bio 211, lecture 13 - cell junctions and matrix

clefting

smooth clump of epithelia, then involutes and forms wedges that split the epithelia into many distinct clumps

steps for the process of forming a neural tube

1. fertilization of the oocyte

2. many rounds of cell division without any growth

3. gastrulation

4. hollow ball of cells

5. epithelia sculpts

6. sides close to form epithelial tube

convergent extension

happens when cells reorganize so that they shrink (converge) in one axis and elongate (extend) in another axis

cadherin receptors

in the PM; bind to other cadherins in neighboring cells

mechanical stress

transmitted from cell to cell by cytoskeletal filaments anchored to cell-matrix and cell-cell adhesion sites

ECM

directly bears mechanical stresses of tension and compression

cell matrix adhesions

found at the bottom of the epithelial cells; connect to actomyosin filaments

desmosomes and hemidesmosomes

• plug into IFs

• provide structural strength to the tissues

• not a big role in generating and transmitting forces

basement membrane / basal lamina

• epithelial cells sit on this

• function is to provide structural support for the tissue

• provide physical barrier to separate outside from inside

what are the 2 ways to attach to the basement membrane?

• actin-linked cell matrix adhesions

• hemidesmosomes

actin-linked cell matrix adhesions

• integrin-containing complexes that plug into talin and vinculin

• transmit forces from actomyosin network to outside, and vice versa

hemidesmosomes

• also attached to ECM at basement membrane

• filament system they’re plugged into is IFs

• job is to provide structural strength

gap junctions

• do not give structural strength

• put the cytoplasm of adjacent cells into communication with each other

• have gap of sufficient diameter that macromolecules can freely flow from one cell to another

relationship between calcium and gap junctions

• Ca concentration in the cytoplasm is increased, and activates Ca-dependent signaling events

• gap junctions allow Ca to flow into adjacent cells

desmosome

• attached to IF on the inside of the cell

• on the outside of the cell, they form junctions on neighboring cells

• give structural strength to the tissue

• mediate strong adhesion

what is the difference between desmosomes and hemidesmosomes?

• desmosomes are junctions involved in intracellular adhesion of epithelial cells

• hemidesmosomes are junctions involved in adhesion of epithelia to basement membranes

tight junctions

• connect the cells so tightly that not even ions can flow past

• prevent the flow if big and small molecules across epithelial layers

tight junctions between the cells

only way for nutrients or other molecules to get across an epithelial layer and reach the bloodstream is to go through the cell

homophillic binding

• cadherins bind to each other

• Ca2+ ions are required in the extracellular space for homophilic binding to occur

E-cadherin

transmembrane glycoprotein which connects epithelial cells together at adherens junctions

P-cadherin

cell adhesion protein that helps maintain tissue structure and polarity

N-cadherin

transmembrane protein that helps cells stick together in a calcium-dependent way

how is calcium involved in making connections?

• outside the cell, there is lots of Ca in the environment

• Ca binds to cadherins at the hinge regions that occur between cadherin domains

• this makes the protein more rigid and less flexible (rigidity is important in forming stable connections to neighboring cadherins)

what happens if you get rid of calcium?

the cadherin connections cannot form, and existing connections rapidly fall apart

“sorting out” — different cadherins

• mixed population of cells containing different types of cad

• after a few days, they will have formed distinct clumps where it is all one cad in one clump and another cad in the other clump

why does sorting out occur? (different cadherins)

• as cells bump into another one with the same cad, it becomes a firm connection, and they don’t let go (essentially an irreversible connection)

• if they bump into a cell with a mismatched cad, they can let go and keep searching

“sorting out” — different levels of cad

cells with different amounts of cad are able to interact with each other, but the cells expressing more cad can form more connections with each other, so they get sorted out

why do the cells with different amount of cad get sorted out?

• to have the most surface area in contact with the high-expressing cells (more connections this way and more stable arrangement)

• cells with less cad will be kept to the perimeter because they form less junctions this way

cells with less cad are (more/less) adhesive

less

what is the overall purpose of sorting out?

allow for a higher degree of organization within the developing epithelial tissue

neural tube formation in later stages

• e-cad is enriched at the surface epithelia and down-regulated as the neural tube begins to form

• neural tube expresses many cads due to “sorting out”

• cells that express the same cads form these clumps within the tissue — segregated from other epithelial cells within this neural tube

what happens if cad expression is messed up?

organization is lost

p120-cat and b-cat

bind the cytoplasmic tail of cad and help traffic the receptor to the PM and stabilize the initial homophilic binding

p120-cat

helps cad get expressed at the PM

b-cat

• helps connect the a-cat to the cad

• considered an adaptor (one side connected to cad and the other side connected to a-cat)

a-cat

• provides the bridge from the cad receptor to the actomyosin filaments

• binds to b-cat and connects e-cad to actin filaments

vinculin

brings additional actomyosin filaments to the complex following the application of force to the complex

applying force from outside the cell

pulling on a neighboring cell and causing tension on the cad

applying force from inside the cell

activate RhoA to activate contractility then tug on the cad form inside the cell

applying force from inside the cell or outside the cell triggers…

a positive feedback loop of force which leads to growth and strengthening of the cad junctions

what does a-cat do in response to force?

• stretches

• reveals new vinculin binding sites that were previously hidden

• allows actomyosin fivers to get plugged into complex and more force to be applied

step 1 of cell-cell adhesion

initial cell-cell contact

• tissues move toward each other and contact each other for the first time

• both express the same cad

• form homophilic interaction across the gap between cells

cadheins cluster at initial contact site

step 2 of cell-cell adhesion

rac activation and arp2/3 nucleation

• initial contact activates Rac1 at those sites

• Rac1 activates arp2/3 at sites of initial contact

• arp2/3 makes branched protrusions

• Rac and Arp2/3 push the PM forward so it can form more connections with the underlying matrix through nascent adhesion and adhesion maturation

local rac activation and rho inhibition leads to loss of cortical tension

step 3 of cell-cell adhesion

rac1 turned off an RhoA turned on to strengthen adhesions

• Rac1 activated, then RhoA turned off through cross-talk pathways

• rac1 finishes expanding zone of contact, and GAP shuts it down (allows RhoA activity to increase at newly expanded site of contact

• RhA activity is also responsible for strengthening adhesions

expansion of adherens junction

when rac1 activity is decreased…

RhoA activity is elevated

what happens when a-cat is extended?

• force-dependent change in conformation reveals the new vinculin binding site

• now have a second actomyosin fiber getting plugged into a-cat

• positive feedback where you get massive expansion of contractility attached to growing/strengthening cads

what are you left with at the end of the cell-cell adhesion process?

epithelial tissue with mature cell-cell junctions that are localized just below the apical surface (close to the top, but not at the very top)

cad role between cells

connected into “belt” of actomyosin contractility, which can generate further force in response to more activation signals

tissue shaping

spatially and temporally regulated actomyosin contractility and adhesion in epithelial cell monolayers

cell-cell contractility

belts of actomyosin bundles connected by homophilic e-cad binding

cell-matrix contractility

integrin-based cell-matrix adhesion connected by actomyosin stress fibers

order of layers in the cell

1. epithelial cells

2. basement membrane

3. collagen and fibroblasts

stroma

• no BM

• more loose and fibrile protein network

• mostly made of type 1 collagen

• cells can move through the fibers easily, unlike the BM

steps of ECM tissue formation

1. heart perfused with detergent to remove cells

2. only the ECM remains

3. adding neonatal cardiac cells to the matrix = beating heart

how do cells move and function in wound healing?

in wound healing, cross-talk between cells depositing matrix and the matrix that the cells deposit tell the other cells where to go, what to do, and how to differentiate

proteoglycans and GAGs

• large, highly charged polysaccharides (GAGs) can be linked to protein (proteoglycan)

• shock absorber

• enriched in cartilage

• fill spaces during development

polysaccharides attract _____ to ______

water, balance the charges

fibrous proteins are found in _____

dermis and BM

type 4 collagen

• fibrous protein

• component of BM

type 1 collagen

component of loose reticular stromal matrix

fibrillar collagen

protein that provides structure and stability to tissues like bone, cartilage, and skin (most abundant protein in the body)

glycoprotein

• found in BMs

• more protein than it is sugar

fibronectin

• critical for development and wound healing

• dynamic matrix protein which can be deposited in the context of wound healing/development to enhance adhesion of fibroblasts

laminin

cooperate with type 4 collagen to weave together the sheet-like BMs

nidogen

helps maintain BM and tissue architecture; also has role in cell adhesion, migration, and signaling

proteoglycans are composed of ….

GAG chains covalently linked to core proteins

linking to form core proteins

1. core proteins are synthesized in the ER

2. polysaccharides added in the Golgi

3. mature proteoglycan secreted into the extracellular space

aggrecan

• enriched in cartilage

• acts like shock absorber to protect joints from mechanical stress due to high-degree of hydration

single collagen fiber

triple helix of 3 different collagen proteins woven together — α, β, γ

bundling of collagen

• bundled into thicker, stronger collagen networks

• one way of being organized into higher-order structure

• fibrils grouped to make fibers

alignment of collagen

• all the bundles are going the same direction

• can switch by layer; controlled by deposition

fibronectin

• interact with integrin (cell)

• interact with collagen and fibrin (makes up blood clots)

• acts like signal (development, wound healing)

fibronectin & wound healing

1. wound: vessel severed

2. fibronectin spills out into the surrounding stroma (matrix surrounding the blood vessels; enriched in type 1 collagen)

3. fibronectin binds to collagen and to nearby cells

4. cells activated and provided with additional adhesive sites to move along the fibro-bound collagen

why does BM look like a sheet?

the 2 matrix proteins that make up the BM (type 4 collagen and laminin) have many different binding sites for other matrix proteins and cells

the more binding sites there are ….

more tightly woven together the network will be

laminin is a ____, and can bind _____

• trimer

• integrins, other laminins, and other matrix proteins (it’s a trimer)

type 4 collagen

• forms many additional connections compared to type 1 collagen

• comes together with other proteins to form the sheet-like structure that is woven together tightly to be a barrier

inherent complexity

the systems are interconnected by cytoskeleton and signaling cross-talk

hyaluronan

large GAG which is produced at the cell surface by a complex of enzymes

why is hyaluronan important?

important in development and wound healing by providing a transient space-filling material that can maintain the shape of a tissue while cells migrate

proteoglycans, GAG chains, core protein

1. core protein synthesized in the ER

2. polysaccharides added in the Golgi

3. mature proteoglycan secreted to the extracellular space