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At focal adhesions, and probably also in the Iessprominent actin-linked cell-matrix adhesions that cells mainly make in normaltissues,activation of the small GTPaseRho plays a part in the maturation of theadhesive complex, by promoting recruitment of actin filaments and integrins tothe contact site.
Artificially mutated integrins that lack an intracellular tail fail toconnect with cytoskeletal filaments, fail to cluster, and are unable to form strongadhesions.ExtracellularMatrixAttachmentsActThroughIntegrinstoControlCellProliferationand SurvivalLike other transmembrane cell adhesion proteins, integrins do more than justcreate attachments. They also activate intracellular signaling pathways andthereby allow control of almost any aspect of the cell'sbehavior according to thenature of the surrounding matrix and the state of the cell's attachments to it.Studies in culture show that many cells will not grow or proliferate unlessthey are attached to extracellular matrix; nutrients and soluble growth factors inthe culture medium are not enough. For some cell t1pes, including epithelial,endothelial, and muscle cells, even cell survival depends on such attachments.lVhen these cells lose contact with the extracellular matrix, they undergo programmed cell death, or apoptosis.This dependence of cell growth, proliferation,and survival on attachment to a substratum is known as anchorage dependence, and it is mediated mainly by integrins and the intracellular signals theygenerate.Anchorage dependence is thought to help ensure that each type of cellsurvives and proliferates only when it is in an appropriate situation.
Mutationsthat disrupt or override this form of control, allowing cells to escape fromanchorage dependence, occur in cancer cells and play a major part in their invasive behavior.The physical spreading of a cell on the matrix also has a strong influence onintracellular events. Cells that are forced to spread over a large surface area bythe formation of multiple adhesions at widely separate sites survive better andproliferate faster than cells that are not so spread out (Figure f 9-5f ).
The stimulatory effect of cell spreading presumably helps tissues to regenerate afterinjury. If cells are lost from an epithelium, for example, the spreading of theremaining cells into the vacated spacewill help stimulate these survivors to proliferate until they fill the gap. It is uncertain how a cell sensesits extent of spreading so as to adjust its behavior accordingly,but the ability to spread depends onintegrins, and signals generated by integrins at the sites of adhesion must play apart in providing the spread cells with stimulation.Our understanding of anchorage dependence and of the effects of cellspreading has come mainly from studies of cells living on the surface of matrixcoated culture dishes. For connective-tissue cells that are normally surroundeda definedamountoff i b r o n e c t i ni ns i n g l ep a t c hC E L LD I E SBY APOPTOSIStlthe samea m o u n to ffibronectind i s t r i b u t e di ns m a l ls p o t sfallC E L LS P R E A D S ,SURVIVES,AND GROWSaJattc50 pmFigure19-51The importanceof cellspreading.In this experiment,cell growthand survivalareshownto dependon theextentof cellspreadingon a substratum,ratherthan the merefact of attachmentorthe cellthe numberof matrixmoleculescontacts.(Basedon C.S.Chenet al.,1997.WithScience276:1425-1428,permissionfrom AAAS.)1176Chapter19:Cell Junctions,Cell Ad:hesion,and the ExtracellularMatrixby matrix on all sides, this is a far cry from the natural environment.
Walkingover a plain is very different from clambering through a jungle. The ty?es of contacts that cells make with a rigid substratum are not the same as those, muchless well studied, that they make with the deformable web of fibers of the extracellular matrix, and there are substantial differences of cell behavior betweenthe two contexts. Nevertheless,it is likely that the same basic principles apply.Both in uitro and in uiuo, intracellular signals generated at cell-matrix adhesionsites, by molecular complexes organized around integrins, are crucial for cellproliferation and survival.IntegrinsRecruitIntracellularSignalingProteinsat SitesofCell-Substratum AdhesionThe mechanisms by which integrins signal into the cell interior are complex,involving several different pathways, and integrins and conventional signalingreceptors often influence one another and work together to regulate cell behavior, as we have already emphasized.
The Rasi MAP kinase pathway (see Figure15-61), for example, can be activated both by conventional signaling receptorsand by integrins, but cells often need both kinds of stimulation of this pathwayat the same time to give sufficient activation to induce cell proliferation. Integrins and conventional signaling receptors also cooperate in activating similarpathways to promote cell survival (discussedin Chapters 15 and 17).One of the best-studied modes of integrin signaling depends on a cltoplasmic protein tyrosine kinase called focal adhesion kinase (FAK). In studies ofcells cultured in the normal way on rigid substrata, focal adhesions are oftenprominent sites of tyrosine phosphorylation (Figure f g-5ZA), and FAK is one ofthe major tyrosine-phosphorylated proteins found at these sites. \A/hen integrins cluster at cell-matrix contacts, FAK is recruited by intracellular anchorproteins such as talin (binding to the integrin B subunit) or paxillin (whichbinds to one type of integrin a subunit).
The clustered FAK molecules crossphosphorylate each other on a specific tyrosine, creating a phosphotyrosinedocking site for members of the Src family of cytoplasmic tyrosine kinases. Inaddition to phosphorylating other proteins at the adhesion sites, these kinasesthen phosphorylate FAK on additional tyrosines, creating docking sites for avariety of additional intracellular signaling proteins. In this way, outside-in signaling from integrins, via FAK and Src-family kinases, is relayed into the cell (asdiscussedin Chapter l5).One way to analyze the function of FAK is to examine focal adhesions in cellsfrom mutant mice that lack the protein. FAK-deficient fibroblasts still adhere toFigure 19-52 Focaladhesionsand therole of focal adhesionkinase(FAK).(A)A fibroblastculturedon a fibronectincoatedsubstratumand stainedwithfluorescentantibodies:actinfilamentsarestainedgreenand activatedproteinsthatcontainphosphotyrosineare red,givingorangewherethe two componentsoverlap.The actin filamentsterminateatfocaladhesions,wherethe cell attachesto the substratumby meansof integrins.Proteinscontainingphosphotyrosinearealsoconcentratedat thesesites,reflectingthe localactivationof FAKandother proteinkinases.Signalsgeneratedat suchadhesionsiteshelp regulatecell(8,C)Thedivision,growth,and survival.influenceof FAKon formationof focaladhesionsis shownby a comparisonofnormaland FAK-deficientfibroblasts,stainedwith an antibodyagainstvinculinto revealthe focaladhesions.(B)Thenormalfibroblastshavefewer focaladhesionsand havespreadafter2 hoursin culture.(C)At the sametime point,theFAK-deficientfibroblastshavemore focaladhesionsand havenot spread.(A,courtesyof KeithBurridge;B,C,fromD.
llic et al.,Nature377:539-544,'1995.With permissionfrom MacmillanPublishersLtd.)n o r m a lf i b r o b l a s t s(B)1 0p mFAK-deficientfibroblasts50 pm1177IN T E G RNISA N D C E L L _ M A T RAI XDHESIONfibronectin and form focal adhesions. In fact, they form too many focal adhesions; as a result, cell spreading and migration are slowed (Figure 19-528 and C).This unexpected finding suggeststhat FAK normally helps disassemble focaladhesions and that this Ioss of adhesions is required for normal cell migration.Many cancer cells have elevated levels of FAK,which may help explain why theyare often more motile than their normal counterparts.IntegrinsCanProduceLocalizedIntracellularEffectsThrough FAK and other pathways, activated integrins, like other signaling receptors, can induce global cell responses,often including changes in gene expression. But the integrins are especially adept at stimulating localized changes inthe cytoplasm close to the cell-matrix contact.
We have already mentioned animportant example in our discussion of epithelial cell polarity: it is through integrins that the basal lamina plays its part in directing the internal apico-basalorganization of epithelial cells.Localized intracellular effects may be a common feature of signaling bytransmembrane cell adhesion proteins in general. In the developing nervoussystem,for example, the growing tip of an axon is guided mainly by its responsesto local adhesive (and repellent) cues in the environment that are recognized bytransmembrane cell adhesion proteins, as discussedin Chapter 22.The primaryeffectsof the adhesion proteins are thought to result from the activation of intracellular signaling pathways that act locally in the axon tip, rather than throughcell-cell adhesion itself or signals conveyed to the cell body. Through localizedactivation of the Rho family of small GTPases,for example (as discussed inChapters l5 and 16),the transmembrane adhesion proteins can control motilityand guide forward movement.
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