Module 4: Cell Adhesion and Communication w ECM

cell-adhesion molecules (CAMs)

mediate direct cell-cell adhesions (homotypic and heterotypic), and adhesion receptors mediate cell-matrix adhesions

• primarily integral membrane proteins, which cytosolic domains that bind intracellular adaptor proteins that link the surface with the cytoskeleton (typically actin and intermediate filaments)

extracellular matrix (ECM)

a dynamic, complex meshwork of proteins and polysaccharides that contributes to the structure and function of a tissue

• cell-matrix adhesion molecules → adhesion receptors bind to ECM components; link the external environment to the internal cytoskeleton

bidirectional information transfer

• outside-in → from CAMs and bound extracellular macromolecules to the cytoplasm

• inside-out → from the cytoplasm through adapter proteins to CAMs and bound extracellular macromolecules

cadherins

bind to each other (homophilic and heterophilic) via domains

CAMs

members of the immunoglobulin superfamily

• form both homophilic and heterophilic interactions

integrins

heterodimeric, consist of α and β chains

• adhesion receptors, bind to large adhesive proteins such as fibronectin in the ECM

selectins

contain a carbohydrate-binding lectin domain that recognizes specialized sugar structures on adjacent cell glycoproteins/glycolipids

CAM cis interactions

referred to as intracellular or lateral interactions

• forms lateral clusters within the plasma membrane of the same cell

• regions that form cis interactions vary among different CAMs

CAM trans interactions

referred to as intracellular or adhesive interactions

• generate strong, velcro-like adhesions between neighboring cells

mutually reinforcing

Trans and cis interactions are ______________.

• cis interactions can increase probability of forming trans interactions

• trans interactions can induce cis interactions, which in turn strengthen trans interactions

factors that regulate adhesive strength

• clustering → at cell junctions where CAMs tend to cluster, the CAMs can generate very tight adhesion when many weak interactions are combined

• binding affinities

• kinetic properties of the CAMs (“on or off” rates) control the association/dissociation properties, and thus the strength of the adhesion

• spatial distribution and density of molecules (ensemble properties)

• biochemical properties and “active” vs. “inactive” states

• external forces such as stretch and pulling

ECM stiffness

varies across tissues

• brain → soft ECM

• muscle → intermediate ECM

• bone → rigid ECM

influences cell behavior

• regulates cell shape, migration, proliferation, and differentiation

mechanical properties from ECM compositions

• collagen provides tensile strength

• proteoglycans provide compressive resistance

• cross-linking proteins (laminin) provide increased stiffness

true

T/F: Cells can sample and “sense” the ECM through transmembrane adhesion receptors (integrins) which then communicate to the cytoskeleton

• instructs cells how to behave within their environment

  • cell convert mechanical properties of the ECM into biochemical signals

mechanotransduction

alters cell behavior; drives gene expression, migration, and fate decisions

extracellular matrix proteins

proteoglycans → unique type of glycoprotein

collagens → form fibers

multi-adhesive matrix proteins → organizers of the ECM

• fibronectin and laminin

  • long, flexible molecules that contain multiple domains

  • bind various types of collagen, other matrix proteins, polysaccharides, and extracellular signaling molecules as well as adhesion receptors

  • interactions w adhesion receptors - regulate cell-matrix adhesion and cell shape and behavior

collagen

most abundant ECM proteins; provide the primary structural scaffold in many tissues

• form fibrils and fibers to create tensile strength and resistance to stretching

• highly organized and cross-linked

  • enables mechanical stability and durability of tissues

type I → skin, bone, tendon

type II → cartilage

type III → basement membrane (network forming)

proteoglycans

act as structural “fillers” for the ECM

• hydration and cushioning

  • (-) charged CAG chains, long unbranched sugar molecules that attract water

  • form hydrated gel-like environments in tissue

  • provide resistance to compression

• act as reservoirs for growth factors and cytokines

  • regulate diffusion of signaling molecules

  • protects them from degradation and controlling availability for cell surface receptors

• contribute to tissue biomechanics

  • control viscosity, porosity, and ECM spacing

  • sensors for mechanical force in tissues (bone, cartilage) in response to physical activity

multi-adhesive glycoproteins

links cells to the ECM and organizes matrix structure

• bind to collagens, proteoglycans, integrins

modular, multi-domain proteins → enable simultaneous interactions with multiple partners and itself (networks)

• examples

  • fibronectin → connective tissue ECM

  • laminin → basement membrane

true

T/F: Cells can contribute to the assembly of the ECM by:

• secreting its components

• directing the assembly of these components into complex, interwoven structures

highly dynamic

Once assembled, the ECM is not a static structure, but rather __________

• chemical, physical, and biological properties can be altered as cells secrete enzymes and other molecules into ICM → ECM remodeling (cross-linking components, protease cleavage of ECM components)

density of cells and ECM

• dense connective tissue → contains mostly ECM containing tightly packed ECM fibers interspersed with rows of relatively sparse fibroblasts (cells that synthesized ECM)

• sparse connective tissue → squamous epithelial cells tightly packed into a quilt-like pattern with little ECM between the cells

brain ECM

sparse and highly specialized, occupying small extracellular spaces between densely packed neurons and glia

• dominated by proteoglycans and glycoproteins (tenascins)

• forms specialized structures → perineuronal nets, organize around neurons to stabilize synapses and limit plasticity

• pia membrane → contains dense basement membrane-like ECM

  • rich in laminin, collagen IV, nidogen, and heparan sulfate proteoglycans

  • provides structural support and barrier function

brain ECM vs. pia ECM

• brain ECM is soft, permissive, signaling-rich

• pia ECM is dense, structured, barrier-forming scaffold

different isoforms

Diversity of cell adhesion molecules arise from _________

• different members of a family (integrins) can be encoded by different genes

• gene products can be alternatively spliced to produce different protein products

tissue morphogenesis

disruptions in cell-matrix and cell-cell interactions interfere with tissue development

• experiment:

  • immature salivary glands → isolated from murine embryos

  • undergo branching morphogenesis in vitro for 10 hours

  • results:

    • absence of added Ab → normal branching

    • presence of Ab (anti-FN) → blocks fibronectin activity

  • conclusion → integrin-fibronectin interaction is required for branch formation

    • inhibition of integrin fibronectin receptor blocks branch formation

disruptions to adhesion and ECM functions

characteristic of various pathologies

• skeletons (mouse) → cartilage (blue) and bone (red)

  • WT → normal

  • collagen II → deficient

  • perlecan → deficient

  • fibronectin → deficient

absence of key ECM components leads to dwarfism, with many skeletal elements shortened and disfigured

fibronectin

a dimer consisting of two polypeptides linked at the C-terminus by disulfide bonds

• contains 3 functional domains → type I/II/III repeats

• combination of repeats on different isoforms allows it to bind multiple ligands

• consists of 20 different isoforms generated via alternative splicing from a single gene transcript

  • interacts with other ECM components like fibrillar collagen and heparan sulfate proteoglycans

  • binds to adhesion receptors (integrins) to influence shape and movement of cells

  • essential for cell migration and differentiating during embryogenesis

RGD motif

a tripeptide sequence in the cell-binding region of fibronectin is required for cell adhesion (Arg-Gly-Asp)

• minimal sequence required for recognition by integrins

• found in a loop that protrudes outward from fibronectin

• upon synthesis, absorption of fibronectin into ECM helps fold the protein and exposes the sequence

do cells bind RGD-containing peptides?

Various peptide sequences with RGD or scrambled RGD synthesized chemically and plated on dishes

Cultured normal rat kidney cell allowed to adhere to the dishes for 30 minutes

• results → cell adhesion increased above the background level with increasing concentration of peptides containing the RGD motif, but not for peptides with scrambled RGD

• conclusion → cell surface receptors (integrins) bind to RGD

true

T/F: All integrins evolved from 2 ancient general subgroups:

• integrins that bind proteins containing the tripeptide sequence (R) Arg - (G) Gly - (D) Asp motif (fibronectin)

• integrins that bind laminin (occurs thru non-RGD recognition motifs)

• made up of α and β subunits

integrin active/inactive state

inactive state → the BA propeller domains are bent (permitting low affinity ligand binding) and cytoplasmic tails are closely intertwined

active state → separation of heterodimers transmembrane and cytoplasmic domains, revealing binding sites for adapter proteins

integrin receptor mediated signaling

Integrins interact via adapter proteins and signaling molecules with a broad array of intracellular signaling pathways

Integrin signaling is activated by both ECM binding and cytoskeletal interaction (bidirectional integrin signaling pathway)

inside-out signaling

Intracellular signals regulate integrin activation

• adapter proteins (talin, kindlin) bind integrin cytoplasmic tails

• induce conformational change to high affinity state

  • increases binding to ECM ligands (fibronectin)

  • enhances adhesion strength and clustering

• links intracellular cell state to ECM

outside-in signaling

Binding of ECM ligands activates integrins

• induces conformational changes in cytoplasmic domains

  • recruits adapter proteins (talin, paxillin, vinculin)

• activates signaling pathways FAK, Src, and ILK

• regulates cytoskeletal organization and cell behavior

• links ECM properties to intracellular responses

integrin link ECM to cytoskeleton

Integrin signaling is coupled to their physical linkage to the cytoskeleton

• integrins physically connect ECM proteins (fibronectin) to actin filaments

• physical linkage enables transmission of mechanical forces

• adapter proteins link integrin cytoplasmic tails to cytoskeleton

• actin filaments terminate at adhesion sites at PM

• integrin clustering organizes adhesion complexes

Cells bind the ECM but fail to transmit forces to the cytoskeleton

A mutation prevents integrins from binding to intracellular adapter proteins (talin). Which of the following is the most likely outcome?

a. Cells cannot bind ECM ligands

b. Cells bind the ECM but fail to transmit forces to the cytoskeleton

c. Cells lose cadherin-mediated adhesion

d. ECM proteins cannot assemble properly

It allows many weak interactions to act together to form strong adhesion

Which of the following best explains why clustering of adhesion molecules strengthens cell adhesion?

a. It increases the affinity of individual binding interactions

b. It reduces the number of ligand-binding sites

c. It allows many weak interactions to act together to form strong adhesion

d. It prevents intracellular signaling

Reduced focal adhesion formation

A cell is placed on a surface lacking fibronectin. Which of the following is most likely to occur?

a. Increased integrin activation

b. Reduced focal adhesion formation

c. Increased actin polymerization at the membrane

d. Enhanced cadherin-mediated adhesion

Intracellular adapter proteins induce conformational changes that increase ligand affinity

Which of the following best explains how integrin activation can be regulated from inside the cell?

a. Intracellular adapter proteins induce conformational changes that increase ligand affinity

b. Binding of ECM ligands causes integrins to be internalized

c. Integrins degrade ECM components to expose binding sites

d. Integrins detach from the cytoskeleton to increase mobility

adhesion sites

integrin signaling is coupled to their physical linkage to the cytoskeleton

• adapter proteins link integrin cytoplasmic tails to the cytoskeleton

• integrins physically connect ECM proteins (fibronectin) to actin filaments

actin filaments terminate at _____________ at the plasma membrane

integrin clustering organizes multi-protein adhesion complexes, establishing a continuous structural linkage across the plasma membrane (signaling)

contractile forces

Cells actively generate ___________ via the actomyosin cytoskeleton

• myosin motor proteins contract actin filaments via (+) end directed motility

• pull inward toward cell center

• force generation is dynamic and regulated by signaling pathways

  • used to probe the mechanical properties of the ECM

  • enables cells respond to their physical environment

integrins transmit forces

integrins link the ECM to the actin cytoskeleton and transmit mechanical forces across the plasma membrane

• contractile forces generated by actomyosin are transmitted to adhesion sites

• ECM resists these forces, generating tension

  • magnitude of tension depends on ECM mechanical properties

• establishes physical pathway for bidirectional signaling between cell and its environment

ECM stiffness

soft ECM → deforms easily, leading to lower tension across adhesions

• smaller, more dynamic adhesions

stiff ECM → resists deformation, leading to higher tension across adhesion

• larger, more stable focal adhesions

• integrins and associated proteins respond to tension → mechanosensing

• mechanical cues are converted into biochemical signals that regulate cell behavior

ECM remodeling

Mechanical tension across adhesion sites is used to produce force

• this force can stretch ECM proteins such as fibronectin, promoting ECM remodeling

  • this is how mechanical forces transmitted through integrins are converted into biochemical signals that control cell’s external environment

force-dependent adhesion remodeling

Fibronectin stretching causes it to unfold → exposes hidden binding sites (type III domain) that form β sheets with other fibronectin molecules to help promote ECM assembly (outside the cell)

• provides molecular mechanism for ________________

• Talin stretching at the c-terminus exposes protein interaction sites at adhesions (inside the cell)

vinculin-binding sites

Mechanical tension stretches talin at integrin adhesions

• Talin unfolding exposes cryptic ______________

• Vinculin → binds talin and links to additional actin filaments

  • reinforces the integrin-cytoskeleton connection

  • promotes assembly and stabilization of actin bundles

  • drives growth and maturation of focal adhesions

true

T/F: Mechanical tension is the primary driver that controls the organization of adhesion complexes (depends on ECM properties and the environment)

true

T/F: Adhesion structure reflects the mechanical environment of the cell (why the external environment is so critical for cell health and function)

Piezo1

a mechanosensitive ion channel embedded in the plasma membrane

• membrane stretch induces channel opening, allowing Ca²⁺ influx into the cell

• Ca²⁺ signaling regulates cytoskeletal dynamics, gene expression, and cell behavior

proteolytic cleavage

_____________ of ECM liberates growth factors and signaling molecules that were previously sequestered within the ECM

metalloproteases (MMPs)

remodel and degrade the ECM; exist as both membrane tethered and secreted enzymes

• 3 classes

  • collagenases

  • gelatinases

  • elastases

collagenases

MMP-1, MMP-8, MMP-13

• cleave fibrillar collagens (types II, II, III)

• initiate breakdown of highly structured collagen fibers during tissues remodeling, wound healing, and development

gelatinases

MMP-2, MMP-9

• degrade gelatin, Type IV collagen, and basement membrane components

• critical for cell migration, angiogenesis, and invasion across basement membranes

elastases

MMP-12, MMP-7

• degrade elastin and elastic fibers

• regulate tissue elasticity (smooth muscle) and remodeling, esp in lung, vasculature, and inflammatory responses

ADAMs

membrane-tethered metalloproteases localized to the plasma membrane

• mediates ectodomain shedding by cleaving extracellular domains of transmembrane proteins

• regulate release and activation of signaling molecules (cytokines, growth factors, receptors, adhesion molecules)

  • function as key interface between ECM and ICM

ADAMTs

soluble (not membrane-bound) MMPs that function in the extracellular space to remodel the ECM

• regulate ECM turnover and mechanical properties

  • control tissue stiffness, elasticity, and cell–matrix signaling environments

• Thrombospondin motifs (TSRs) mediate ECM interactions and substrate specificity

  • they cleave key ECM components such as proteoglycans (e.g., aggrecan) and other matrix proteins,

  • key roles in development and tissue organization, including bone/cartilage and vasculature (affected in disease)

metalloproteases (MMP) regulation

MMP activity reflects a balance between activation and inhibition

• synthesized as inactive zymogens (pro-MMPs)

  • pro-domain blocks the catalytic site to prevent premature ECM degradation

• activation → by proteolytic cleavage or conformational change

• inhibition → by TIMPs, bind active MMPs to limit proteolysis and maintain ECM balance

macrophage-mediated ECM remodeling

regulates brain-CSF perfusion

• macrophages in periarterial spaces secrete matrix metalloproteases (MMPs) to remodel ECM

  • MMP-mediated degradation of collagen and laminin maintains an open periarterial pathway

• Efficient ECM remodeling supports robust brain–CSF perfusion along periarterial spaces

• aging or dysfunction → reduced macrophage activity leading to ECM accumulation and narrowing of flow pathways

  • impaired CSF perfusion decreases waste clearance, contributing to accumulation of toxic proteins and tissue dysfunction

polarization

Cells that build tissues show ___________, with adhesion molecules generating and maintaining distinct surfaces

simple columnar epithelia

• Elongated cells – including mucus-secreting cells (lining of

  the stomach and cervical tract) and absorptive cells (lining

  of the small intestine)

• Microvilli – on apical surface

simple squamous epithelia

• Thin cells – including cells lining blood vessels (endothelial

cells/endothelium) and many body cavities

stratified squamous (nonkeratinized) epithelia

• Line surfaces such as the mouth and vagina

• Resist abrasion

• Generally prevent material absorption/secretion into or

  out of lined cavity

basal lamina

• Thin fibrous network of collagen and other ECM components

• Connects epithelia to underlying connective tissue

tight junction

• Surrounds the cell below the microvilli – connects to all neighboring cells

• Regulates paracellular transport of substances between the intestinal lumen and internal body fluids (blood) via the extracellular space between cells

• Boundary between apical and basolateral regions of the plasma membrane

gap junctions

allow movement of small molecules and ions between cytosols of adjacent cells

• form pores for cells of certain size diffuse into cell

adherens junction

• Continuous junction with all neighboring cells

• Circumferential belt of actin and myosin filaments associated with the adherens junction – functions as a tension cable that can internally brace and control cell shape

• desmosomes and hemidesmosomes

desmosomes

spot cell-cell junctions

hemidesmosomes

spot cell-ECM junctions, similar to focal adhesion

• anchor epithelium to underlying ECM

• basal surface (ECM)

cadherins

mediate cell-cell adhesions in adherens junctions and desmosomes

• preferentially mediate homophilic adhesion (E-cad/E-cad)

• require Ca²⁺ for binding

  • in presence of calcium, mouse fibroblasts do NOT self adhere

  • E-cadherin transgene, calcium added → cells adhere and clump together

  • cadherin, no calcium → cells fail to adhere together

E-cadherin

mediates adhesive connections in cultured MDCK epithelial cells

• clusters mediate initial attachment of cells into sheets

• experiment results:

  • mediates initial attachment and subsequent zippering together of the epithelial cells

  • forms bicellular junctions and tricellular junctions

exop