Cell-ECM Adhesion Flashcards
cells in an anima live a crowded life
-we tend to study them as individual but in the animal they live in a crowded life surrounded by other cells and ECM
-if they’re epithelial cells, there’s a BM
-soluble and insoluble signals being exchanged
four major families of cell adhesion receptors
-cadherins
-IgCAMs
-integrins
-selectins- bind between immune cells and endothelium
integrins mediate cell-ECM adhesion
-some ECM protein bound by dimer of integrins- heterodimer with alpha and beta subunits
-cross a cell membrane and tail will accumulate adaptor proteins connected to a cytoskeleton
-epithelial tissue context- cells closely adhering to each other sitting on BM and major point of contact with ECM is with BM specifically
-immune cells and fibroblasts that sit in the mesenchyma stroma- relatively cell sparse with few cells per unit area volume
-cell-ECM adhesion is more than just tickling of receptors in a ligand, AJs needs to connect to cytoskeleton to achieve those strong changes in cell behavior
inactive (closed) and active (open) conformations of integrin dimers
closed- tucked away then domains swing open and much higher affinity for ligand in open state
integrin activity is regulated by both dimer conformation and receptor clustering
-is it open and touching its neighbor?
-important AAs that regulate cis or lateral interactions between integrin dimers in the membrane
integrins link the BM to the intermediate filament cytoskeleton at hemidesmosomes
-hemidesmosome is named b/c it looks like half of a desmosome in EM –> does not use the same adaptor proteins or adhesion receptors and doesn’t bind the same ligand on the other side
-ECM-integrins-adaptor proteins connecting to intermediate filaments or keratin cytoskeleton
integrins link the stromal ECM to actin cytoskeleton at focal adhesions
-immune cells/fibroblasts moving around- more dynamic adhesions
-thinking about ECM, integrins, adaptor proteins linking to actin cytoskeleton –> couples actin-myosin contractility directly to integrand ligation
when are anchoring junctions dynamic?
-cell migration, embryonic development, and tissue remodeling and repair
-if you want to change the structure of tissue, have to change the structure of the ECM –> junctions need to be dynamic
assembly and disassembly and focal adhesions during cell migration
-single cell on a substrate –> how does it move forward?
-important pocs organized as focal adhesions and basic rhthym of moving is you extend and make new adhesions then move cell body forward and release prior adhesion
-whole set of interactions to strengthen, mature, and disassemble focal adhesion
maturation and disassembly of focal adhesions is tightly coordinated during cell migration
-1st contact with integrin and ECM- start to recruit adaptor proteins and it matures –> start to recruit actin –> forms large, mature focal adhesion that produces significant forces –> starts to disassemble and fall apart
-cycles of assembly and disassembly that are constantly running underneath as a cell is migrating across a substrate
-in 3D environments, you might be pushing in different directions but still fundamentally need to reach out, grab, exert force, and release
what are the core components of the focal adhesion?
ECM, integrin, FAK, talin, vinculin, paxillin, and actin
regulation of cell adhesion receptors
-cell type specific expression- dozens of cadherins and integrins- change ones that re there and get different binding properties
-trafficking to or away from the cell surface- adhesion receptors in the membrane and endocytosis can remove them from the membrane individually or remove large clusters of them from the membrane to give almost immediate effect of reduction or increase in binding ability
-lateral interactions within membrane also matter- whether they’re mono dispersed in uniform fashion or clustered at discrete points organized into junctions
-state of activation- more important for integrins than adherins
ECM
-composed of macromolecules, proteins, and polysaccharides (sugars)
-synthesized and secreted by cells
-connective tissue (fibrillar) and BM (dense sheet)
-collagens in both groups
integrn binding to the ECM has signaling consequences inside the cell
-thought of as being simple places you attach- just mechanical contact
-cell notices whether integrins are ligated- if you have integrin bound to ECM, on the inside that accumulates proteins like FAK and Src
–> once you have FAK and Src, you can get to MAPK
–> once you’re at Src and MAPK, you can regulate any cell behavior- proliferation, migration, differentiation
-integrins themselves do not have enzymatic activities (no kinases or proteases)- they borrow enzymatic activity of intracellular kinases so ILK, FAK, and Src
–> binding of integrin FAK induces its auto phosphorylation and causes phosphorylation of a number of targets
FAK-mediated pathways target the cell cycle
-once you get right to FAK then you can control Src and JNK and MAPK and cyclin D1 and go to cell cycle progression
-Src can also get you to RAC –> actin cytoskeleton –> cell migration
what are the major classes of ECM?
-adhesive glycoproteins
-collagens
-proteoglycans
fibronectin
-when you look at the domain structure, you have heparin and fibrin binding, collagen binding, fibrin binding, cell binding, heparin binding –> great logic to build a sheet b/c every one of these individual domains binds to others and you get dense lateral interactions that make the ECM strong
-RGD loop is where cells bind and other domains are about positioning proteins in the right place relative to their protein neighbors and making RGD motif conditionally available depending on orientation
-fibronectin is single gene with alternatively spliced isoforms- present in ECM as fibrils and blood as soluble
laminins
-major compnent of BM- binds collagen IV, GAGs and integrins
-logic of binds type IV collagens, heparins, and integrins –> great way to organize that has lost of lateral interactions
-at least 15 ways to make laminin from combos of alpha, beta, and gamma laminin genes
collagens
-synthesized, secreted, and organized by cells
-major structural component of ECM and provides tensile strength to tissue
-regulates variety of cellular activities via integrins
-sheet forming (type IV) and fibrillar (type I)
collagen I: main fibrillar collagen
-most abundant protein in the body
-major structural component and provides tensile strength
biosynthesis of fibrillar collagen I
-fibers bundled into bigger bundles into bigger bundles
-make a polypeptide then synthesize pro-collagen alpha chains then hydroxylate them then you have to glycosylate them –> initiate folding of 3 pro-alpha chains –> pro collagen alpha helix that gets secreted –> cleave polypeptides and polypeptides form basic unit that assembles first into fibrils then fibers
proteoglycans
-in the ECM, they’re structural and mechanical support for tissues, allow diffusion of soluble factors and migration of cells, and they regulate activities of growth factors and secreted proteins
-co-receptors for integrins and growth factor receptors in the membrane
hierarchical structure of proteoglycans
-GAGs- major structural component of ECM and you have high density of negative charges that coordinate water-sugar with amino group
Ex. cartilage in knees resists compression when you run b/c of presence of GAGs
-have protein core decorated with GAGs and each of the red lines has a lot of negative charges that coordinates water then you put big strands with lots of these and all of them individually accumulating a lot of water then you have resistance to compressive strength
MMPs cleave ECM and other extracellular proteins
-pro domain and when it’s present the MMPs are inactive
-catalytic domain that actually does cutting
-once it’s active, these can do multiple things
-large gene family
-MMPs go out in front of migrating cells, degrade ECM, and make space for cell migration
-clear Exs. in development and cancer where MMP clears space through BM or stromal ECM
-they can also alter microenvironment to release signaling molecules
-change availability and activity of growth factors and other proteins
-ECM can act as memoory where you can secrete a lot into ECM and it will bind there and be unavailable to the cell until you release an enzyme to get it then all of the proteins become available —> provides way for cells to respond quickly
-accumulate in ECM for extended time then you release MMP you’re able to rapidly make them available to cell
-you can slo alter the balance of protease activities b/c they’re pro domains and they require cleavage to become active, one MMP can cleave the domain of another MMP and make it active
-endogenous inhibitors that allow cells to put away this activity –> make a lot of MMP activity and put it away by making more TIMPs
deletion of individual MMPs has tissue specific consequences
-MMP that cleaves lots of stuff without one-to-one relationship to substrate
-each MMP can cleave and arrange different substrates
-take normal development of mammary gland the ducts elongate and branch
-take away MMP2 they don’t elongate as well but side branch more
-take away MMP3 they elongate as well or better but don’t side branch
-get very specific defects in overall tissue architecture as result of gain or loss of specific MMPs- ECM environment in front of one of these buds is quite different from the ECM along the trailing ducts
–> takes different MMP activities to allow space for change
overexpression of MMP3 is sufficient to induce breast cancer in mice
-gave rise to various forms of hyperplasia –> early adenocarcinoma –> late adenocarcinoma –> metastasis
-changed people’s minds of what could be oncogenes
salivary gland branching is a model system to study morphogenesis and tissue remodeling
-b/c in the early embryo, forming bud that cleaves to give rise to a variety of branch structures and trailing ducts
-exchange of cell-cell interactions (cadherin-mediated) for cell matrix interactions (integrin mediated)
role of MMPs in morphogenesis
-whole salivary gland- elongates then branches
-increase MMPs- elongates fine but doesn’t branch
-decrease MMPs- lots of extra side branching
changing the ECM composition by adding fibronectin also changes branching
-tells cells what to do- is that enough? how could it matter to change one protein?
-already started clefting at later stage and branching more in the control
-add fibronectin and you get even more branching- cells are acutely listening to the amount of protease activity and type and amount of ECM protein
salivary gland branches by exchanging cell-cell for cell-matrix interactions
-cells come in –> exchange progressitvely of cell-cell to cell-matrix –> fibroblasts migrate into cleft and help expand it –> reinforces with additional ECM proteins
-ultimately these are cell-cell being exchanged for cell-matrix
expt. #1: ideal fibroblast with single type of ECM receptor…analyze the effects on cell migration relative to WT when one class of integrand dimers is deleted
-deleted so there’s no integrin activity
-cells might float away- doesn’t have the ability to interact with surface- this if this is the only way that it can touch the surface
-without integrins, worse at spreading could make it worse at cell migration
expt #2: real fibroblasts with multiple types of integrin dimers
-WT fibroblasts plated on different ECM proteins…what could be the differences in behavior?
-change shape and differentiation state
-rate of proliferation
-change in migration speed
-cell death rates
-altered cytoskeleton contractility
-altered signaling
expt. #3: real fibroblasts in 3D ECM…effects of loss of one integrin gene?
-reduced rates of migration
-changed signaling
-defect in migration
-blinder or more sensitive to specific pairing
-nothing could happen
-cells could die
-proliferation could also change
-differentiation
expt. #4: real fibroblasts in 3D ECM…effects of loss of all integrin function?
-interfere with cyclins you could affect cell cycle and get cell cycle arrest
-failure to migrate
-cell death
-faster migration without integrin activity –> they’re about making strong connections and exerting force on substrate
-could limit or gain proliferation
-lose integrin-based adhesion to the matrix
expt. #5: real fibroblasts in 3D ECM…effects of loss of one MMP gene?
-nothing happens
-cell dies
-limit migration
-could be master regulator and get strong phenotypes
-could have strong effect on ECM organization
-proliferation
-differention
expt. #6: real fibroblasts in 3D ECM…effects of loss of all MMP genes?
-defect in ECM remodeling
-fail to access various growth factors or signaling molecules
-get confused and possibly die
-affects on migration are context dependent: if spacing between fibers in the ECM is large, migration might be minimally affected but in a densely cross linked ECM, migration might be blocked entirely
expt. #7: human genetics
-WT/null of the collagen I gene- nothing may happen since you have another WT allele but may not get 100% function
-WT/super sensitive collagen I- body doesn’t know it’s producing a super sensitive copy of collagen, so every strand of collagen I will be sensitive –> structural integrity of tissues will be compromised
expt. #8: mouse genetics…effects of loss of MMP14 gene?
-could have issues with the neural crest migrating in embryonic development
-MMPs regulate access to signals and may lose this access to signals
-no cell migration
-may be able to degrade BM
-as it grows too much, the organs don’t accommodate for increased growth
-in the early embryo there’s very little ECM and comes later on in fetal development
