activate I-kB
kinase by an unknown mechanism (step 2b). Following
phosphorylation of I-kB by I-kB kinase and binding of E3
ubiquitin ligase (step 3), polyubiquitination of I-kB (step
4) targets it for degradation by proteasomes (step 5).
The removal of I-kB unmasks the nuclear-localization
signals (NLS) in both subunits of NF-kB, allowing their
translocation to the nucleus (step 6). Here NF-kB
activates transcription of numerous target genes (step
7), including the gene encoding the subunit of I-kB,
which acts to terminate signaling.

Lodish et al., Molecular Cell Biology, W.H. Freeman

Integration of Cellular Responses to Multiple Signaling Pathways: Insulin Action
■ Insulin and glucagon work together to maintain a stable blood glucose level
■ A rise in blood glucose triggers insulin secretion from the 𝛃 islet cells
■ In fat and muscle cells, insulin triggers fusion of intracellular vesicles containing the GLUT4
glucose transporter to the plasma membrane
■ Insulin inhibits glucose synthesis and enhances storage of glucose as glycogen

Secretion of insulin in response to a rise in blood glucose. The entry of

glucose into pancreatic β cells is mediated by the GLUT2 glucose
transporter (step 1). The conversion of glucose into pyruvate is thus
accelerated, resulting in an increase in the concentration of ATP in the
cytosol (step 2). The binding of ATP to ATP-sensitive K+ channels in the β
cells closes those channels (step 3), thus reducing the efflux of K+ ions
from the cell. The resulting small depolarization of the plasma membrane
(step 4) triggers the opening of voltage- sensitive Ca2+ channels (step 5).
The influx of Ca2+ ions raises the cytosolic Ca2+ concentration, triggering
the fusion of insulin-containing secretory vesicles with the plasma
membrane and the secretion of insulin (step 6).
Lodish et al., Molecular Cell Biology, W.H. Freeman
FURTHER READING
Induction of gene transcription by activated MAP kinase.
In the cytosol, MAP kinase phosphorylates and activates the kinase p90RSK,
which then moves into the nucleus and phosphorylates the SRF transcription
factor. After translocating into the nucleus, MAP kinase directly phosphorylates
the transcription factor TCF. Together, these phosphorylation events stimulate
transcription of genes (e.g., c-fos) that contain an SRE sequence in their
promoter.

MAP kinase pathways that are triggered by activation of various

receptor classes including GPCRs

Lodish et al., Molecular Cell Biology, W.H. Freeman

Two mechanisms for terminating signal transduction from the EpoR
(a) SHP1, a protein tyrosine phosphatase, is present in an inactive form in unstimulated cells. Binding of an SH2 domain in SHP1
to a particular phosphotyrosine in the activated receptor unmasks its phosphatase catalytic site and positions it near the
phosphorylated tyrosine in the lip region of JAK2. Removal of the phosphate from this tyrosine inactivates the JAK kinase.

(b) SOCS proteins, whose expression is induced in erythropoietin-stimulated erythroid cells, inhibit or permanently terminate
signaling over longer time periods. Binding of SOCS to phosphotyrosine residues on the EpoR or JAK2 blocks binding of other
signaling proteins (left). The SOCS box can also target proteins such as JAK2 for degradation by the ubiquitin proteasome
pathway (right). Similar mechanisms regulate signaling from other cytokine receptors.
Lodish et al., Molecular Cell Biology, W.H. Freeman
- Many cytokine (and RTKs) receptors can initiate the IP3/DAG signaling pathway by
activating PI-3 kinase and PLC (a different PLC isoform than the one activated by GPCRs)

Overview of signal-transduction pathways triggered by ligand binding to the erythropoietin receptor

(EpoR), a typical cytokine receptor. (c, d) Two phosphoinositide pathways are triggered by recruitment of
phospholipase C and PI-3 kinase to the membrane following activation of EpoR. Elevated levels of Ca2+
and activated protein kinase B also modulate the activity of cytosolic proteins that are not involved in
control of transcription.
Lodish et al., Molecular Cell Biology, W.H. Freeman
 Inhibition of Signaling Cascades in Myeloma Cells

metinib
Selu

ib
olitin
Rux

b
tilisi
Pic

Antibodies targeting IL-R6 (e.g. Tocilizumab, Sarilumab) or TNFa (Adalimumab)

*Multiple myeloma is a B-cell malignancy that is characterized by an excess of monotypic plasma cells in the bone marrow
Nat Rev Cancer. 2002 Dec;2(12):927-37; https://www.selleckchem.com/
Integration of Cellular Responses to Multiple
Signaling Pathways: Insulin Action

Insulin stimulation of fat cells induces translocation of GLUT4 from intracellular

vesicles to the plasma membrane. In fat and muscle cells, insulin signaling acts in
multiple steps to increase the level of GLUT4 at the plasma membrane. In resting
cells, the majority of the GLUT4 protein is localized to specialized GLUT4 storage
vesicles, tethered to Golgi matrix proteins by the TUG protein. Binding of insulin to
the insulin receptor leads to activation of a protease (step 1) that cleaves the TUG
protein, releasing GLUT4-containing vesicles (step 2), which then move along
microtubules, powered by a kinesin motor, to the cell surface. Insulin also activates
PKB (step 3). PKB then phosphorylates the Rab GAP protein AS160 (step 4),
inhibiting its ability to accelerate GTP hydrolysis by Rab proteins, which accumulate
in their active GTP-bound states (step 5) and allow the GLUT4 storage vesicles to
move along microtubules to the cell surface (steps 6a and 6b). Finally, these
vesicles fuse with the plasma membrane (step 7). This step is catalyzed by the
exocyst and also by another monomeric GTP-binding protein, RALA. PKB stimulates
this membrane fusion event by phosphorylating and thus inactivating the RALA GAP
protein RGC (step 8), allowing RALA to accumulate in its active GTP-bound state
(step 9). The resultant increase in plasma membrane GLUT4 allows the cell to
incorporate glucose from the extracellular fluids at a rate about 10 times that of
unstimulated cells (step 10). Following removal of insulin, the plasma membrane
GLUT4 is internalized by endocytosis (step 11) and eventually transported to
vesicles (step 12). Many other proteins, not shown here, participate in these
signaling and vesicle budding and fusion events.
Lodish et al., Molecular Cell Biology, W.H. Freeman
 Regulation of glycogen metabolism by cAMP in liver and muscle cells

Active enzymes are highlighted in darker shades; inactive forms, in lighter shades. (a) An increase in cytosolic cAMP activates PKA, which inhibits glycogen
synthesis directly and promotes glycogen degradation via a protein kinase cascade. At high cAMP, PKA also phosphorylates an inhibitor of phosphoprotein
phosphatase (PP). Binding of the phosphorylated inhibitor to PP prevents this phosphatase from dephosphorylating the activated enzymes in the kinase
cascade or the inactive glycogen synthase. (b) A decrease in cAMP inactivates PKA, leading to release of the active form of phosphoprotein phosphatase. The
action of this enzyme promotes glycogen synthesis and inhibits glycogen degradation.
Lodish et al., Molecular Cell Biology, W.H. Freeman
Regulation of glycogen metabolism by cAMP in liver and muscle cells

Lodish et al., Molecular Cell Biology, W.H. Freeman

 Next Class…

• Module:

A. Cell and Tissue Biology

 Todays’ Summary

Integration of Cellular Responses

Gene silencing and Regulation
Architecture and functional role of tissues:

- epithelial, connective, muscular and nervous tissues.

 Gene silencing
- Interruption or suppression of the expression of a gene at transcriptional (=epigenetics*) or post-transcriptional levels

- When genes are silenced or knockdown, their expression is reduced or even abolished (is often confused with gene
knockout)

*Epigenetics: “the study of heritable changes in gene function that do not involve changes in the DNA sequence”

Epigenetic changes are visible in tortoiseshell female cats. Depending upon which X-chromosome
becomes inactivated, some skin cells give rise to orange fur while others give rise to black fur.

http://californiaagriculture.ucanr.edu/landingpage.cfm?article=ca.v060n03p132&fulltext=yes
 Transcriptional gene silencing (TGS)
- is the result of histone modifications (methylation, acetylation, phosphorylation, ubiquitylation, and
sumoylation) and DNA methylation, creating an environment of heterochromatin around a gene that
makes it inaccessible to transcriptional machinery.

- DNA methylation of a gene promoter typically acts to repress its transcription

- Histone modifications act in transcriptional activation/inactivation
 Post-transcriptional gene silencing (PTGS)
is the result of degradation of a specific mRNA preventing its translation. A usual mechanism of post-transcriptional gene
silencing is RNAi.
Gene silencing mechanisms of siRNA and miRNA

siRNA: dsRNA (either transcribed or artificially introduced) is

processed by Dicer into siRNA which is loaded into the RISC. AGO2,
which is a component of RISC, cleaves the passenger strand of
siRNA. The guide strand then guides the active RISC to the target
mRNA. The full complementary binding between the guide strand
of siRNA and the target mRNA leads to the cleavage of mRNA.

miRNA: Transcription of miRNA gene is carried out by RNA

polymerase II in the nucleus to give pri-miRNA, which is then
cleaved by Drosha to form pre-miRNA. The pre-miRNA is
transported by Exportin 5 to the cytoplasm where it is processed by
Dicer into miRNA. The miRNA is loaded into the RISC where the
passenger strand is discarded, and the miRISC is guided by the
remaining guide strand to the target mRNA through partially
complementary binding. The target mRNA is inhibited via
translational repression, degradation or cleavage.
Nucleic Acids (2015) 4, e252; doi:10.1038/mtna.2015.23
 Post-transcriptional gene regulation by mRNA modifications

The recent discovery of reversible mRNA methylation has opened a new realm of post-transcriptional gene regulation in eukaryotes.

The identification and functional characterization of proteins that specifically recognize RNA m6A (N6-methyladenosine) unveiled it as a
modification that cells utilize to accelerate mRNA metabolism (processing in the nucleus to translation and decay in cytoplasm) and
translation

Nat Rev Mol Cell Biol. 18:202-210 (2017)

 Post-transcriptional gene regulation by mRNA modifications

Post-transcriptional Processing of mRNA

Garrett & Grisham, Biochemistry, Saunders College Publishing

 Tissue Biology
A tissue is a collection of cells and ECM that
perform a given function.

Organs are composed of parenchyma, made up by

cells responsible for the function of the organ and
stroma which is the supporting tissue
> 200 cell types (7 main types) in mature human body

Epithelial cells:
-Usually grow in contiguous 2D sheets
-Tightly connected with their neighbors (cannot migrate)
-They have polarity
-They are bound to a basal lamina

Mesenchymal cells:
-Usually exist alone
-Have a bipolar shape
-They can migrate
-Their growth is contact-inhibited
-They can differentiate into osteoblasts, chondrocytes,
fibroblasts
 Epithelial tissue
Epithelial tissues are composed of closely aggregated polyhedral cells with very little extracellular substance but showing
strong adherence to each other (tight junctions, desmosomes, adherens junctions, gap junctions). Are not irrigated by blood
vessels.

Almost all epithelia are separated

from the connective tissue by the
basal lamina (or basement membrane)

Main functions:
Covering and lining of surfaces (e.g. skin, intestines), absorption (e.g. intestines), secretion (e.g. glands), sensation
(e.g. olfactory neuroepithelium)

Epithelia classification is based on:

- Number of cell layers: simple (one sheet) or stratified (multilayered)

- Cell (and also nucleus) shape: squamous (“pavimentoso”) (flattened), cuboid, columnar or pseudostratified (has only one
cell layer but looks like more)
- Presence of cell surface specializations (Microvilli that increase the cell surface area; and Cilia that allows a current of
fluid to be propelled in one direction)
Junqueira & Carneiro, Basic Histology, McGraw-Hill
 Common types of covering epithelia in the human body
Type Cell form Examples of distribution Main function

Squamous Lining vessels Facilitates the movement of

(endothelium). Serous lining viscera (mesothelium), active
of cavities: pericardium, transport by pinocytosis (meso-
pleura, peritoneum & endothelium), secretion of
(mesothelium) biologically active molecules
(mesothelium)
Cuboid Covering the ovary, thyroid Covering, secretion, ciliated
Simple epithelia in female reproductive
system

Columnar Lining the intestine, Protection, lubrication,

Junqueira & Carneiro, Basic Histology, McGraw-Hill

stomach, gallbladder absorption, secretion

Pseudostratified Some columnar Lining of trachea, bronchi, Protection, secretion, cilia-

and some cuboidal nasal cavity mediated transport of particles
trapped in mucus
 Common types of covering epithelia in the human body

Type Cell form Examples of distribution Main function

Surface layer squamous Epidermis Protection, prevents water

keratinized (dry) loss

Surface layer squamous Mouth, esophagus, larynx, vagina, Protection, secretion,

nonkeratinized (moist) anal canal prevents water loss
Cuboid Sweat glands, developing ovarian Protection, secretion
Stratified follicles
Transitional (urothelium) Bladder, ureters, renal calyces Protection, distensibility; it
is cuboidal when is not
stretched or squamous
when the organ is
distended
Columnar Conjunctiva Protection

Junqueira & Carneiro, Basic Histology, McGraw-Hill

Endothelium lines blood and lymph Mesothelium lines certain body cavities
vessels (pericardium, pleura, peritoneum)

Section of a vein containing red blood cells. All The simple squamous epithelium that
blood vessels are lined with a simple squamous covers the body cavities (the abdominal
epithelium called endothelium (arrowheads) cavity in this case) is called mesothelium

Junqueira & Carneiro, Basic Histology, McGraw-Hill

 Connective tissue (“conjuntivo”)
Connective tissues are composed mainly of ECM (unlike other tissues). The wide variety reflects
variations in the composition and amount of cells and ECM. Are originated from the mesenchyme
(an embryonic tissue) that develops from mesoderm.

Main functions:
Provide and maintain form in the body, and structural and metabolic aid for other tissues

Blood
 Cells of the connective tissue
Cell type Function
Fibroblast, chondroblast, osteoblast Production of ECM - Structural
Plasma cell Production of antibodies – Immunological
Lymphocytes Production of immunocompetent cells - Immunological
Eosinophils Allergic, vasoactive, inflammatory processes – Immunological
Neutrophils Phagocytosis - Defense
Macrophages Secretion of cytokines - Defense
Mast cells and basophils Liberation of active molecules (e.g. histamine) - Defense
Adipose (fat) cell Storage of fats – Energy reservoir, heat production

Note: Adipocyte, megakaryocyte, and osteoclast cells are significantly larger than the other cells illustrated. Junqueira & Carneiro, Basic Histology, McGraw-Hill
Fibroblasts synthesize and secrete ECM proteins, GAGs and proteoglycans and also
growth factors (involved in growth and differentiation).

In adults, fibroblasts rarely divide unless additional fibroblasts are needed

Active (left) and quiescent (right) fibroblasts. Quiescent fibroblasts are elongated cells
Fibroblasts that are actively engaged in synthesis are with thin cytoplasmic extensions
richer in mitochondria, Golgi complex, and rough ER
than are quiescent fibroblasts (fibrocytes).
Junqueira & Carneiro, Basic Histology, McGraw-Hill
Macrophages and the mononuclear phagocyte system

Macrophages are polymorphic phagocytic cells

Electron micrograph of a macrophage, lysosomes (L),

nucleus (N), nucleolus (Nu). The arrows indicate
phagocytic vacuoles.

Cell type Location Main function

Monocyte Blood Precursor of macrophages
Macrophage Connective tissue Production of cytokines involved in inflammation,
antigen processing and presentation
Kupffer cell Liver Same as macrophages
Microglia cell Nerve tissue of CNS Same as macrophages

Langerhans cell Skin Antigen processing and presentation

Dendritic cell Lymph nodes Antigen processing and presentation
Osteoclast Bone Digestion of the bone
Multinuclear giant cell Connective tissue Segregation and digestion of foreign bodies
Junqueira & Carneiro, Basic Histology, McGraw-Hill
Plasma cells (plasmocytes) are derived from B lymphocytes and produces antibodies

Ultrastructure of a plasma cell. The cell contains

a well-developed rough ER, with dilated Electron micrograph of a plasma cell showing an
abundance of rough ER (R). Note that many cisternae
cisternae containing immunoglobulins
(antibodies). In plasma cells, the secreted are dilated. Four profiles of the Golgi complex (G) are
observed near the nucleus (N). M, mitochondria.
proteins do not aggregate into secretory
granules. Nu, nucleolus.
Junqueira & Carneiro, Basic Histology, McGraw-Hill
Adipose tissue is rich in adipocytes which are also found isolated or in small groups in other
connective tissues is highly vascularized. It is the largest organ in the body… (in men 15-20% and in
women 20-25% of body weight)

Yellow/white (unilocular) adipose tissue stores energy as

triglycerides, 9.3 kcal/g (muscles and liver also store
energy but in glycogen form, 4.1 kcal/g).

Brown (multilocular) adipose tissue produces heat and

is abundant in newborns (and hibernating animals)

Photomicrograph of multilocular adipose tissue (lower portion) with its

characteristic cells containing central spherical nuclei and multiple lipid
droplets. For comparison, the upper part of the photomicrograph shows
unilocular tissue (showing adipocytes’ nuclei of compressed against the
cell membrane).

Junqueira & Carneiro, Basic Histology, McGraw-Hill

Cartilage tissue is characterized by chondrocytes and an ECM enriched with GAGs and
proteoglycans. Variations in the ECM composition originate hyaline, elastic and fibrous
cartilages.

Photomicrograph of hyaline cartilage. In embryo serves as a temporary skeleton. In adults is located

in the articular surfaces of movable joints, in the walls of the larger respiratory passages (nose,
larynx, trachea, bronchi), in the ventral ends of ribs and at the ends of bones (epiphyseal plate)
Chondrocytes are located in matrix lacunae. The upper and lower parts of the figure show the
perichondrium stained pink. Note the gradual differentiation of cells from the perichondrium into
chondrocytes.
Junqueira & Carneiro, Basic Histology, McGraw-Hill
Bone tissue supports fleshy structures, protects vital organs (cranial and thoracic cavities) and harboursthe
bone marrow where blood is formed. Is highly vascularized and metabolically active. It serves as a reservoir of
ions (calcium, phosphate, etc).

Has a mineralized ECM and inside lacunae, osteocytes/osteoblasts which synthesize the organic ECM, and
osteoclasts which make reabsorption and remodeling of the bone tissue.

Photomicrograph of bone. The lacunae and canaliculi

filled with air deflect the light and appear dark, showing Schematic drawing of a long-bone diaphysis. At the right
the communication between these structures through is a haversian system showing lamellae, a central blood
which nutrients derived from blood vessels flow. capillary (there are also small nerves, not shown).
Junqueira & Carneiro, Basic Histology, McGraw-Hill
 Bone resorption

Lysosomal enzymes packaged in the Golgi complex and protons are released into the bone
matrix. The acidification facilitates the dissolution of calcium phosphate from bone and is
the optimal pH for the activity of lysosomal hydrolases (e.g. collagenases). Bone matrix is
thus removed and the products of bone resorption are taken up by the osteoclast’s
cytoplasm, probably digested further, and transferred to blood capillaries.

Junqueira & Carneiro, Basic Histology, McGraw-Hill

Blood consists of cells (erythrocytes, platelets (thrombocytes), and leukocytes) and plasma (ECM):
albumins, g-globulins, lipoproteins, prothrombin and fibrinogen, signaling molecules, water and ions.
(serum is plasma without the coagulated proteins)

Cell type Main products and functions

Erythrocyte Hemoglobin - CO2 and O2 transport
Platelet Blood-clotting factors - Clotting of blood
Neutrophil Rich in specific granules - Phagocytosis of bacteria
Eosinophil Rich in specific granules - Defense against parasites; modulation of inflammation
processes
Basophil Rich in specific granules - Inflammation mediation
Monocyte Rich in specific granules - Phagocytosis of protozoa and virus and senescent cells
B lymphocyte Immunoglobulins - Production of antibodies
T lymphocyte Killing of virus infected cells and modulation of other leukocytes (interleukins)
Natural killer cell Attacks some tumor and virus-infected cells

“Granules” are vesicles and lysosomes rich in enzymes, proteins carbohydrates and signaling molecules

Junqueira & Carneiro, Basic Histology, McGraw-Hill

Main components and functions of blood

Junqueira & Carneiro, Basic Histology, McGraw-Hill

 Bone marrow (myeloid tissue) is found in the hollow interior of bones. It constitutes 4% of total body
weight, and is responsible for hematopoiesis (erythropoiesis, granulonopoiesis, monocytopoiesis,
megacaryocytopoiesis or thrombopoiesis).

- Network of stromal cells (fibroblasts, macrophages, adipocytes, osteoblasts,

osteoclasts, endothelial cells forming the sinusoids) and hematopoietic cells
- Produces 2.5x109 erythrocytes and 2.5x109 platelets and 50-100x109
granulocytes per day and per kg of body weight!
- Removes (like liver and spleen) damaged erythrocytes
- Is the place for B lymphocytes maturation

Section of active red bone marrow showing

some of its components. Six blood sinusoid
capillaries containing many erythrocytes are
indicated by arrowheads.

A femur showing its red bone marrow and a focus of yellow bone marrow consisting mainly of fat
cells (progressively substitutes red marrow in adults)
Junqueira & Carneiro, Basic Histology, McGraw-Hill
Lymphoid tissue. Circulating lymphocytes originate (lymphopoiesis) mainly in the thymus
and the peripheral lymphoid organs (spleen, lymph nodes, tonsils). Some migrate to thymus
where they become T-lymphocytes and other differentiate at bone marrow, B-lymphocytes

The lymphoid organs and lymphatic vessels are widely distributed in the body. The lymphatic
vessels collect lymph from most parts of the body and deliver it to the blood circulation primarily
through the thoracic duct.
Muscle tissue is divided in 3 types:

Cardiac muscle is composed of

irregular branched cells bound
together longitudinally by
intercalated disks.

Smooth muscle is an agglomerate of

fusiform cells.

Skeletal muscle is composed of

large, elongated, multinucleated
fibers.
- Skeletal muscle contracts quickly, forcefully and under voluntary control
- Long multinucleated fibers (cells), up to 35 cm in length and 10-100 µm in diameter, form
bundles, and result from the fusion of embryonic mononucleated myoblasts
- Can undergo limited regeneration (from inactive myoblasts)

Striated skeletal muscle in

longitudinal and cross
Longitudinal section of striated sections. The nuclei can be
muscle fibers. The blood vessels
seen in the periphery of
were injected with a plastic material
the cell.
before the animal was killed.
Junqueira & Carneiro, Basic Histology, McGraw-Hill
Schematic representation of the thick (myosin) and thin
filament (actin, tropomyosin, and troponin (TnI, TnC, and TnT))

Junqueira & Carneiro, Basic Histology, McGraw-Hill

 Sequential activation of gated ion channels at a neuromuscular junction
(or motor end-plate)

Arrival of an action potential at the terminus of a presynaptic motor neuron induces opening of voltage-gated Ca2+ channels
(step 1) and subsequent release of acetylcholine, which triggers opening of the ligand-gated nicotinic receptors in the
muscle plasma membrane (step 2). The resulting influx of Na+ produces a localized depolarization of the membrane, leading
to opening of voltage-gated Na+ channels and generation of an action potential (step 3). When the spreading depolarization
reaches T tubules, it triggers opening of voltage-gated Ca2+ channels and release of Ca2+ from the sarcoplasmic reticulum
into the cytosol (step 4). The rise in cytosolic Ca2+ causes muscle contraction (see next slide).
Lodish et al., Molecular Cell Biology, W.H. Freeman
 Molecular mechanism of contraction

Muscle contraction, initiated by the binding of Ca2+ to the TnC unit of troponin, which exposes the myosin binding site on
actin (cross-hatched area). In a second step, the myosin head binds to actin and the ATP is hydrolysed yielding energy,
which produces a movement of the myosin head. As a consequence of this change in myosin, the bound thin filaments
slide over the thick filaments reducing the distance between the Z lines, thereby shortening of the whole muscle fiber.

Junqueira & Carneiro, Basic Histology, McGraw-Hill

- Cardiac muscle contracts vigorously, rhythmically and under involuntary control
- Cells are 85-100 µm in length and 15 µm in diameter
- Has almost no regenerative capacity beyond early childhood.

Photomicrograph of cardiac muscle. Note the cross-

striation and the intercalated disks (arrowheads)
which are enriched in gap junctions to assure
ionic continuity between adjacent cells. Thus,
muscle to act as a syncytium allowing the signal to
contract to pass in a wave from cell to cell.
Junqueira & Carneiro, Basic Histology, McGraw-Hill
- Smooth muscle contracts slowly and under involuntary control
- Lengths from 20 to 500 µm
- Contraction does not rely in a paracrystalline organization of actin and myosin and
depends on the phosphorylation of myosin and of calcium binding protein, calmodulin, but
not dependent of tropomyosin
- Smooth muscle is capable of active regeneration

Photomicrographs of smooth muscle cells in cross

section (upper) and in longitudinal section (lower).
Note the centrally located nuclei.
Junqueira & Carneiro, Basic Histology, McGraw-Hill
 Nerve tissue is composed of nerve cells (neurons) which sense, process and respond to features of both
internal and external environment, glial cells (neuroglia) which occupy space between neurons and modulate their
functions. Each neuron has many interconnections with other neurons.

General functional organization of CNS and PNS

Junqueira & Carneiro, Basic Histology, McGraw-Hill

Neurons are responsible for the reception, transmission, and processing of stimuli, the triggering of certain cell
activities, and release of neurotransmitters. Generally, receive information via their dendrites and transmit
information via their axons to other neurons or other cells forming synapses.

Where neurons and their target cells

meet, information is transmitted
across synapses by the release of
neurotransmitters.
 Membrane potential
Neurons have an electric charge difference across their plasma membranes. The difference in voltage across
membrane is called membrane potential. In an unstimulated neuron is called resting potential. Nerve impulses
are also called action potentials and travel along the plasma membrane

- The negative resting potential is created by the 3Na+/2K+ ATPase and K+ and Na+ ion channels.
- Na+- K+ pump moves 2 K+ ions inside the cell as 3 Na+ ions are pumped out.
- K+ ions diffuse out of the cell at a faster rate than Na+ ions diffuse into the cell because neurons have more K+ leakage channels than
Na+ leakage channels.
Action potentials (speed up to 100 m/s) result
from rapid changes in voltage-gated Na+ and K
+
channels.

An action potential is a rapid reversal in charge

across a portion of the plasma membrane
resulting from the sequential opening and
closing of voltage-gated sodium and potassium
channels. These changes in voltage-gated
channels occur when the plasma membrane
depolarizes to a threshold level.
Synapses are functional connections
for communication between neurons or
neurons and other cells

Synaptic transmission
begins with the arrival
of an action potential

Synapses can be
excitatory or
inhibitory (the
neuromuscular is always
excitatory)
 Molecular mechanism of vesicle fusion

Synaptotagmin

SNAREs can be divided into two

categories: vesicle or v-SNAREs,
which are incorporated into the Ca2+
membranes of transport vesicles SNARE complex
during budding, and target or t-
SNAREs, which are associated with
nerve terminal membranes.

But other proteins are involved!

Annu. Rev. Biophys. 2015. 44:339–367
FURTHER READING
The next 6 slides are to help you to remember Transcription, Post-transcription, Translation,
Post-translation mechanisms which are essential for understanding cell-cell signaling

Garrett & Grisham, Biochemistry, Saunders College Publishing

 Transcription

Post-transcriptional Processing of mRNA

Garrett & Grisham, Biochemistry, Saunders College Publishing

Protein Synthesis

Garrett & Grisham, Biochemistry, Saunders College Publishing

Protein sorting (“distribuição”)

Lodish et al., Molecular Cell Biology, W.H. Freeman

 Protein Folding

Protein Translocation

Garrett & Grisham, Biochemistry, Saunders College Publishing

 Protein Degradation

- Some protein degradation pathways are nonspecific. But there is also a selective, ATP-dependent pathway for
degradation - the ubiquitin-mediated pathway

Garrett & Grisham, Biochemistry, Saunders College Publishing

DNA methylation at the 5 position of cytosine has the specific effect of reducing gene expression. In adult somatic cells, DNA methylation
typically occurs in a CpG dinucleotide context; non-CpG methylation is prevalent in embryonic stem cells, and has also been indicated in
neural development. Furthermore, non-CpG methylation has also been observed in hematopoietic progenitor cells, and it occurred
mainly in a CpApC sequence context. (https://en.wikipedia.org/wiki/DNA_methylation)

Cytosine 5-Methylcytosine

By Christoph Bock (Max Planck) wikimedia commons

N6-methyladenine increased levels leads to transcriptional silencing and constitutes a crucial component of the epigenetic regulation
repertoire in mammalian genomes.
Nature 532: 329-333 (2016)

Adenine N6-methyladenine
Chromatin modifications

Protein & Cell 4: 656-663 (2013)

 Epigenetic regulation of haematopoietic stem cell function

Key epigenetic regulators (pink) are shown superimposed

on hematopoietic stem cell (HSC) differentiation and
maintenance events.

5-mC, 5-methylcytosine
CBX, chromobox
CLP, common lymphoid progenitor; CMP
common myeloid progenitor
DNMT, DNA methyltransferase
GMP, granulocyte−monocyte progenitor
MEP, megakaryocyte−erythrocyte progenitor
MPP, multipotent progenitor
NK, natural killer
PRC, Polycomb repressive complex
TET, ten-eleven translocation
TrxG, Trithorax group

a) HSCs give rise to all major cell types in peripheral blood and bone marrow. Epigenetic regulators involved in HSC proliferation, self-renewal and lineage
commitment during differentiation are indicated.
b) DNA methylation levels change in a dynamic and highly locus-specific manner during hematopoietic lineage commitment
DNMT1, the DNA methyltransferase that is responsible for maintaining the pattern of DNA methylation after replication, by depositing 5-mC on hemi-methylated DNA. DNMT3A and DNMT3B are responsible for establishing
the de novo pattern of DNA methylation on unmethylated cytosines during cell fate determination in embryonic development and adult homeostasis.
TrxG complex counteracts the activity of Polycomb group proteins to drive gene expression, mainly by marking genes with histone 3 Lys4 (H3K4) methylation.
TET2 converts 5-mC to 5-hydroxymC, which can function as a first step towards DNA demethylation or can have a stable functional role in gene regulation.
PRC is involved in stabilizing gene repression during development and adult homeostasis by marking genes with histone 2A Lys119 monoubiquitylation (H2AK119Ub1) or with histone H3 Lys27 trimethylation (H3K27me3).

Nat Rev Mol Cell Biol 17: 643-658 (2016)

 Epigenetic drugs in the market

Epigenetic drugs make it possible to reverse the aberrant gene expression which leads to various disease states. The inhibitors,
DNA methyltransferase (DNMT) and Histone Deacetylase (HDAC) are responsible for regulating the cellular expression.
Currently, several drugs are approved by FDA and are commercially available. For an overview of epigenetics drugs see Genetics & Epigenetics
2014:6 9–19 doi:10.4137/GEG.S12270.

“A recent study found that 1% of all drugs that have been approved by the US FDA show significant epigenetic activity and
silence promoters in colon cancer cells”, in EMBO reports 16: 276-279 (2015)

Target Drug Company Indications

Myelodysplastic Syndrome (MDS)
DNMT inhibitor Vidaza (azacitidin) Celgene
Acute myeloid leukaemia (AML)
Eisai/Johnson Myelodysplastic Syndrome (MDS)
DNMT inhibitor Dacogen (decitabine)
& Johnson Acute myeloid leukaemia (AML)
HDAC inhibitor Zolinza (vorinostat) Merck Cutaneous T Cell Lymphoma (CTCL)

HDAC inhibitor Istodax (romidepsin) Celgene Peripheral T-cell lymphoma (PTCL)

HDAC inhibitor Farydak (Panobinostat ) Novartis Multiple myeloma

 The role of microRNAs in cell fate determination of mesenchymal stem cells:
balancing adipogenesis and osteogenesis

miRNAs that control signaling governing osteogenesis and adipogenesis

BMP (TGFß superfamily) and Wnt signaling pathways have been demonstrated to preferentially induce
the osteogenesis of MSCs at the expense of adipogenesis. miR-17-5p/miR-106a and miR-30c/miR-30d
inhibit BMP signaling by targeting key components of the pathway, such as BMP2 and Smad1,
respectively. miR-30e inhibits Wnt signaling via the repression of LPR6, a key co-receptor of Wnt.

BMB Rep. 2015; 48(6): 319-323

MicroRNA-34/449 controls mitotic Correct orientation of the mitotic spindle determines the plane of cellular
spindle orientation during cleavage and is crucial for organ development. In the developing cerebral
mammalian cortex development cortex, spindle orientation defects result in severe neurodevelopmental
EMBO J. 35: 2386–2398 (2016) doi:10.15252/embj.201694056 disorders, but the precise mechanisms that control this important event
are not fully understood. Here, we use a combination of high-content
screening and mouse genetics to identify the miR-34/449 family as key
regulators of mitotic spindle orientation in the developing cerebral cortex.
By screening through all cortically expressed miRNAs in HeLa cells, we show
that several members of the miR-34/449 family control mitotic duration
and spindle rotation. Analysis of miR-34/449 knockout (KO) mouse
embryos demonstrates significant spindle misorientation phenotypes in
cortical progenitors, resulting in an excess of radial glia cells at the expense
of intermediate progenitors and a significant delay in neurogenesis. We
identify the junction adhesion molecule-A (JAM-A) as a key target for miR-
34/449 in the developing cortex that might be responsible for those
defects. Our data indicate that miRNA-dependent regulation of mitotic
spindle orientation is crucial for cell fate specification during mammalian
neurogenesis.
 Post-transcriptional gene regulation by mRNA modifications
m6A regulators in humans

Nat Rev Mol Cell Biol. 18:202-210 (2017)

 Post-transcriptional gene regulation by mRNA modifications

m6A and other mRNA post-transcriptional modifications

Qualitative distribution profiles of N1-methyladenosine (m1A; purple) and N6-methyladenosine (m6A; red) in mRNA. m1A
is found primarily near translation start codons and first splice sites, whereas m6A is primarily found in long exons and
within 3ʹ untranslated regions (3ʹ UTRs). In addition to m6A and m1A, other chemical modifications found on eukaryotic
mRNA with emerging regulatory functions include 5-methylcytosine (m5C), pseudouridine (ψ) and 2ʹ-O-methylation
(2ʹOMe). CDS, coding DNA sequence.

Nat Rev Mol Cell Biol. 18:202-210 (2017)

 The major organs systems of mammals
System Tissues and organs Functions

Nervous system Brain, spinal cord, sensory organs, peripheral nerves Receives, integrates, stores information and controls
muscles and glands

Endocrine system Glands: pituitary, thyroid, parathyroid, pineal, adrenal, A system of glands releases chemical messages
testes, ovaries, pancreas (hormones) that control and regulate other tissues

Muscle system Skeletal muscle, smooth muscle, cardiac muscle Produces forces and motion

Skeletal system Bones Provides structural support for the body

Reproductive Female: ovaries, oviducts, uterus, vagina, mammary glands Produces sex cells and hormones necessary to procreate
system Male: testes, sperm ducts, accessory glands, penis and nurture offspring

Digestive system Mouth, esophagus, stomach, intestines, liver, pancreas, Acquires and digests food, absorbs and stores nutrients,
rectum, anus then make them available to the cells of the body
Respiratory system Airways, lungs, diaphragm Exchanges respiratory gases with the environment
Circulatory system Heart and blood vessels Transports respiratory gases, nutrients, hormones, and
heat around the body
Lymphatic system Lymph and lymph vessels, lymph nodes, spleen Brings extracellular fluids back into the circulatory
system; helps the immune system fight invading
organisms

Immune system Many type of blood cells Fights invading organisms and infections

Skin system Skin, sweat glands, hair Protects the body from invading organisms and harsh
physical conditions, helps regulate body temperature
Excretory system Kidneys, bladder, ureter, urethra Regulates the composition of the extracellular fluids;
excretes waste products
 Common types of
covering epithelia in
the human body

Simple columnar epithelium formed by long cells with elliptical nuclei.

The epithelium rests on the loose connective tissue of the lamina
propria. A basal lamina (not visible) is interposed between the
epithelial cells and the connective tissue. The round nuclei within the
epithelial layer belong to lymphocytes that are migrating through the
epithelium (arrows)
Stratified squamous nonkeratinized (moist)
epithelium of the esophagus

Pseudostratified columnar epithelium of the trachea, formed

by long and short cells. As some cells do not reach the surface
of the epithelium their nuclei are present in different heights of
Stratified transitional epithelium of the urethra. The red-stained
the epithelial layer. Mucus-secreting cells, called goblet cells
basement membrane between the epithelium and the underlying
(arrow)
loose connective tissue is indicated by arrows
Junqueira & Carneiro, Basic Histology, McGraw-Hill
Loose (“frouxo”) connective tissue – supports
many structures that are normally under Dense connective tissue is adapted to
pressure and low friction. It is flexible, well offer resistance and protection. Have
vascularized and not very resistant to stress e.g. fewer cells than loose connective tissue
between muscle cells, supports epithelial tissue and a clear predominance of collagen
and forms a layer that sheathes the lymphatic fibers. There are 2 types: irregular and
and blood vessels. regular dense connective tissues

Section of loose connective tissue. Many fibroblast nuclei Section of immature dense irregular collagen tissue.
are interspersed with irregularly distributed collagen This figure shows numerous fibroblasts (arrow) with
fibers. Small blood vessels are indicated by arrows many thin cytoplasmic extensions (arrowheads).

Junqueira & Carneiro, Basic Histology, McGraw-Hill

Section of rat skin. The subepithelial connective tissue (dermis) is loose connective tissue rich
fibroblasts. The deepest part of the dermis consists of dense irregular connective tissue, which
contains many randomly oriented thick collagen fibers, scarce GAGs, and few cells.
Junqueira & Carneiro, Basic Histology, McGraw-Hill
 Process of lipid storage and release by the adipocyte
!"#$%&!'()' *!''

capillary

Triglycerides are transported in blood from the intestine and liver by lipoproteins known as chylomicrons and VLDLs. In
adipose tissue capillaries, these lipoproteins are partly broken down by lipoprotein lipase, releasing fatty acids. The free fatty
acids diffuse from the capillary into the adipocyte, where they are re-esterified to glycerol phosphate, forming triglycerides.
These resulting triglycerides are stored in droplets until needed. Norepinephrine from nerve endings stimulates the cAMP
system, which activates hormone-sensitive lipase. Hormone-sensitive lipase hydrolyzes stored triglycerides to free fatty acids
and glycerol. These substances diffuse into the capillary, where free fatty acids are bound to the hydrophobic moiety of
albumin for transport to distant sites for use as an energy source.
Junqueira & Carneiro, Basic Histology, McGraw-Hill
Thermogenin dissipates the proton electrochemical gradient
Photomicrograph of elastic cartilage, stained for Photomicrograph of fibrocartilage. Note the
elastic fibers. Cells are not stained. This flexible rows of chondrocytes separated by collagen
cartilage is present, for example, in the auricle of fibers. Fibrocartilage is frequently found in the
the ear and in the epiglottis. insertion of tendons on the epiphyseal hyaline
cartilage.

Junqueira & Carneiro, Basic Histology, McGraw-Hill

Epiphyseal plate showing details of the endochondral ossification. Cartilage matrix (purple) is covered
by recently formed bone tissue (red). Bone marrow and fat cells fill up the space left by the new bone.

Junqueira & Carneiro, Basic Histology, McGraw-Hill

CO2 and O2 transport by erythrocytes and plasma

80% of the CO2 is transported

as HCO3- (2/3 of it in plasma)

In tissues, CO2 is produced

Blood clotting
SMOOTH MUSCLE CONTRACTION AND RELAXATION

contraction
Typical intracellular and extracellular ions concentrations

Lodish et al., Molecular Cell Biology, W.H. Freeman

A potassium (or other ion) equilibrium potential exists when the tendency of K+ ions to
diffuse out of the neuron is balanced by the negative charges pulling them back in. This
potential can be calculated using the Nernst equation.
 Glial cells physically support neurons and perform many housekeeping functions
Glial cell type Location Main functions
Oligodendrocyte CNS Myelin production, electric insulation
Schwann cell Peripheral nerves Myelin production, electric insulation
Astrocyte CNS Structural support, repair processes, blood-brain
barrier, metabolic exchanges
Ependymal cell CNS Lining cavities of CNS
Microglia CNS Macrophagic activity

All these cells are derived from progenitor cells in neural tube except microglia which is formed at bone marrow

Junqueira & Carneiro, Basic Histology, McGraw-Hill

In myelinated axons, action potentials appear to jump between nodes of Ranvier
(patches of axonal plasma membrane that are not covered by myelin)
 Next Class…

• Module:

A. Cell and Tissue Biology

 Todays’ Summary

Tissue Dynamics:

- homeostasis, tissue repair, tissue mechanics

- morphogenesis
- cell differentiation and metabolism
 Tissue Dynamics

The three dynamic states of tissues and the underlying cellular-fate processes
 Tissue homeostasis (equilibrium): the normal steady-state function of tissue

- Some tissues produce cells (bone marrow, skin) as their main function, while others produce a secreted product (glands).
Some tissues primarily carry out mass-transfer operations (lungs, kidneys) while others are biochemical “refineries” (liver) or
can adapt to physiological need (hypertrophy of muscle)

Tissue repair: wounded tissue displays a healing

process that is relevant to tissue engineering
- A biopsied piece or a graft of tissue is expected to initially
display a healing -type response after being placed in culture
or engrafted.
Tissue repair occurs in phases: Early in the process (days),
there is a coordination between cell proliferation, adhesion
and migration. Remodeling of the wound occurs later (weeks
to years) as a result of cell differentiation and ECM proper
formation.
The healing response is faster in fetus and slower in adults

Tissue formation: the formation of tissue involves developmental biology including morphogenesis (describes
the evolution and development of form). Morphological changes are important in the formation and subsequent
function of the tissue and are fundamental to tissue formation and repair
 Cell Differentiation

is a process by which a cell undergoes phenotypic changes to a defined specialized

type (is the process where a cell changes from one cell type to another).

After fertilization of an egg, several cell divisions and

differentiation take place. This spherical mass reorganizes
forming blastocyst containing a cavity, and starting
gastrulation which is a large-scale morphogenic process.

or embryonic stem cells (ESCs)

 Schematic representation of
the human embryo during
early gastrulation

a) Early human primitive streak has gradients of Nodal and WNT morphogens. The dorsal view of three different stages of embryonic development is shown. On the
left is a day 15 human germ disc with a gradient of WNT ligands shown in blue. Nodal (green) is secreted by the primitive node, which migrates in a posterior-
to-anterior direction. In the middle is a day 18 human germ disc, illustrating the anterior migration of the primitive node. On the right is a day 19 human embryo,
with blood islands first appearing in the anterior yolk sac.
b) Cartoon representing a transverse section of the human germ disc from days 15 to 20 of gestation. Migrating mesodermal cells pass
through the primitive streak (centre) at various times throughout gestation. As cells pass through the streak, they are exposed to high (anterior) or low (posterior)
concentrations of Nodal and to high or low concentrations of WNT ligands. Lateral plate mesoderm, which is exposed to low Nodal and high WNT signalling, will give
rise to the definitive haematopoietic programme. (Nodal belongs to the TGFβ superfamily) Nature Reviews Molecular Cell Biology 18, 56–67 (2017)
Morphogenic processes proceed on a characteristic time scale
The underlying molecular-control mechanisms of morphogenic processes are not known in detail but they are
dependent on the cell-fate processes (division, differentiation, death, movement and adhesion) that rely on
changes in chemical, mechanical and electrical factors involving cells.

Cell-cell communication is an essential hallmark of multicellular organisms and can be mediated through direct
cell–cell interactions (e.g., the number of neurons synapses is dependent of glial cells interactions) or by
signaling molecules and ECM components as we studied before.
An overview of some of the
major signal transduction
pathways in mammals

By Roadnottaken, Wikimedia Commons

ECM and mesenchymal induction Induction of mesenchymal or lung bud
of salivary gland differentiation epithelium differentiation
 Mechanisms of epithelial–mesenchymal transition
Epithelial–mesenchymal transition (EMT) is a cellular program crucial for embryogenesis, wound healing and malignant
progression.

During EMT, cell–cell and cell–ECM interactions are remodeled, which leads to the detachment of epithelial cells from each
other and the underlying basal lamina, and a new transcriptional program is activated to promote the mesenchymal fate.

*Transcriptional factors Nat Rev Mol Cell Biol 20, 69-84 (2019)
 Signaling pathways that activate EMT

Nat Rev Mol Cell Biol 20, 69-84 (2019)

Several cell-intrinsic signaling pathways cooperate to induce the expression of epithelial–mesenchymal transition
(EMT)-inducing transcription factors (ZEB, SNAIL and TWIST) that act pleiotropically to induce the transition to the
mesenchymal or partially mesenchymal cell state.
 Metabolism and differentiation

Microbes and cells from multicellular organisms have similar metabolic phenotypes under similar environmental conditions.
Unicellular organisms undergoing exponential growth often grow by fermentation of glucose into a small organic molecule such
as ethanol. These organisms, and proliferating cells in a multicellular organism, both metabolize glucose primarily through
glycolysis, excreting large amounts of carbon in the form of ethanol, lactate, or another organic acid such as acetate or butyrate.
Unicellular organisms starved of nutrients rely primarily on oxidative metabolism, as do cells in a multicellular organism that are
not stimulated to proliferate. This evolutionary conservation suggests that there is an advantage to oxidative metabolism
during nutrient limitation and nonoxidative metabolism during cell proliferation.
Science 324: 1029-1033 (2009) doi:10.1126/science.1160809
 Schematic representation of the differences between oxidative phosphorylation, anaerobic glycolysis, and aerobic glycolysis
(Warburg effect).
In the presence of oxygen, nonproliferating (differentiated) tissues first metabolize glucose to pyruvate via glycolysis and then completely oxidize
most of that pyruvate in the mitochondria to CO2 during the process of oxidative phosphorylation. Because oxygen is required as the final
electron acceptor to completely oxidize the glucose, oxygen is essential for this process. When oxygen is limiting, cells can redirect the pyruvate
generated by glycolysis away from mitochondrial oxidative phosphorylation by generating lactate (anaerobic glycolysis). This generation of lactate
during anaerobic glycolysis allows glycolysis to continue (by cycling NADH back to NAD+), but results in minimal ATP production when compared
with oxidative phosphorylation. Warburg observed that cancer cells tend to convert most glucose to lactate regardless of whether oxygen is
present (aerobic glycolysis). This property is shared by normal proliferative tissues. Mitochondria remain functional and some oxidative
phosphorylation continues in both cancer cells and normal proliferating cells. Nevertheless, aerobic glycolysis is less efficient than oxidative
phosphorylation for generating ATP. In proliferating cells, ~10% of the glucose is diverted into biosynthetic pathways upstream of pyruvate
production. Science 324: 1029-1033 (2009) doi:10.1126/science.1160809
 Cellular mechanotransduction mechanisms

Gene 391: 1–15 (2007) doi: 10.1016/j.gene.2007.01.014

Mechanical loads can induce signal transduction by directly transmitting forces from the ECM to integrins, the cytoskeleton, and the
nucleus, eventually resulting in changes in gene transcription and protein translation. Also, mechanical stretching of cells opens stretching-
activated channels (SACs), causing influx of ions (e.g., Ca+) and thus a series of downstream signaling events. Still, mechanical loads acting
on a cell may unfold a domain of the extracellular protein (M) and expose a cryptic site that may serve as an activating ligand for a cell
surface receptor, which results in a series of signaling events. Additionally, mechanical load applied to a “force receptor” (FR) may initiate
signal transduction, which results in transcription, followed by translation. As a result, soluble factors are secreted into the ECM which act
on the receptor (R) and then initiate a cascade of signaling events. Other signaling molecules that are involved in mechanotransduction can
include CD44 transmembrane protein and its intracellular domain (CD44ICD), which is translocated into the nucleus causing gene
transcription.
Model for the interaction of talin with its binding partners in stiff (A) and soft (C) ECMs

The force applied to talin depends on both the force of

actomyosin contraction and the rigidity of the underlying
substrate.

Very stiff ECM substrates promote firm anchoring of the talin

molecule to the cell membrane via integrin. Simultaneous
actomyosin contraction concentrates tension along the talin rod,
thereby unfolding it. This may decrease the affinity with
previously bound adaptor proteins, such as RIAM and DLC-1,
which allows vinculin binding on newly opened VBSs. Vinculin
binding to talin further stabilizes the focal adhesion and leads to
focal adhesion maturation.

In very soft substrates, the matrix deformation is such that the

force talin is exposed to is reduced, which leads to an even lower
number of exposed VBSs; thus, RIAM and DLC-1 remain bound to
the structured rod.
FASEB J. 30:2073-2085 (2016)
 Although a number of experiments have shown that mechanical cues can also
modulate gene expression, the underlying mechanisms are far from clear.

The mechanical state of a cell modulates nuclear morphology and with it the 3-dimensional organization of chromosomes, thereby establishing specific patterns
of chromosome intermingling. Such intermingling regions harbor different genes that are spatially clustered by their corresponding transcription factors, such as
serum response factor (SRF; left panel) or p65 (right panel), and associate with active RNA polymerase II. This suggests that the spatial clustering and expression
of target genes of particular transcription factors are optimized for the mechanical state of a cell.
Nat. Rev. Mol. Cell Biol. 18: 717–727 (2017)
 The morphogenetic field

(a) Cell activity is guided by a complex, spatially distributed set of signals from the host organism mediated by diffusing chemical,
extracellular matrix, tension/pressure, and bioelectrical properties.

(b) This morphogenetic field orchestrates cell behavior toward large-scale anatomical programs during development and
regeneration; its influence is subverted during oncogenic transformation and aging. Mastery of the information stored in this field,
and of the mechanisms by which cells interact with it, will result in the ability to reprogram large-scale tissue and organ shape, with
transformative implications for the fields of birth defects, regenerative medicine, cancer, and synthetic bioengineering.

WIREs Syst Biol Med 2013, 5:657–676

A Verger's Dream

Saints Cosmas and Damian performing a miraculous cure by

transplantation of a leg. Master of Los Balbases, 1495

“Saints Cosmas and Damian were early Christian martyrs who, according to legend,
practiced medicine without payment and therefore were represented to the public
as medical ideals. In this Spanish altarpiece, the saints appear in a vision, dressed in
the full finery of academic doctors as they perform the miracle of transplanting a
leg. The vision is described in a book of 1275 by Jacobus de Voragine, Legenda
aurea (The golden legend). The vision was received in the Church of Saints Cosmas
and Damian, in Rome, by a verger who had a disease that was eating away the flesh
of his leg. One night he dreamed that the two saints came and cut off his bad limb,
and in its place transplanted the leg of a dead African who had just been buried in a
nearby churchyard. When he awoke, the verger found that he had a healthy black
leg, while it was discovered that the African's body now lacked a limb. The
conclusion: "Then let us pray unto these holy martyrs to be our succor and help in
all our hurts, wounds and sores, and that by their merits after this life we may come
to everlasting bliss in heaven. Amen." The painting was probably once in the Church
of Saints Cosmas and Damian in Burgos, in northern Spain. The painter is called the
Master of Los Balbases after a nearby town in which there is an altarpiece by him in
the Church of Saint Stephen.” From https://www.wdl.org/en/item/3251/
Organ printing
 Tissue engineering
- Organ printing, or computer-aided layer-by-layer assembly of biological tissues and organs

- A cell printer that can print gels, single cells and cell aggregates has been developed.

(a) Computer aided design-based presentation of model of cell printer. (b) Bovine aortic endothelial cells were printed in 50 µm size drops in a line. (c) Cross-
section of the matrix gel showing the thickness of each sequentially placed layer. (d) Picture of the real cell printer and part of the print head with nine nozzles.
(e). The printer is connected to a bidirectional parallel cable together with 9 jets extent of mixing. Endothelial cell aggregates ‘printed’ on collagen before (f) and
after their fusion (g). Trends Biotech. 21, 157-161 (2003)
 Tissue engineering

Roadmap for organ printing

 Trachea transplantation
http://news.bbc.co.uk/2/hi/7735696.stm

1 Trachea is removed from dead donor patient

2 It is flushed with chemicals to remove all existing cells
3 Donor trachea "scaffold" coated with stem cells from the patient's hip bone marrow. Cells from the airway lining added
4 Once cells have grown (after about four days) donor trachea is inserted into patient's bronchus
http://www.dailymail.co.uk/sciencetech/article-2384715/At-tastes-meat--Worlds-test-tube-artificial-beef-Googleburger-gets-GOOD-review-eaten-time.html
The Hottest Tech in Silicon Valley Made This Meatball
April 2016

http://fortune.com/2016/04/25/memphis-meats-lab-grown-meat/
 https://shiokmeats.com

The company estimates that it can make a kilogram of shrimp meat for $5,000 !!!

https://techcrunch.com/
FURTHER READING
 Signaling pathways that activate EMT

Nat Rev Mol Cell Biol 20, 69-84 (2019)

In the context of nonneoplastic cells, the transforming growth factor-β (TGFβ), WNT and NOTCH pathways are activated during embryonic development and wound healing. Activation of EMT downstream of these
pathways is crucial for palatal fusion and formation of the primitive streak , mesoderm and node in developing embryos. Additionally , TGFβ and WNT-induced EMT promotes wound healing and fibrosis. The canonical
WNT pathway is activated upon binding of canonical WNT ligands to the Frizzled family of membrane receptors. This leads to the release of β-catenin from the GSK3β (glycogen synthase kinase-3β)–AXIN (axis inhibition
protein)–APC (adenomatous polyposis coli protein) complex. β-Catenin then translocates to the nucleus, where it binds to the transcription factors TCF (T cell factor) and LEF (lymphoid enhancer-binding factor) to
activate genes that drive EMT. The NOTCH pathway is activated upon binding of the Delta-like or Jagged family of ligands to the NOTCH receptor. This binding triggers a series of proteolytic cleavage events that
culminate in the release of the active, intracellular domain of the NOTCH receptor (NOTCH-ICD), which enters the nucleus to function as a transcriptional co-activator. Binding of the TGFβ proteins to the TGFβ family of
receptors leads to receptor phosphorylation and activation of SMAD complexes, which activate the EMT programme. SMAD proteins also interact with β-catenin and NOTCH-ICD (dashed arrows), serving as central
nodes for crosstalk between the TGFβ, WNT and NOTCH pathways. The TGFβ pathway also collaborates with the PI3K AKT pathway, which in turn triggers the activation of the mTOR complex and nuclear factor-κB (NF-
κB), the p38 MAPK pathway and the RAS–RAF–MEK–ERK signalling axis. The aforementioned pathways are also triggered upon binding of growth factors to their cognate receptors. Moreover, binding of several
cytokines to their receptors triggers the phosphorylation and activation of Janus kinases (JAKs) and signal transducer and activator of transcription proteins (STATs); STAT dimers activate the transcription of genes
encoding EMT transcription factors.
 Transcriptional gene regulation (Epigenetics): DNA methylation

Dynamics of 5mC/5hmC/5fC/5caC in paternal and maternal genomes during preimplantation development

DNA demethylation of the zygote, gauged by 5mC levels, occurs by a passive mechanism in the female pronucleus, diluting the marks with the
passage of every cell cycle. The male pronuclear genome becomes demethylated actively by the action of the Tet enzymes. Tet3 is expressed in
the oocyte and zygote. After fertilization, Tet3 is relocated from the cytoplasm to the paternal nucleus to convert 5mC to 5hmC/5fC/5caC.
Subsequently, paternal and maternal genomes undergo replication-dependent dilution of 5hmC/5fC/5caC in males and 5mCin females. It is
possible that replication-independent active DNA demethylation may occur in a loci-specific manner in zygotes, but the exact mechanism is
currently unclear. DNA methylation patterns in ICM are reestablished by de novo DNA methyltransferases DNMT3a and DNMT3b at the
blastocyst stage.
TET proteins catalyze the steps of the demethylation of 5mC to 5-hydroxymethyl cytosine (5hmC), then converted to 5-formylcytosine (5fC), then 5-
carboxylcytosine (5caC). Cold Spring Harb Perspect Biol 2014; 6:a019133
 The transfer of signaling molecules (proteins, RNAs, etc) between cells is also mediated
by extracellular vesicles (EVs) (exosomes, microvesicles, ectosomes, oncosomes, etc)

Overall composition of extracellular vesicles (EVs)

Schematic representation of the composition and
membrane orientation of EVs. Examples of tetraspanins
commonly found in EVs include CD63, CD81, and CD9.
Note that each listed component may in fact be present
in some subtypes of EVs and not in others. For instance,
histones and proteasome and ribosome components are
probably secreted in large plasma membrane–derived
EVs and/or apoptotic vesicles rather than exosomes.
Abbreviations: ARF, ADP ribosylation factor; ESCRT,
endosomal sorting complex required for transport;
LAMP, lysosome-associated membrane protein; MHC,
major histocompatibility complex; MFGE8, milk fat
globule epidermal growth factor-factor VIII; RAB, Ras-
related proteins in brain; TfR, transferrin receptor.

The composition of EVs also changes during cellular differentiation and pathological processes in which are involved.
Therefore, EVs can also be used as biomarkers to assess tissue function and disease progression.
Annu. Rev. Cell Dev. Biol. 2014. 30:255–89
Exosomal miRNAs summarizing miRNA expression as biomarkers in gastrointestinal cancer

Cancer Gene Therapy (2017) 24, 48–56

Metabolic pathways active in proliferating cells
are directly controlled by signaling pathways
involving known oncogenes and tumor
suppressor genes.

This schematic shows our current understanding of how

glycolysis, oxidative phosphorylation, the pentose phosphate
pathway, and glutamine metabolism are interconnected in
proliferating cells. This metabolic wiring allows for both NADPH
production and acetyl-CoA flux to the cytosol for lipid
synthesis. Key steps in these metabolic pathways can be
influenced by signaling pathways known to be important for
cell proliferation. Activation of growth factor receptors leads to
both tyrosine kinase signaling and PI3K activation.

Via AKT, PI3K activation stimulates glucose uptake and flux through the early part of glycolysis. Tyrosine kinase signaling negatively regulates
flux through the late steps of glycolysis, making glycolytic intermediates available for macromolecular synthesis as well as supporting NADPH
production. Myc drives glutamine metabolism, which also supports NADPH production. LKB1/AMPK signaling and p53 decrease metabolic flux
through glycolysis in response to cell stress. Decreased glycolytic flux in response to LKB/AMPK or p53 may be an adaptive response to shut off
proliferative metabolism during periods of low energy availability or oxidative stress. Tumor suppressors are shown in red, and oncogenes are in
green. Key metabolic pathways are labeled in purple with white boxes, and the enzymes controlling critical steps in these pathways are shown in
blue. Some of these enzymes are candidates as novel therapeutic targets in cancer. Malic enzyme refers to NADP+-specific malate
dehydrogenase [systematic name (S)-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating)].

Science 324: 1029-1033 (2009) doi:10.1126/science.1160809

Summary of how metabolism can control cellular
signals that regulate cell differentiation.

Red circles show metabolites that directly affect signaling,

blue circles their target proteins, and red lines their
interactions. Metabolic enzymes are in red type. Blue signals
affect differentiation, or control metabolism itself.

Abbreviations: GLS, glutaminase; HAT, histone acetyltransferases; HDAC, histone deacetylases; IDH*,
mutant form of isocitrate dehydrogenase; JmjC KDM, JmjC domain-containing histone demethylases;
LDH, lactate dehydrogenase; PDH, pyruvate dehydrogenase; PK, pyruvate kinase.

Trends Cell Biol 23: 484-492 (2013) doi:10.1016/j.tcb.2013.05.004

The ways metabolic changes may be linked to cell differentiation. (A) Growth factors (GF), transcription factors (TF), and other cell signaling
proteins (blue) direct cells to stop proliferating and differentiate. Cell cycle exit changes demand for metabolites (red), for example by reducing the rate of
nucleotide and amino acid incorporation into macromolecules. By altering local equilibrium, or through allosteric effects on metabolic enzymes, this change in
demand redirects metabolic flux. (B) Cellular signaling directs cells to differentiate and in parallel regulates metabolic enzyme activity. The resulting rewiring of
metabolic pathways serves the altered demands of the differentiated cells, for example through reduced rates of nucleotide and amino acid production. (C)
Changes in metabolic flux alter the concentration of specific metabolites, which regulate cellular signaling proteins, and consequently influence cell
differentiation. The mechanisms by which this might take place are summarized in next slide.
Trends Cell Biol 23: 484-492 (2013) doi:10.1016/j.tcb.2013.05.004
Effects of signals that control cell proliferation and differentiation on metabolism*
*effects of these pathways on metabolism and on cell proliferation and differentiation depend on the tissue and context.

favor anabolism

favor catabolism

Trends Cell Biol 23: 484-492 (2013) doi:10.1016/j.tcb.2013.05.004

Relocation of gene loci within the nucleus during stem cell differentiation

(a) In stem cells, pluripotent and housekeeping genes

are actively transcribed within the nuclear interior
while lineage-specific genes are silenced at the
nuclear periphery.

(b) With lineage commitment, lineage-specific genes

are detached from the nuclear lamina and relocated
to the nuclear interior. In contrast, pluripotent genes
are silenced and attach to the nuclear lamina.

(c) Additionally, some lineage-specific genes remain

inactive despite being displaced from the nuclear
envelope and are transcribed only after terminal
differentiation.

J Biomech Eng. 2017 Feb 1;139(2). doi: 10.1115/1.4035350

 Vmem at the level of single cells: its transduction impacts cell states

(a) A sample survey of many cell types and recent functional data reveals that at the level of single cells, Vmem determines cell plasticity and proliferation potential. Depolarized cells tend to
be rapidly proliferating and undifferentiated (e.g., embryonic, stem, or tumor cells), while terminally differentiated somatic cells tend to be highly polarized. Importantly, cell state can be
functionally altered (switched between these two classes, in either direction) by artificial change of V mem (b) A range of mechanisms have now been characterized that transduce
alterations of Vmem into downstream effector cascades (transcriptional changes). These include signaling proteins with a voltage-sensitive conformation (e.g., integrins and voltage-sensitive
phosphatases) and transporters of small signaling molecules whose activity is regulated by Vmem (such as gap junctions, voltage-gated calcium channels, and solute carriers, which allow Vmem
changes to signal via serotonin, Ca2+, butyrate, and likely many other yet-to-be discovered compounds).
WIREs Syst Biol Med 2013, 5:657–676
 Molecular force transduction by ion channels
- Ion channels can act as both sensors and effectors, providing the necessary fluxes to relieve osmotic pressure, shift the
membrane potential or initiate chemical signaling

- For a channel to be mechanosensitive it needs to respond to mechanical stresses by changing its shape between the closed
and open states. In that way, forces within the lipid bilayer or within a protein link can do work on the channel and stabilize its
state.

Mechanosensing in different dimensions

(A) A one-dimensional sensor, such as talin, can unfold and

elongate with tension. Such a conformational change could
expose cryptic binding sites within the protein.

(B) A membrane channel that opens in response to

membrane tension is an example of a two-dimensional
sensor that increases its in-plane area (A).

(C) A hypothetical elastic structure that decreases its volume

under osmotic pressure is an example of a three-
dimensional mechanosensor.

Journal of Cell Science 125, 3075–3083 (2012)

(https://www.youtube.com/watch?v=EWR7hPl1EQo)
 Southern Fried Chicken Duck à l'Orange

The team expects to continue reducing production costs dramatically, with a target launch of its products to
consumers in 2021
http://fortune.com/2016/04/25/memphis-meats-lab-grown-meat/
https://static1.squarespace.com/static/5a1e69bdd7bdce95bf1ec33b/t/5b4dea66aa4a99d6f5bc406b/1531832935083/PRESS+RELEASE_Mosa+Meat_17+July+2018.pdf
 Next Class…

• Module:

A. Cell and Tissue Biology