Lecture #13 - Cell-cell Adhesion and Tissue Morphogensis

Question 1

Organs

Answer 1

Organs are modular combinations of the fundamental tissues

Question 2

Xavier Marie Bichat

Answer 2

Found all organs are composed of a common set of fundemental tissues
- Have specific set of parts and combine the parts in diferent ways to get different organs

KEY Said there were 21 fundamental tissues (THIS part was wrong there are NOT 21 fundamental tissues)
- He was not using a microscope = could not see clearly enough to see differences (HE was not using a microscope)

Worked 40 years before cell theory

Question 3

Virchow

Answer 3

Took the 21 fundamental tissues and said there were 5 fundamental tissues + organized epithelium into types

5 Fundemental tissues –> epithelium + muscle + blood + nerve + connective tissue

Thought of epithelium and connective tissues as the true fundemental tissues BUT Muscle + nerve + blood were so recurent he HAD to include them

Used the latest microscope (was able to look more clearly at the tissues)

NOTE - language around histology + organ/tisue organization = agreed upon LONG ago (during darwin ; before cell theory and mendle)

Question 4

Fundemental tissues

Answer 4

Epithelium + Muscle + Blood + nerve + connective tissie

NOTE - epithelium is one of the fundemntal tissues

Question 5

Answer 5

Question 6

Epithelial tissue (overall)

Answer 6

Epithelial tissue are a primary component of most organs

Epithlial tissue is very important

Example - can clearly tell the image is of a lung just by looking at the epithelial cells

Question 7

Epithlial cells + disease

Answer 7

Epithelial cells are the cell of orgin in 90% of human cancers

Question 8

Organization of cells into tissues

Answer 8

Connective Tissue = has few cells embedded in extensive extracellular matrix (Highly vascularized)

Epithelia: abundant cell-cell contact + little extracellular matrix + no blood vessels
- . Epithelium: lining of glands, skin, intestines

Endothelium = lines blood vessles ; mesothelium lines pleural/pericardial spaces

Question 9

What can we use Epithlia to study

Answer 9

Epithelium can be used to understand the cellular organizatin of a tissue

Epithelium can be used to understand the concept of function arsing from cellular anatomy

Epithelium can be used to understand the molecular basis of adhesion and compartimelization (How do you make one part of an organ seperate from another)

Question 10

Epithelial cells vs. Epithelium

Answer 10

Epithelial Cells is NOT an epithelium

T cell alone can function BUT epithlieal cell alone does NOT complete the function of epithelium (NEEDS other cells –> epithelial cells exist in social context)

Question 11

What do you need to be an epithelium?

Answer 11

1. Cells are tightly adhered to each other (close cell-cell contact)
2. Cells need to be apico-basally polarized (cells need to have a different top vs. bottom)
   
   • Basal = bottom of cell (near BM) ; Apical = top of cell (near lumen or free surface)
3. Epithelial cell exist on top of basement membrane
4. Epithelial tissues are avascular
   
   • There are NO blood vessels between the epithelial cells in the epithelium
     - Need the epithelium to be next the connective tissue because the epithelium gets blood form the vessles in the connective tissue
5. Specialized intercellular junctions

Question 12

Basment membrane

Answer 12

Basment membrane = specilized ECM

Basement membrane sites between the epithelium and the connective tissue

Basement membrane needs to be selectively permeable BECAUSE need blood to get from connective tissue through the basement membrane to the epithelium
- Vessels that supply the nutrients and remove the waste to the epithelium are on the other side of basement membrane (in the connective tissues)

Question 13

What are the functions of the epithelium

Answer 13

1. Barrier between host and envirnment
2. Compartimezation
3. Secretion
4. Absorbption

Secretion vs. Compartmilizaton vs. Barrer have different organization of the epitheloum to be able to accomplish the three functions

Question 14

Epithelium as a barier

Answer 14

Function = be a selective barrier between two environments (makes two envirnments)

Example – Skin + intestine
- IF put hand in bacteria culture you would be ok because of the barrier the epithelia in skin provide

Skin can act as barrier because it has a layer of sacrificial cornified dead cells in the epidermis
- Below the dead cells are differentiating cells (can be lost) –> amplifying cells –> then stem cells (more important cells that won’t be lost because protected by the less important cells above them)

Overall - Dead cells are physical barrier AND have a capacity to make new cells

Question 15

How do you get barrier and make two different environments

Answer 15

Example - Making barrier of inside of stomach Vs. outside of stomach (make two different environments)

2 ways to achieve a differenence in the 2 environments (2 ways molecules can cross a barrier)
1. Paracellular - molecules moves between cells using intercellular junctions)
- Molecule needs to get through the junctions BUT water can go around the junction
2. Trancelluar - molecules move across cells

Question 16

Trancellular Transport

Answer 16

Use the poalirty to tune to how you want to move the molecules
- Don’t want transporters in equal amounts at apical/basal surface because then would burn ATP but would not change anything

2 ways of doing trancellular transport:
1. Vesicle mediated transport (aka transcytois)
2. Membrane carier/chanels/transporters

BOTH methods require apical/basal polarity

Question 17

Trancytosis

Answer 17

Trancytosis = endocytosis endocytosis on one surface and exocytosis on another surface (transport is coupled from one surface to another)
- Use - transepithelial transport of proteins

Example use – Bring antibodies into the gut put antibodies back in on the other side
- Find the cargo in the basal surface and deliver the cargo to the other side OR find on apical surface and bring it in

Question 18

Transcellular transport using Membrane carrier/chanels/transporters

Answer 18

Directly and selectively bring in moecules

To establish and maintain directional gradients the cell itself need to be directionally polarized (have apico-basal polarity)

Example – transepithelial transport of glucose (vectoral transport of glucose)
- Bring Na and glucose in the apical side and out the basal side
- Na leaves on basal side using a Na/K pump (pump creates the energy to send glucose form high to low on the basal side)

Question 19

Epithelium for compartimelization

Answer 19

Example – endothelium and lymphatics

Compartments seperate regions from each other inside of the body (compartemntolize regions within organs)

Example of comprtmilization – Selective permeability (acheived in 2 ways):
1. Controlling the movement of fluid/movement of small or large molecules between cells using intracellular junctions (primary way)
2. By adding new cells
- Example - Artery wraps smooth muscle around the endothelium to have more compartimilization beyond the intracellular junction so the artery won’t loose blood Vs. smaller vessles have holes in the smooth muscle so they are permeable
- END - things can diffuse out of blood vessels to tissue BUT won’t leave artery

Question 20

Epithliuem Function - Secretion

Answer 20

Example – Mammry + salivary + prostate glands

Glands will make things and are constutley releasing them or releasing the contents ONLY in response to specific stimuli (selectivley released)

Secretion = uses specilized granuals
- Example – Goblet cells (specilzied for secretion) - Know specilized for secretion because most of the volume of the cell is develoted to mucus granuals
- When goblet cells have signal the cell will push mucus out

Question 21

Absorbption in the Epithelium

Answer 21

For absorbtion you always want a higher Surface area

Example - Microvilli in the small intestine
- To increase surface area –> make the intestine longer (has limited gain)
- To increase the Surface area even more = instead of having a flat surface there are microvilli that increases the surface area (have a larger contact area for absorption)

Question 22

How is epithelial structure organized to acheice organ specific function

Answer 22

Overall – use specialized anatomy for specific function

Question 23

Defining epithelial types

Answer 23

Epithelial types are defined by the number of cell layers and the shape of the cells in the top layer

–> Cell layers
- Single layer = simple epitheliam
- Looks Multilayer BUT all of the cells are connected to the BM = Pseudostratified
- Multiple cell layers where some of the cells are not connected to the basement membrane = Stratified
- In between (some areas have one layer and some areas have multiple layers) = Transitional epithelium

–> Defined by the cell shape on the apical layer
- Flat (thin) shape = squamous
- Cube shape = cuboidal
- Tall shape = Columnar

EXAMPLE - Top (free cell layer) = thin sqamous ; have multiple layers of cell = strafified –> Statified squamous

Question 24

Answer 24

Question 25

Apico-basal polaerity of epithelia cells

Answer 25

Epithileial cells have top to bottom polarity in reference to the basement membrane Have polariety among cells in stratified epithelia + have polarity within cells in simple epithelia

Question 26

Polarity among cells in stratified epithelial

Answer 26

Polarity is AMONG the cells - Some layers are at or near the basal layer (close to blood) VS. some layers are close to the apical side (close to the free surface of the lumen) Cells in different layers have different structures and functions Example - Skin (shows idea of polarity in stratified epithelium) - Cells in apical layer = dead --> then differentiating --> then dying --> then amplifying --> stem cells (closest to the BM) - layers go are proliferative near basement membrane vs. differentaed near the aplical surafce

Question 27

Polarity within cells (polarity within cells in simple epithelia)

Answer 27

Simple epithelia = only have 1 layer of cells between the basement membrane and the lumen (ONLY have polarity within epithelial cell) Single cell has a apical surface + basolateral surface (intercellular junctions are between the apical and basal surfaces) - Basal side interacts with the BM through hemidesmosomes in the basement membrane

Question 28

Cell Adhesion

Answer 28

Cell adhesion is a way for cells to communicate Types of cell adhesion: 1. Homophilic cell-cell adhesion - Two of the same cell type sticking to each other 2. Heterophilic cell-cell adhesion --> Different cell types sticking to each other 3. Cell-extracellular matrix adhesion

Question 29

Classes of intercellular junctions

Answer 29

1. Occluding junctions (tight junctions) - Function – control movement of water and molecules between cells 2. Anchoring junctions (has 2 subtypes) - Type 1 - cell-cell anchoring junctions (adherens junctions and desmosomes) - Type 2 - Cell-matrix anchoring junctions (focal adhesions and hemidesmosomes) - Function – conects cells to each other (type 1) or to the ECM (type 2) 3. Communicating junctions (Gap junctions) - Function - Connects one cell to another AND allows the movement of small molecules between the cytoplasm of coennected cells AND allows cells to talk to each other

Question 30

Cells junctions in epithelium

Answer 30

Junctional complex of mature epithelia has oculing junctions (tight junctions) + Anchoring junctions (adherans junctions and desmosomes + hemidesmosomes) + communicating junctions (gap junctions) - Epithelium has multiple classes of adherans junctions - Desmosomes and hemidemsomes have plaques and intermediate filaments coming out - Intercellular junctions are on the lateral surface between cells Location - Tight junctions (apical most) --> THEN adherans junctions --> then desmosomes --> then gap junctions --> then hemidesmosomes (basal side)

Question 31

What do junctions in the epithelium connect to?

Answer 31

Most junctions in epithelium are connected to the Cytoskleton Tight junctions and Adherance junctions --> connect to actin Desomsomes and hemidesosomes --> connect to intermediate filaments

Question 32

Tight junctions

Answer 32

Overall - Tight junctions form a seal that prevents the free movement of molecules between outside and inside environments

Question 33

Tight Junction experiment

Answer 33

Experiment shows shows that tight junctions control paracellular permeability Dip pen in ink well --> put ink in media on one side of a epithelial layer -> look at how far ink goes THEN put ink on the other side of the epithelial cell and ask how far it goes Ink goes to the same place when put on luminal or basal side --> When put ink on the lumenal side – the ink will stop at the apical most point of cell contact AND When put ink of the bottom side– ink stops at the apical most point of cell contact

Question 34

Why are tight junctions so good

Answer 34

Why is tight junction so good --> because tight junction makes a network of interconnection and branching strands that form a belt around a point of contact between the cells Because of belt there is no place for the water or ions to run by = have to accomidate (has to go THROUGH the tight junction it cant go around)

Question 35

Tight junction composition

Answer 35

Tight junctions are intramembranous particles Composed of Claudines + Occulin (adhesion receptors on the membrane that connect to one another across the membrane) - Claudines + Occulin have 4 tranmembrane regions

Question 36

Claudines

Answer 36

Claudines = large gene family Function - Provide the Tight juncton strands and the aqeous pores ALSO provide tissue and cell type specificity in tight junctions - Use the many different types of claudins for specificity (cant do with occulins because there are only 2 types) NOTE (for logic of specificity) - If have many types of 1 protein and few types of the oher protein the protein that has many types gives sepcificty and the one with few types is constitutuve (always there) but does not give specificity

Question 37

Tight junction connection to Cytoskelaton

Answer 37

Claudines and occulins bind to cytoplasmic proteins --> the cytoplasmic proteins bind to actin (Claudin - cytoplasmic protein - actin) Cytoplasmic protein = adapter protein - Cytoplasmic proteins connect to adhesion reecptor (claudins and occulins) to the cytoskalaton - Adhesion receptor is not connected dircetley to the cytoasklaton

Question 38

Types of contact at tight junctions

Answer 38

1. Have contact across the membrane mediated by claudin and occulin (Contact between the claudins and occulins) 2. Claudins and occulins connect to the cytoskalaton through cytoplasmic proteins (Claudines and occulins bind to cytoplasmic proteins --> the cytoplasmic proteins bind to actin) SHOWS Tight Junction is NOT just about the clusinds and occulins binding BUT each other BUT also about the cluadins and occulins binding to adapter proteins and apdater proteins bind to the cytoskeleton - THIS IS what allows you to control cell behavior

Question 39

Zo-1

Answer 39

First Tight junction associated protein Zo1+ caludins + occulins + cytoplasmic adapter proteins has PdZ domain + SH3 domain + GUK domain (Domains = known for mediated protein-protein interactions) - Domains gives the ability to accumulate structural and signaling proteins at that spot

Question 40

Answer 40

Question 41

Dual Function of tight junction

Answer 41

1. Gate --> Controls paracellular permeabiliy (Controlling the anchorage point so things can’t get through) - Seen in India Ink experiment 2. Fense --> partition the plasma membrane into apical and basolateral domains (organizing center for polarity and signaling) - Makes it harder for membrane proteins to go between apical and basal sides (would be hard for protein to get past the tight junction)

Question 42

Shift in concept for tight junctions

Answer 42

Before = Tought of only as a structural barrier NOW = Know tight junctions are also signaling center - Based on BioID (proximity labeling) and mass spec --> found many signaling molecues at the tight junction)

Question 43

Types of anchoring junctions

Answer 43

1. Cell-cell anchoring junction --> connect cells to each other and to the cytoskeleton (Adhering junctions = Adherens Junctions + Desmosomes) 2. Cell matrix anchoring junction --> connect cells to the ECM (hemidesmosomes) Cytoplasmic proteins in the junctions connect to the cytoskalaton

Question 44

Type Adhering Junctions

Answer 44

1. Adherens Junctions 2. Desmosomes BOTH involved in cell-cell adhesions

Question 45

Cadherins

Answer 45

Cadherins = used in the Adhering junctions (desomones and Adherens junctions --> used for cell-cell adhesion) Single transmembrane pass Forms homodimer (2 E-cadherins bind) Have Homotypic binding (homodimer on one cell binds to homodimer of another cell) Large gene family Two types: 1. Classical cadherins (in adherens junctions) 2. Desomosmal cadherins (in desmosomes)

Question 46

Crystcal structure of E-cadheran 1-2

Answer 46

EC1 domain reaches across and binding to the other E-Cadherin on other cell - Makes the zipper model E-cadherin homodimer is formed (Cis interaction) --> THEN dimers interact across the membrane (dimer on one cells binds to dimer on other cell)

Question 47

Affect of 2 Cadherin dimers binding

Answer 47

Dimer on one cells binding to dimer on other cell has no effect on cell behviors because the binding is a small amount of energy compared to what the cell can make with actin/myosin contractility Dimer on one cells binding to dimer on other cell is the start of intercaton BUT NOT what the final junction looks like

Question 48

Adhesion belt

Answer 48

Adherens junction form an adhesion belt --> make a belt of actin around the top of the cell Example – Adherens junctions organzie the actin into microvilli

Question 49

Core elements of adhrens junction

Answer 49

F-Actin + alpha and beta catenin + E-cadherin - Cadherins = adhesion receptors - Alpha and beta catenin = 2 intercellular adpater proteins that connect Cadherins to actin cytoskalaton E-cadherin bind to adoater proteins (alpha and beta katenin) --> adpater proteins bind to the cytoskalaton (actin)

Question 50

Loss of Adhrens Junctions an cadherin

Answer 50

Adhrenes junction = essential for the formation of the epithelium - Dysregulation of adherens junctions can induce loss of other anchroing junctions Lose E-cadherans = fail to compact (fails in morula state) E-cadherin levels are often chnaged in epiltehlial cancer --> changes in E-cadherin affects the amount of adherens junctions in cancer because E-cadherin is used in Adherens junctions

Question 51

Functions of Beta Catenin

Answer 51

2 Functions: 1. Structural role at the PM - In Adherens junction = intercellular adpater protein 2. Signaling role in cytoplasm - In nucelus = regulates transcrioion as a part of Wnt signaling pathway MEANS loss of Adherenes junction can have signaling consequences (because loss of adherens junctions would affect beta catenin) - NOTE it is hard to know if beta catenin going to the nucelus was the result of normal transcription of beta catenin of if the beta catenin was lost for the adhrens junction

Question 52

Answer 52

Question 53

Integrins

Answer 53

Function - Matrix linking protein --> connect cells to the basement membrane in hemidesmosoms - Adhesion recpetor for Cell-ECM in hemidesmosomes Structure – function as a dimer of alpha and beta units (heterodimer) Large gene family (can confer specificity) Integrin activation is confirmation dependent

Question 54

Integrins Vs. Cadherins

Answer 54

Cadherins have small confirmational changes when dimers bind Vs. Intergins = Have BIG confirmational change during activation - Integrins are Confirmation dependent (Tucked away closed version when inactivated ; open when activated)

Question 55

Binding affinity of adhesion receptors (Includes cadherins + integrins)

Answer 55

Adhesion receptors binding across the cell membrane have weak binding affinity Have weak affinity in: - Adhesion receptors for cell-cell adhesion = cadhrins (in adherens junctions and desomsomes) - Adhesion receptors for cell-ECM adhesion = integrins (in hemidesmosomes) Example - Cadherin dimer pairs interacting (in cell-cell adhesion) OR Integrin binding to the ECM (cell-ECM adhesion) are weak = has no effect on cell behavior

Question 56

How do you get strength in Anchoring junctions (Adhrens and desmosomes) - how do you get strong adhesions

Answer 56

Make junction stronger by: 1. Many adhesion receptors per junction (many cadherins/integrins) - 1000s of adhesion receptors binding per junction increases the strength - Avidity – low affinity becomes high energy by gaining avidity (lots of interactions in parallel) 2. Cluster the interaction among the transmebrane adhesion receptors (ex cadherin) - Function of the adhesion receptor (cadherins or integrins) changes because when cluster they will have cis interactions with 1000s of receptors at that site 3. Linking of adhesion receptors to cytoslaton through intracelular adapter proteins - Connects recpetprs to contractility if connect to actin

Question 57

Strengthening of cell-ECM junction

Answer 57

Uses the same method as strengtening the cell-cell anchring junctions Example - Integrins bind to adapter proteins and the adapter proteins bind to the cytoskeleton

Question 58

Summary of Intercellular Junctions (Linker protein and the cytoskaltal compoennet the junction attatches to)

Answer 58

Question 59

Answer 59

Question 61

What do tumor cells need to invade?

Answer 61

For tumor to invade the BM and metastsize: 1. Pile cell layers up 2. Loss/change in cell-cell adhesion (need to let go of neighbors and break off an individual cells) 3. Break through the epithelial basement membrane to get to the connective tissue - Cells can shred the BM or use proteases to digest the BM (or can mix both) 4. Invade connective tissue - Normal epithelial cells are NOT in the connective tissue 5. Survive in new ECM envirnment (now surrounded by new ECM and immune cells) - Survival is one of the hardest thing cells need to do to metastisize 6. Invade through the vscular BM to access circulation and leave organ

Question 62

Desmosomes (overall)

Answer 62

Desmosomes = type of adherens junction (adhesion spots between cells) Desmosomes – big and thick (have many proteins there)

Question 63

Location of desmosomes

Answer 63

Desomoes are found where mechanical stress is high - Hold tissues together in places with high mechanical stress Example - Epihelial layer of skin (epidermis) has many desmosomes

Question 64

Molecular basis of desmosomes

Answer 64

Desmocolin + desmoglin = desmommal cadherins (adhesion receptor/linking cells) Desmplakin + plakoglobulin + Plakophilin = Cytoplasmic Adapters proteins - Adapter proteins bind to Intermediate filaments Attatchment to Cytoskelaton - Desmogelin or Desmocolin bind to plakophilin/plakoglobulin/desmoplakin --> desmoplakin binds to IF

Question 65

Gap Junctions

Answer 65

Function - Connects one cell to another AND allows the REGULATED movement of molecules between the cytoplasm of connected cells Location of gap junctions: In epithelium + smooth muscle + cardiac muscle + nervous tissue + bone (osteocytes) To image Gap junctions – use freeze fracture (look at face of contact between cells) --> see many cylinders

Question 66

What goes through Gap Junctions

Answer 66

Gap junction = intercellular chanel allowing ions/small molecules to go from one cell to another - Often move Ions + sugars + amino acids + cAMP - Junctions can be organized to be bigger for bigger moelcules to pass ( selectively move small proteins through) ; in plants can move transcription factors between cells Gap junction be as restrictive as 100 Da - Example – Ionic transport (can have have signaling consequences by selectivly releasing Calcium to other cell)

Question 67

Structure of Gap Junctions

Answer 67

Gap junctions = composed on connexins 6 Connexins join to form a connexon - have a gap in the middle of the connexon (ion goes in the middle gap) - Connexon can be homomeric or heteromeric - Connexons cross the intercellular space

Question 68

Confirmations of Gap Junctions

Answer 68

Gap junctions can be in open or closed confirmation Shows gap junctions are REGULATED pores between cells Two levels of regulation in Gap Junctions: 1. Composition of connexins determines how big the pore is (determines the size of molecules that can pass) 2. Gating activity of gap junction is regulated (Open vs. shut is regulated)

Question 69

Answer 69

Question 70

Are epithelial tissue static or dynamic

Answer 70

Many epithelial tissues are dynamic (change regularly) Dynamic nature requires the remodeling of epithelial junctions Example of dynamic epithelial tissue - Skin - Dynamics = Skin sheds the dead cells and makes new cells that will differentiate and then shed - Cycle - stem cells (bottom) --> progenitor cells --> expansion of popultion --> terminal differentiation (leave cell cycle) --> final stage of differentiation --> dying (Cycle requires an exchange in intracellular junctions) - Can’t leave one layer and go to the next without movement + proloferation + change in junctions

Question 71

What is needed for epithelial cells to change neighbors

Answer 71

IF Need to change neighbors = need to change junction Example - need to go to a different layer of skin = need to change intercellular junctions

Question 72

Example #2 of Dynamic epithelial tissue

Answer 72

Renewal of intestinal epithelial cells Villi (apical side) sheds cells into the lumen (no cells divide) At crypt (basal) cells divide - Have stem cells --> stem cells go up from the basal crypt to the villi where they will be shed There are SOME cells that are stationary (Ex. goblet cells) BUT the epithlial cells are not

Question 73

What does metastisis require

Answer 73

Metastasis requires changes in each fundamental properties of an epithelium Metastists = biggest set of changes in epithelial cell behavior

Question 74

Steps in metasisis

Answer 74

Start - Have epithelial cell --> rapid epithelial cell division --> cell receruits vasculature cell goes on walk about (detatches and survives)--> cell goes through the basment membrane --> cell goe sto the endothilum (Intavasion) --> cell is in blood --> cell survives and goes through endothelium to leave the blood vessel (Extravication) --> go through basement membrane --> micromestasis --> profilerfate and evade immune system/coapt immune and recurit vasculature --> grow to significance Needs exchanges in cell-cell and ell-ECM adhesions to accomplish this

Question 75

What happens when the cell goes through the endothelum and is in blood

Answer 75

Cell is in a fluid with immune cells and shear stress - Cell needs to evade apoptosis due to new envirment + immune cells + shear stress VERY new envirnment for cell (cell has never been a single cell and it has never been in a fluid)

Question 76

What happens when the cell forms a micrometasis in new location

Answer 76

Cell is in a new envirnment AGAIN Cell in in a ’mismatch envirnment’ (has worng intergin pairs for wrong ECM)

Question 77

Steps in metastisis

Answer 77

Primary tumor --> detatchment --> intravastion --> migration --> extravasion --> micrometasisis --> macrometastisis In macrometasisis = have colinization + proliferation + angiogensis

Question 78

Burden of cancer on huamans

Answer 78

The overwhelming burden of cancer on humans is epithelial cells giving rise to caner (carcinomas) and most people die from metastasis Most death due to epithelial cancers is due to metastasis MEANS that very few cells that can do the whole cycle and grow to metastasis at the end that drive 90% of cancer deaths

Question 79

The central challenges in metastasis

Answer 79

The central challenges in metastasis are: 1. Escape from the primary site into the adjacent connective tissue 2. Enter/Survive/exit vasuclature or lymphatics 3. Evade immune surveillance 4. Survive and proliferate at a distant site Each of these steps requires careful control over cell-cell and cell-matrix adhesion

Question 80

IF take kidney cells and put in a dish --> can you get organ/tissue level physiology and structure

Answer 80

NO 2D can be good BUT it is NOT helpful if you want tissue or organ architecture Issue = In 2D culture 2 different cell types look the same - Example - Epithelial cells vs. Mesenchymal cell in a 2D dish --> look the same

Question 81

Modeling tissue morphogensis and cancer in 3D

Answer 81

Can model tissue morphogensis and cancer in 3D If put epithelial cells and mesenchymal cells in a 3D envirnment the epithelial cells organize into epithlum (with junctinos+ basement membrane + polarity) AND meshceychymasl cells stay inside the ECM

Question 82

3D culture Overall

Answer 82

3D culture – common technique 3D culture models have been developed for most organs and many disease states There is no 1 perfect technique --> need a good match to the question you want to answer and the methods you are using

Question 83

What can you culture in 3D?

Answer 83

1. Whole organ/organ slice 2. Tissue organoid - Example - peice of kidney epithelium and ECM 3. Stem cell organoid (isolate stem cells and expand them) 4. Primry cells - Primary kidney epithelium in barier culture

Question 84

Forms of 3D culture

Answer 84

Can put any of the cell inputs into any culture forms Types of culture: 1. 2D cells 2. 2D cells + ECM (add ECM under or ECM in the media) 3. 2.5D/Drip culture (embdeding the cells in jello) 4. Culture inserts - Grow cells on cologen + fibroblasts - Gives the air liquid interface (strong signal for differentiation) - Ex. make skin used for grafting from keratinciyin and collagen

Question 85

Use of 2D culture

Answer 85

Use 2D culture to answer sub cell level question (might only need to think about what are the right cells to put into a petri dish)

Question 86

Example of Organ Culture

Answer 86

Use whole organ culture to study kidney branching morphogensis in real time Use 1 – can get live imaging of kidney branching morphogenesis in 3D organ culture Use 2 – Map epithelial branching in real time (how epithelial cells keep brancing) - Limitation = not at cellular resolution - Can map the developmental program by which kidney achieve adult structure

Question 87

and is the interaction strong or weak

Answer 87

Answer – Have adhesion of cells through transmembrane receptors (likey cadherins superfamily) What is the first step when cells contact each other (NO junction yet) --> get adhesions (start to get contact between the cadherins with one pair of dimers toucing) --> THEN more cadherins dimers to bind to other dimers (more cadherins bind to ecah other) - Have cadherin dimers in the membrane --> cadherins will bind in diferent cells (one pair of dimers will inercat) IS this string or weak --> Answer = weak (individual cadherin interactions are weak) - The first dimers binding has week energy of interactions

Question 88

Answer 88

Answer – to reinforce the connection recruit cytoplasmic proteins and cytoskelataon + ALSO recrit more cadherin compelxes to the junction - Have more dimers intercating THEN more dimers are recruited and more adpaters are recruited (adpaters will link to the cytoskalaton)

Question 89

Answer 89

Question 90

Question – what happens if deplete E-cadherin in single cells

Answer 90

No depletion = then the cells would be able to form a juncton No E-cadherin --> no contact (maybe the cells would undergo apatosis) - Cells won’t ahdere (never get strong interactions) - Might affect other things aside from adhesion

Question 91

Scnerio – have adhesion (dimers in contact) THEN no E-cadherin

Answer 91

Answer – Loss of E-cadherin in an imature adhesion (just after adhesion) would lead to loss of cell to cell contact What would happen = cells would separate and maybe undergo apoptosis - Formed junction would fall apart Cells could stay in the same configuration mono layer of cells that were connect might still look connected but they wouldn't actually have molecular connection) - Monolayer woudl look in plate BUT they are not actually connection - If the cells were sparse when you lose the ocnnection then the cells would fall apart

Question 92

Answer 92

NOW – have E-cadherin protein BUT use shRNA to Knockdown the RNA Answer – already existing protein stays and makes the junction BUT not making new proteins (not making new junctions) Depending on the rate of protein degredation MIGHT keep the junctions for a while HIS answer – depends on the depeltion method + contecxt + time scale - Could fal apart if protein turnover rate is high or stay intact if have a low protein turnover rate

Question 93

NOW have multiple receptors (now have multiple adhesion molecules) ; NOW have many types of adherens receptors

Answer 93

IF deplete E cad in single cells --> cells can fuse other molecles (make the phentype less sever - Could have less sever of an affect --> can use the other things instead - Could have affects on diferentatiion and affect cells behavior Princple of answer = in image

Question 94

First expeinet done to see if loss of E-cadherin alon disrupts adhesion in cells

Answer 94

Used siRNA – should kill adhesios and cell should fall apart --> when added siRNA nothing changed (no change in qRTPCR) AFTER - Did Calcium switch because these are calcium dependnet adhesion receptors - Depeleted Ca -->junctions fell apart --> cells released off teh surface - When add Calcium back the cells had trunover so much of their cadherin that they cound’t stick - Cells will rely on calcium depent adhesion when lose E cad

Question 95

Answer 95

In embryo: Embyro would die (no implantation) - might have the first 4 or 8 clles BUT No compaction = no morula = no blastocyte In skin: - Lose E cad then now prone to mechanical rupturing - Barrier disrupted = more immune cells can get in= inflammation - Deletion of tight junctions is perinatal lethal because can’t control movement of water (dehydrate because skin protects loss of water and mice cant drink faster enough to balance loss of water through epidermis)

Question 96

Summary

Answer 96