Cell-Cell Adhesion and Tissue Morphogenesis

Question 1

what are the five fundamental tissues?

Answer 1

1. epithelium
2. connective
3. blood
4. muscle
5. nerve

Question 2

epithelium as a model system

Answer 2

1. cellular organization of a tissue
2. concept of function arising from cellular anatomy
3. molecular basis of adhesion and compartmentalization- how to make one part of the organ away from other organs

Question 3

epithelium in the “social” context

Answer 3

-epithelia cell is not epithelium –> can’t do its work alone
-need group of cells tightly adhered to each other
-need to have different tops and bottoms- bottom is in reference to the basement membrane (basal lamina) and top is in reference to free surface (apical layer) –> apico-basal polarized
-need to exist on top of basal lamina
-tissues are avascular- do not have blood vessels running between epithelial cells –> always need epithelium and connective tissue b/c the vessels supplying nutrients are on other side of basement membrane
-basement membrane is selectively permeable- need ways to move across it

Question 4

what are the typical functions of epithelia?

Answer 4

1. barrier between host and environment (Ex. skin)
2. compartmentalization (Ex. endothelium and lymphatics)
3. secretion (Ex. mammary, salivary, and prostate glands- make things that are constitutively released or released in response to specific stimuli)
4. absorption (Ex. small intestine)

Question 5

barrier to the environment: cornified cells in the epidermis

Answer 5

-layers of sacrificial dead cells on top
-layers of differentiating cells that aren’t that important before you get to layers of transmit amplifying cells before you get stem cells

Question 6

compartmentalization: selective permeability

Answer 6

-primary way is by controlling the movement of fluid and small or large molecules between the cells- jobs of specialized intercellular junctions
-also add additional cells
Ex. carotid artery- you want no losses of blood or blood components out but when you get capillary nexus, want to be able to have things move much more freely in tissue
-in the arteries, they wrap smooth muscle very densely around the endothelium –> additional layers of compartmentalization beyond those intercellular junctions
-as you get out to smaller and smaller vessels, smooth muscle or pericyte layer becomes fenestrated and changes in composition of junctions allow you to have considerable permeability

Question 7

secretion: specialized granules

Answer 7

goblet cell is used for secretion since it has devoted most of its volume to mucus granules that hold them until signal tells them to release them

Question 8

absorption: microvilli of intestinal epithelium (increased surface area)

Answer 8

surface area is key- you can increase by making intestines longer (limited gain) or instead microvilli dramatically increase the surface area in contact with fluid of the lumen and therefore allow a much larger contact area for absorption

Question 9

epithelial types are defined by the number of cell layers

Answer 9

-single cell layer- simple epithelium
-appears to be multiple cell layers but they’re all getting to the BM- pseudostratified
-multiple cell layers without contact to BM- stratified
-goes in between- transitional

Question 10

epithelial types are defined by the apical most shape

Answer 10

-square or cube- cuboidal
-thin- squamous
-tall- columnar

Question 11

polarity in stratified epithelia (among cells)

Answer 11

-top-bottom polarity with respect to the BM
-in stratified epithelium, this is a question of polarity among the cells
-b/c there are multiple layers that are at or close to the basal layer, medium closer to apical, apical-most cell layers –> tend to be different in structure and function
Ex. skin- dramatic Ex.- apical most cell layer is dead then next layer are differentiating then dying then amplifying then you have stem cells at the bottom

Question 12

polarity in simple epithelia (within cells)

Answer 12

-only one cell layer between the BM and free surface of lumen
-polarity within the epithelial cell
-single cell has apical and basolateral surfaces with intercellular junctions in between

Question 13

cell-cell adhesion- fundamental cell communication strategy

Answer 13

-homophilic cell-cell adhesion- like cells sticking to each other Ex. mammary epithelial cell sticking to mammary epithelial cell
-heterophilic cell-cell adhesion- blood cell like macrophage or neutrophil sticking to inner surface of the endothelium

Question 14

what are the three classes of intercellular junctions?

Answer 14

1. tight junctions in mammals-control the movement of water and molecules between cells
2. anchoring junctions- adherens junctions and desmosomes (cell-cell anchoring junctions) and focal adhesions and hemidesmosomes (cell-matrix anchoring junctions)
3. communicating junctions- gap junctions

Question 15

cell junctions in epithelium

Answer 15

-tight junction- always apical most- membranes are very close together, lead to hypothesis that they were controlling movement of fluid b/c it was skinny point
-gap junction- recognizable from repeating connections and allow cells to talk to each other directly
-multiple classes of adhering junctions- adherence junction itself is unimpressive in EM but desmosomes and hemidesmosomes have plaques then intermediate filaments are big and static coming out of them

Question 16

most junctions are connected to the cytoskeleton

Answer 16

-tight and adherins junction to acitn
-desmosomes and hemidesmosomes connecting to intermediate filaments

Question 17

selective transport of molecules/ions across the barrier occurs by one of three routes

Answer 17

-2 transcellular (across) and 1 paracellular (between)
-if you want Ca high on one side and low on the other, the molecule can move between the cells or across the cells

Question 18

paracellular transport

Answer 18

-through intercellular junctions- has to somehow get by them
-multiple of these junctions are foci on 3D interface between the cells

Question 19

tight junctions form the seal that prevents the free movement of molecules between outside and inside environments

Answer 19

-India ink pen and put it on one side of a confluent epithelial layer and saw how far it went then put ink on the other side to see where it went
-luminal side- it stops at apical most end where cells seem to be at kissing point
-bottom side- it runs really quickly up between the cells and stops at the apical most point of very close cell contactI

Question 20

In the EM, TJs are seen as a network of interconnected and branching strands formed by intramembranous particles

Answer 20

forms belt all the way around the point of contact of cells –> isn’t a place for the water, ions, glucose, or antibodies to run by it –> has to accommodate

Question 21

TJ intramembranous particles are composed of 2 four-transmembrane spanning proteins

Answer 21

-claudins- large gene family with >20 family members
-occludins- small gene family with 2 members
-machine built of 2 parts and one has 20 flavors while the other has 2- one with 20 will give cell and tissue specificity b/c it has lots of copies in the genome so you can turn on claudin 4, 7, 13, etc. but occludin you’re stuck with it
–> whatever gene family you’re looking at, if there’s dozens of them vs one, the one is constitutive

Question 22

both claudins and occluding associate with cytoplasmic proteins that bind to actin filaments

Answer 22

-you’ve got contacts across the membrane mediated by transmembrane proteins
-you also have layers of proteins accumulating that connect those adhesion receptors to the cytoskeleton –> allows you to control the cell behavior

Question 23

ZO-1 was the first TJ-asociated protein IDed and is a founding member of PDZ domain-containing proteins

Answer 23

-zona occludins 1 (zona occludins being old name for TJs) was the first TJ-associated protein
-contains lots of PDZ domains, SH3 domains, GUK domains that are well known for mediating protein-protein interactions
–> if you’ve got an adhesion receptor and its cytoplasmic partner is full of PDZ and SH3 domains, it will stick everything and give you ability to accumulate structural and signaling proteins at this spot

Question 24

dual function of the tight junction

Answer 24

1. gate- controls paracellular permeability- controlling that anchorage point saying you can’t get through this
2. fence- partitioning the plasma membrane so that it’s at least a lot harder for something that’s a membrane protein on the apical side to roll down to basal lateral –> a lot harder to get past the TJ than stay within the domain

Question 25

shift in concept: structural barrier to signaling center

Answer 25

-discovery of the PDZ domain -nice, clear picture of tight junctions with claudins, occludins, ZO-1 and actin but then people started doing BioID and then you see everything is there -shift from thinking of this as purely a structural barrier to signaling center --> if you could put PTEN and CDC42 and Rho-A and RAC1, you could change lots of cell type behavior -clearly controls cellular permeability and accumulating signaling molecules

Question 26

what are the transcellular routes?

Answer 26

1. vesicle-mediated transport (cargo is usually a macromolecule, also called transcytosis)- endocytosis with exocytosis on a different surface --> transcytosis 2. membrane carrier/channels/transporters- can directly and specifically bring in a specific species --> both require apico-basal polarity -if you have transporters sitting on both the apical and basal lateral surface in equal amount with equal activity, you can burn a lot of ATP dumping things in and out

Question 27

transepithelial transport of proteins by transcytosis

Answer 27

-in the gut there are mechanisms to bring antibodies in- specialized machinery to find them on basal surface and deliver it to the other side or find it on the apical side and bring it in -uses same machinery that you'd use to pull up any other cargo but it's coupling it from one polarized surface to another

Question 28

transepithelial transport of glucose

Answer 28

-carriers, transporters, arms importers, uniporters --> all are ways of bringing things in on one side and taking advantage of natural gradients or creating gradients by burning energy Ex. glucose high in the lumen of the gut- you can bring in things on that surface -if you have natural differences in ions, you can just bring them in or use that to bring something else in -you can also burn energy with pumps to exchange different ionic species --> to do this properly and establish and maintain directional gradients, the cell itself needs to be directionally polarized

Question 29

anchoring junctions

Answer 29

1. cell-cell anchoring junctions connect cells to each other 2. cell-matrix anchoring junctions connect cells to the ECM 3. cytoplasmic proteins in the junctions connect to the cytoskeleton

Question 30

the other two junctions in the "junctional complex" of mature epithelia have adhesive functions

Answer 30

2 types of junctions involved in cell-cell adhesion: adherens junction and desmosomes

Question 31

most frequently studied cell-cell linking proteins: Cadherins

Answer 31

-ancient and highly conserve gene family -single-pass transmembrane homodimer- one pass through the membrane and E-cadherin binds to the dimer on the other cell surface -large gene family- cell and tissue specificity -classical cadherins are in the adherens junction -desmosomal cadherins are in the desmosome

Question 32

based on the crystal structures of EC1-2 fragments of classical cadherins: bidning is between EC1-2 in classical cadherins: a zipper model

Answer 32

-fina domain- EC1, perhaps EC2, bridges across and you get a zipper model- 2 E-Cadherins floating around and form by cis interactions a dimer and dimers interact across the membrane -from dimer binding you have approximately zero effect on cell behavior -one dimer binding one dimer is small amount of energy compared to actin-myosin contractility

Question 33

most frequently studied cell-matrix linking proteins: integrins

Answer 33

-ancient and highly conserved -dimer of alpha and beta subunits- always going to be one and one -large gene family- cell and tissue specificity -integrin activation is conformational dependent- for cadherins, there are subtleties to how the structure changes when it binds but for integrins, not subtle with huge switch in conformation -integrins connect cells to basement membrane at the hemidesmosome

Question 34

adhesion receptors have weak ligand binding affinity

Answer 34

-receptors that are adhesion binding across a membrane and adhesion receptors binding to the ECM -weak affinity- one of the dimer pairs interacting or binding ECM- no effect on cell behavior --> how do you get strong adhesion?

Question 35

anchoring junctions

Answer 35

-put many adhesion receptors there- 1000 or 10,000 per junction -clustered interactions among them --> their function changes from the fact that they're having interactions with thousands of receptors at that site -link to cytoskeleton through intracellular adaptor proteins -1st game is avidity- low affinity becomes high energy by gaining avidity (lots of interactions in parallel) and you connect through adaptor proteins to cytoskeleton and that can, in the case of actin, connect you to contractility

Question 36

cell-ECM junctions have similar logic

Answer 36

integrins, adaptor proteins, and cytoskeleton

Question 37

adherens junction (aka zonula adherens) form an adhesion belt

Answer 37

makes its own belt of actin around the top of the cell and organizes actin up into structures like microvilli

Question 38

E-cadherin based adherens junction

Answer 38

-E-cadherin (adhesion receptor), beta catenin and alpha catenin (adaptor proteins), and F-actin (connects to cytoskeleton) -essential for formation of epithelium- if you delete E-cadherin in a mouse, it fails at morala stages and does not compact -if you dysregulate adherens junctions, it can lead to loss of other anchoring junctions -E-cadherins levels are frequently changed in epithelial cancers

Question 39

loss of AJ can also have signaling consequences since beta catenin has dual functions

Answer 39

-beta catenin can go to nucleus and once you get to c-myc, you can do anything -difficult to show that this particular molecule of beta catenin is released b/c of disruption of adherins junction and then goes to nucleus in normal cells, you would degrade beta catenin almost instantly --> degradation of beta catenin must be faulty -difficult to show at single molecule level to distinguish between a transcript giving rise to beta catenin in the cytoplasm which goes to the nucleus and beta catenin falling off adherens junction and going to nucleus --> reusing protein in structural role at PM and signaling role in cytoplasm and can be changing places

Question 40

functional properties of tumor invasion

Answer 40

Ex. mammary epithelium- single epithelial layer on top of basement membrane -tumor invasion requires many cell layers and need to break through basement membrane and migrate out into the connective tissue and breaks off as individual cells -need loss or change of cell-cell adhesion -need to break through, digest, and somehow accommodate the epithelial BM, bring proteases and digest it, or they could do both -survive in new ECM environment- epithelial cells need to adapt to not being around its normal cell-cell contacts and ECM contacts -invade through vascular BM to access circulation

Question 41

desmosomes are found where mechanical stress is high

Answer 41

-places you want skin to hold and not rupture -epithelial layer of skin has abundance of these- big and thick with a lot of proteins present

Question 42

molecular basis of desmosomes

Answer 42

-desmoglein and desmocolin- desmosomal cadherins -desmoplakin, plakoglobin, plakophilin- cytoplasmic proteins -intermediate filaments- cytoskeleton

Question 43

gap junction

Answer 43

-freeze fracture- break cells at point of contact and look at face of contact you see many cylinders -cylinders regulating the movement between cells- as restrictive as 100 Da (ionic transport) up to 20,000 (small proteins) -intercellular channel that allows ions or small molecules to go from one cell to the other- ions, sugars, AAs, cAMP, but can be bigger

Question 44

gap junctions are composed of connexins

Answer 44

connexins --> 6 group of connexon --> connexons can be homomeric (all the same type) or heteromeric and connect across the intercellular space -things pass through the gap in the middle

Question 45

gap junctions are gated channels with open and closed conformations

Answer 45

-not just a hole --> regulated pore between the cells -2 levels of regulation: composition of the right genes- composition of the connexons will determine how big that pore is and second level is it open or shut

Question 46

renewal of the epidermis of the skin

Answer 46

-constantly proliferating and moving upwards -shedding dead cels, creating new cells, differentiating, and shedding -stem cells at the bottom releasing to progenitor cells expanding then terminally differentiating and dying -exchange of intercellular junctions- can't leave one layer and go to the next without some combo of movement, proliferation, and changing junctions

Question 47

renewal of intestinal epithelial cells

Answer 47

-villi with no cell division and cells are sloughing off in the lumen -at the base of the crypt, you have stem cells and they're going up

Question 48

metastasis requires changes in each of the fundamental properties of an epithelium

Answer 48

-cell in the epithelium divides out of control and recruits some vasculature --> survives going on walkout and gets through BM endothelium into the blood --> now it's in a fluid environment and adapted --> survives high shear stress --> gets through endothelium and BM and escapes then be in a different environment it's mismatched for --> evade immune detection and attack or co-opt immune detection --> recruit vasculature and grow to significance -few cells that survive all of this and grow to clinical significance that drive 90% or more cancer deaths

Question 49

ECM proteins allow epithelial organization

Answer 49

-epithelial cells organize into epithelium with junctions, lumen, apical basal polarity, BM -if you put them in combo with mesenchymal cells in 3D, epithelium forms BM and mesenchymal cells stay inside ECM

Question 50

inputs into 3D culture

Answer 50

-good match of question you're trying to answer and experimental methods -might sometimes want to do whole organ or organ slice or isolate stem cells -take primary human epithelium

Question 51

formats for 3D culture

Answer 51

-many times 2D culture is the easiest, especially at the subcellular level -might want to embed them or put them on cultural insert (strong signal for differentiation) or make skin to be used for grafting -might want to put whole organ into cultural insert

Question 52

setup: two cells in a petri dish: single cell-cell anchoring system...what is the first step on contact?

Answer 52

1. dimers start to touch across membrane- adhesion through transmembrane receptor 2. cell might shrug and break connection or more could get attracted it

Question 53

setup: two cells in a petri dish: single cell-cell anchoring system...what happens to reinforce and strengthen the connection?

Answer 53

recruitment of adaptor proteins that serve as scaffold, additional dimer pairs being recruited to backend

Question 54

what are the key elements in the adherens junction and desmosome?

Answer 54

adherens junction adhesion receptor: E-cadherin key cytoplasmic proteins: beta catenin and alpha catenin cytoskeleton: actin desmosome adhesion receptor: desmoglein and desmocolin key cytoplasmic proteins: desmoplakin, plakoglobin, plakophilin cytoskeleton: intermediate filaments

Question 55

genetic expt. 1: ideal cell with one adhesion receptor, E-cadherin...what happens if you delete the E-cadherin in single cells?

Answer 55

cells will disconnect

Question 56

genetic expt.2: ideal cell with one adhesion receptor, E-cadherin...what happens if you delete the E-cadherin just after adhesion?

Answer 56

-if it's a monolayer and had these tight connections, may still have cobblestone configuration but lack molecular-specific connections -sparse- likely will fall apart

Question 57

genetic expt. 3: ideal cell with one adhesion receptor, E-cadherin...what happens if you delete the E-cadherin in fully adherent cells?

Answer 57

-beta catenin might still get to the nucleus -if the RNA is absent, can't make new protein but depending on rate of protein degradation, there may or may not be any immediate effect on cell adhesion

Question 58

genetic expt. 4: real epithelial cell with multiple adhesion receptors...deplete E-cadherin at each of the three stages?

Answer 58

-cells would have other molecules to rely on -compliment of adhesion receptors and how hey bind affects who the cell thinks it is

Question 59

principles from expt 4

Answer 59

1. E-cadherin is only one of many adhesion receptors and the AJ is only one of several cell-cell junctions 2. experimentally unknown whether loss of E-cadherin alone, in cultured cells, would be sufficient to disrupt adhesion @ different stages 3. reasonable hypothesis is that the consequences of loss of E-cadherin would be greatest when the adhesions are forming or remodeling

Question 60

genetic expt. 5: delete E-cadherin in vivo

Answer 60

-lack of E-cadherin affects their positional sense and they end up in the wrong place and a lot of inductive cues in the embryo happen the same way every time since neighboring connections are the same -fail to differentiate since E-cadherin is essential to inductive cue -four cell and eight cell stages are fine but could get issues in compacted morula --> primary phenotype is that they can't compact and implementation doesn't go well so they die -what about skin? barrier's disrupted and immune cells have easier time getting in, cells could go to dermis from epidermis, loss of sense of who or where you are -increase in desmosomes, hyperplasia, and loss of AJs