Deconstructing membrane traffic

Millennium issue

Deconstructing membrane traffic Regis B. Kelly Intracellular movement of proteins and lipids between organelles is usually described in terms of cargo, carriers, traffic and docking, familiar terms that imply parallels to human activities. Over the past century, scientists have been criticized for constructing hypotheses that reflect too much of their current political and cultural values. In this article, concepts of membrane traffic are re-examined to see whether they reflect the cellÕs view of the world or our own. hen humans plan production, they move the product down assembly lines. Cells do not care for such geographical planning. In the secretory pathway, proteins go from the peripheral endoplasmic reticulum (ER) to the juxtanuclear Golgi complex and then return to the periphery to reach the plasma membrane. In a related geographical perversity, cells constitutively internalize plasma membrane components to recycling endosomes that first move to the juxtanuclear Golgi region and then return, apparently unaltered, to the plasma membrane. Such seemingly unnecessary membrane movements resemble inefficient military planning more than an efficient industrial complex. Since the military does not know when the next crisis will occur, it keeps a large and usually useless ‘rapid deployment force’ on hand. By analogy, trafficking of membranes to the Golgi region, the origin of cytoplasmic microtubules, might allow their rapid redeployment along microtubular tracks to where they are needed. Whatever the explanation, we do not look to the cell, or the military, for efficiency. Membrane traffic is also profligate in its rate. Traffic from the cell surface to juxtanuclear endosomes and back occurs at about 1% per minute for cell-surface lipids and proteins1. Thus, a typical plasma membrane protein with a half-life of 10–20 h will

W

Donor

make at least ten round trips to the Golgi region per lifetime. This means that only 10% or less of the traffic from the Golgi region to the cell surface is newly synthesized proteins, with the remainder being endosomal recycling traffic. Presumably, it is easy for the cell to sustain large amounts of membrane traffic and so it does so with abandon, not with efficiency. High volumes of membrane traffic can carry a lot of cargo. For example, the ER lumen in exocrine pancreas cells contains large amounts of the secreted enzyme amylase. Vesicle traffic carries the amylase without concentration to the ER-to-Golgi intermediate compartment (ERGIC). A considerable retrograde flow of empty vesicles back to the ER from the intermediate compartment results in concentration of the amylase2. Associating a forward flow of cargo protein with a reverse flux of empty vesicles appears to be a common motif in membrane traffic (Fig. 1). It occurs during flux through the Golgi stacks3, in maturation of immature secretory granules4 and in the flow of cargo such as low-density lipoproteins (LDLs) through the endosome to the lysosome5. Before we applaud the cell’s wonderful commitment to recycling empty bottles, we need to recall that the LDL pathway is constitutive, not induced by cargo. Cells do not care whether bottles go out empty or full – as long as they return

Regis B. Kelly rkelly@biochem. ucsf.edu

Intermediate

Acceptor TCB•TIBS•TIG

FIGURE 1. A recurrent theme in membrane traffic is the retrograde flow of empty vesicles. Carrier vesicles carrying soluble cargo proteins leave a donor compartment. They fuse to form a larger organelle, which then buds off empty vesicles that return to the donor. The remaining organelle, enriched in cargo proteins, travels on to fuse with an acceptor, which can go on to be Golgi cisternae, plasma membrane or lysosomes.

© 1999 Elsevier Science Ltd. All rights reserved. For article fee, see p. IV.

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Dept of Biochemistry and Biophysics, Hormone Research Institute, University of California, San Francisco, CA 94143-0448, USA.

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Millennium issue empty. Perhaps it is easier for a cell to make a vesicle that is completely empty of protein than it is to concentrate soluble protein into their lumen.

Fusion of carrier vesicles The canonical traffic pathway (Fig. 1) requires that vesicles fuse with each other or with a target or acceptor organelle. Fusion requires that the lipid bilayer of the carrier vesicle becomes continuous with that of the acceptor membrane, thus increasing the surface area of the acceptor membrane. Simple fusion events can sometimes be measured as a jump in the electrical capacitance of the cell surface6. Occasionally, jumps in capacitance are followed rapidly by decreases of equal magnitude, implying that membrane fusion is being reversed by membrane fission. This ‘flickering’ of capacitance implies that the two membrane states, before and after fusion, must have approximately equal energies. A second unexpected observation is that the first step in fusion is the formation of a ‘fission pore’, a small channel linking the inside of the vesicles to the outside of the cell. Contents of the vesicle dribbling out through this tiny hole could be detected by the sensitive technique of amperometry. Although the fusion/fission pore is well described physiologically, we know little about the molecular mechanisms of pore formation. We do know, however, how two membranes can be packed into such close proximity that lipid rearrangements become possible. Membrane proteins, usually called SNARE proteins7, attached to both carrier vesicle and acceptor membranes, can oligomerize spontaneously to form a helical bundle that drives the two membranes together8. Energy is put into the system when the SNARE complex is disassembled by a hexameric ATPase9. For most cell biologists, the discovery of the SNARE proteins essentially solved the central problem in membrane fusion. Different acceptor membranes have different SNARES, which provides at least a partial explanation of the specificity of carrier-vesicle fusion. We still have a great deal to learn about the further regulation of fusion in space and time. Fortunately, there is no lack of accessory factors. Golgi carrier-vesicle fusion is constrained by tethering proteins10. Carrier vesicles seem to be guided to their appropriate plasma membrane fusion site by the large proteinaceous ‘exocyst’ complex11. All self-respecting cell-biological events seem to have their attendant, Ras-like, GTPase, and fusion is no exception. The fusion-specific Rab GTPases are clearly important and might also control the location of fusion events. In yeast, the effector protein for the Rab4p complex is the exocyst12. A search for proteins that control the specificity of vesicle fusion might prove to be frustrating as we do not know how frequently a carrier vesicle fuses inappropriately. A cell might not need to organize its delivery system with the precision of Federal Express, but it might allow itself to make an occasional mis-delivery. There is no evidence that organelles need to control their composition with compulsive precision.

Carrier-vesicle formation In addition to fusion, membrane traffic requires the budding of carrier vesicles from donor membranes (Fig. 1). The most popular paradigm for this process remains the recognition of a cargo membrane protein by an adaptor protein complex that links cargo to clathrin triskelions13. The clathrins then polymerize into a sphere, imparting curvature to the carrier-vesicle membrane. Much of this model is based on classical electron-microscopy studies of the recycling of synaptic vesicle membranes by Heuser and Reese14. It is always tempting to equate the first discovery in a field to an archetype, interpreting subsequent discoveries as variations. However, recycling of synaptic vesicles is not typical of vesicle formation as these small clathrin-coated vesicles bud from

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the plasma membrane, lose their coats, and then fuse back again with the plasma membrane15. Synaptic vesicles, therefore, are not true carrier vesicles, meaning that small, homogeneously sized clathrin vesicles might not be the norm. Budding of small vesicles cannot explain all membrane traffic because the cargo is sometimes too big for a small vesicle. The formation of dense-core secretory granules in the Golgi stacks of endocrine and exocrine cells is preceded by condensation of the protein cargo into aggregates that are too large to fit into vesicles coated with any of the conventional coats16. We now know that the immature secretory granules are large, irregular membrane sacs that contain condensed cargo. Newly synthesized collagen fibrils also constitute a cargo that is too large to fit into small carrier vesicles. The passage of large collagen fibrils through the Golgi stacks has been used to argue that cisternae move through the Golgi stacks intact and that the characteristic membrane composition of different regions of the Golgi stack is maintained by a retrograde flow of machinery in small vesicles17. By the same argument, budding from the trans-Golgi network (TGN) complex must put the collagen into large secretory vesicles and not into the small 50–80 nm vesicles generated by conventional coating mechanisms. Compared with the formation of small vesicles, the pinchingoff of large vesicles has, as yet, little popular following among cell biologists. After they are pinched off, the large vesicles are cleaned up by vesicular retrieval of the donor membrane’s machinery, using the same basic mechanism encountered earlier (Fig. 1). At this moment it is not clear whether large and small carrier vesicles are made by separate mechanisms. The formation of small and large vesicles is not the only way donor membranes can bud. Early endosomes have characteristic tubules extending from them that are enriched in recycling proteins18. Carrier vesicles could arise by the pinching off of such tubules. Another bizarre example separates membranes in endosomes that are destined for lysosomal degradation from those that will recycle to the plasma membrane. Membranes that contain the condemned proteins involute to give rise to multivesicular bodies19,20. The next decade should reveal how tubulation and involution occur and why cells use these processes.

Coating mechanisms We know much more about the formation of small vesicles than we do about the pinching off of large vesicles, tubulation and involution. Vesiculation seems quite simple for intracellular donor membranes21. A coat forms that consists of COPI, COPII or one of the clathrin adaptors (AP1, AP2, AP3), plus clathrin. A small, Ras-like GTPase is involved in vesiculation – either an ARF protein or Sar1p. The energy required to impart curvature on the membranes seems to come from coat polymerization because adding pure coats to pure liposomes can result in the formation of coated vesicles22–24. Perhaps an uncoating ATPase restores the coat to an unpolymerized, high-energy state. Vesicle formation usually requires negatively charged lipids and is sometimes best achieved with phosphorylated phosphatidylinositols. The proclivity with which coating molecules vesiculate liposomes could mean that membranes need mechanisms to prevent spontaneous vesiculation. Alternatively, coat formation in vitro could be triggered by high concentrations of phosphorylated phosphatidylinositols. Cargo proteins could also stimulate the coating mechanism directly25. One puzzle is why the formation of small vesicles from intracellular membranes is relatively simple but is much more complex when the donors are plasma membranes. For example, vesicles bud from endosomes when only the small GTPase ARF1 and the adaptor AP-3 are added26, whereas vesicle budding from plasma membranes involves amphiphysin, syndapin, endophilin, dynamin, synaptojanin, AP180, intersectin/DAP160 and perhaps even actin,

Millennium issue

Regis B. Kelly ¥ Membrane trafficking

YXXµ2 Dynamin

µ2

Amphiphysins I and II α

Synaptojanin

AP-2 β Clathrin

Eps15 Syndapin N-WASP Actin

Endophilin Synapsin

AP180 Intersectin Epsin

SH3

PRD

NPF

EH

DPF/W

Tryp840 domain

PtdIns(4,5)P2 recognition TCB•TIBS•TIG

FIGURE 2. Proteins involved in endocytosis from the plasma membrane. A membrane protein is recognized via a tyrosine-based motif (YXXf) by the m subunit of the AP-2 adaptor complex. AP-2 binds to clathrin via its b subunit. The AP-2 a subunit can bind to four different proteins with DPF/W (aspartate/proline/phenylalanine or tryptophan) domains. These four proteins can interact with other proteins using interactions of NPF (arginine/proline/phenylalanine) domains with Eps15-homology (EH) domains or via SH3 (Src-homology region 3) domains binding to proline-rich domains (PRDs). Two roles for these accessory proteins are to link endocytosis to the actin cytoskeleton and to the composition of the phospholipid bilayer. (Adapted from Fig. 3 in Ref. 13.)

in addition to AP-2 and clathrin13 (Fig. 2). If this complexity is real, then an explanation is required. One possibility is that it is difficult to impart curvature to the flat plasma-membrane vesicle. When the donor membrane is already highly curved, as it is for the tubules and cisternae of the ER, Golgi and endosomal membranes, there might be less need for coating factors. An alternative explanation invokes the presence of cortical actin at the plasma membrane, suggesting that this must be removed or modified to allow vesicle formation. Early in the new millennium, we can expect a rapid elucidation of why all these proteins are involved in endocytosis from plasma membranes and whether intracellular membranes do not need them or whether current assays have just missed them.

Sorting of proteins Recognition is so apparently effortless to humans that we tend to under-appreciate the complexity of the processes involved. As a result, we sometimes assume incorrectly that cells can recognize proteins with great ease. The difficulty cells have in recognizing proteins is illustrated by protein degradation. Cells degrade proteins with an exponential time course. Implicit in this simple observation is that old and newly minted cell proteins are degraded with equal probability. Presumably, cells degrade proteins they have just made because they have no easy means of distinguishing old from young proteins. The difficulty that cells have in recognizing proteins and sorting them is also illustrated by the selective packaging of peptide hormones into secretory granules. Rather than sort peptide hormones individually, for example, endocrine cells take advantage of the tendency of proteins to self-aggregate4. Where sorting is well established, for example to the ER or the nucleus, sorting signals could be much less precise than the now

familiar e-mail addresses. Sorting might only concentrate a protein by a factor of ten or so more than when it is randomly distributed. For example, during endocytosis from the plasma membrane, receptors for both LDL and transferrin are concentrated in coated pits, but only by approximately tenfold – an efficiency of delivery that would paralyse the Internet. Sorting of membrane proteins might be relatively inefficient because it involves the interaction of very small linear sorting motifs in cargo proteins with subunits of the coating machinery. For example, the m subunits of the adaptor complex AP-2 (Ref. 27) recognize short tyrosine-containing motifs in extended conformations, rather than in tight turns as originally predicted, and with low affinity. Targeting with higher precision could arise by combining simple sorting domains. Targeting from endosomes to synaptic vesicles, for example, requires signals both for targeting to secretory granules and for targeting to late endosomes28. The preference of molecules to aggregate with themselves, rather than with strangers, is also encountered in membrane-lipid sorting. The phospholipid sphingomyelin is concentrated during internalization into endosomes29. On leaving the endosome, lipids that have more-fluid fatty-acid tails go to recycling endosomes, whereas those with solid, that is highly interacting, tails go to late endosomes30. In the biosynthetic pathway, glycolipids tend to aggregate into rafts that, in epithelial cells, are targeted preferentially to apical membranes31. In the latter case, some proteins are known to associate with these lipid rafts and go with them to the plasma membrane. Soluble proteins are also sorted. As membrane trafficking is carried out by organelles with internal volumes, some soluble proteins such as amylase2 are carried by bulk flow. However, the pancreatic cell concentrates chymotrypsin in the ERGIC about

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Millennium issue

Regis B. Kelly ¥ Membrane trafficking

(a)

(b)

v-SNARE µ2

AP-2

α

β

TCB•TIBS•TIG

FIGURE 3. Two possible steps in coat recruitment. In the first (a), the coating complex recognizes some element of the fusion machinery. In this example, based on the finding in Refs 34 and 35, the coat binds to a SNARE protein that will become a cargo protein in the newly formed carrier vesicle. The recognition domain on the SNAREs also appears to be in a functional domain required for the formation of SNARE complexes. In the second step (b), coat polymerizes, increasing the concentration of low-affinity cargo-binding sites, thus recruiting membrane cargo.

ten times more than it concentrates amylase, implying active sorting, presumably by a receptor. Similarly, yeast have a sorting receptor in the ER for an amino acid permease32. An active sorting mechanism is more efficient than bulk flow, but efficiency of transport might not be very important to a cell that can just make more protein. If we drop our puritanical predilection for efficiency, we might uncover some exciting alternative explanations. Vesicles carrying and sorting the membrane and soluble cargo proteins must also carry fusion machinery if they are not to accumulate as inert vesicles in the cell. Sorting of fusion machinery into vesicles seems to use sorting signals different from those used for cargo33. Furthermore, removing the sorting signals with a bacterial neurotoxin inhibits coat recruitment and the formation of vesicles, implying that the fusion machinery in some way nucleates the coating and budding event34. Parallel observations of ER sorting in yeast35,36 also showed a role for SNAREs in nucleating vesicle formation and that the sequences involved in sorting are unlike the canonical cargo-sorting motif. This leads to a model of sorting that distinguishes a nucleating step, using the fusion machinery, from a subsequent recruitment of cargo using different domains of the coating machinery (Fig. 3).

References 1 Steinman, R.M. et al. (1983) Endocytosis and the recycling of plasma membrane. J. Cell Biol. 96, 1Ñ27 2 Martinez-Menarguez, J.A. et al. (1999) Vesicular tubular clusters between the ER and Golgi mediate concentration of soluble secretory proteins by exclusion from COPI-coated vesicles. Cell 98, 81Ñ90 3 Allan, B.B. and Balch, W.E. (1999) Protein sorting by directed maturation of Golgi compartments. Science 285, 63Ñ66 4 Tooze, S.A. (1998) Biogenesis of secretory granules in the trans-Golgi network of neuroendocrine and endocrine cells. Biochim. Biophys. Acta 1404, 231Ñ244 5 Mellman, I. (1996) Endocytosis and molecular sorting. Annu. Rev. Cell Dev. Biol. 12, 575Ñ625 6 Albillos, A. et al. (1997) The exocytotic event in chromaffin cells revealed by patch amperometry. Nature 389, 509Ñ512 7 Sollner, T. et al. (1993) A protein assemblyÑdisassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation and fusion. Cell 75, 409Ñ418 8 Fiebig, K.M. et al. (1999) Folding

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And so? The behaviour of cells would be considered unacceptable in polite, human society. They destroy proteins, organelles and even themselves with ruthless abandon, with no attention to age or potential usefulness. To remain flexible they waste resources on futile pathways irrespective of immediate need and care little about recycling. Their synthetic factories are inefficient, and their distribution systems error prone. Although we know that cells are not like us, we easily forget and lapse into making models as if we were engineers rather than biologists. But to be politically correct, I must now deconstruct myself. Is a unifying model of an exuberant, wasteful cell, productive but sloppy and poor at recognizing proteins, a valid one or a reflection of my own liberal youth, physics training, too much California and the accumulation of years? Are all models undecidable anyway? Thanks a lot, Jacques Derrida*. *The work of the French philosopher Jacques Derrida inspired the school of deconstruction. By subverting narrative assumptions about text, he deconstructed the works of previous scholars to show that language is constantly shifting37.

intermediates of SNARE complex assembly. Nat. Struct. Biol. 6, 117Ñ123 Nichols, B.J. et al. (1997) Homotypic vacuolar fusion mediated by t- and v-SNAREs. Nature 387, 199Ñ202 Sonnichsen, B. et al. (1998) A role for giantin in docking COPI vesicles to Golgi membranes. J. Cell Biol. 140, 1013Ñ1021 TerBush, D.R. et al. (1996) The exocyst is a multiprotein complex required for exocytosis in S. cerevisiae. EMBO J. 15, 6483Ñ6494 Guo, W. et al. (1999) The exocyst is an effector for Sec4p, targeting secretory vesicles to sites of exocytosis. EMBO J. 18, 1071Ñ1080 Marsh, M. and McMahon, H.T. (1999) The structural era of endocytosis. Science 285, 215Ñ220 Heuser, J.E. and Reese, T.S. (1973) Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction. J. Cell Biol. 57, 315Ñ344 Murthy, V.N. and Stevens, C.F. (1998) Synaptic vesicles retain their identity through the endocytic cycle. Nature 392, 497Ñ501 Farquhar, M.G. (1977) Secretion and

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crinophagy in prolactin cells. Adv. Exp. Med. Biol. 80, 37Ñ94 Bonfanti, L. et al. (1998) Procollagen traverses the Golgi stack without leaving the lumen of cisternae: evidence for cisternal maturation. Cell 95, 993Ñ1003 Geuze, H.J. et al. (1987) Membranes of sorting organelles display lateral heterogeneity in receptor distribution. J. Cell Biol. 104, 1715Ñ1723 Odorizzi, G. et al. (1998) Fab1p PtdIns(3)P 5-kinase function essential for protein sorting in the multivesicular body. Cell 95, 847Ñ858 Fernandez-Borja, M. et al. (1999) Multivesicular body morphogenesis requires phosphatidyl-inositol 3-kinase activity. Curr. Biol. 14, 55Ñ58 Schekman, R. and Orci, L. (1996) Coat proteins and vesicle budding. Science 271, 1526Ñ1533 Matsuoka, K. et al. (1998) COPII-coated vesicle formation reconstituted with purified coat proteins and chemically defined liposomes. Cell 93, 263Ñ275 Spang, A. et al. (1998) Coatomer, Arf1p, and nucleotide are required to bud coat protein complex I-coated vesicles from large synthetic liposomes. Proc. Natl. Acad. Sci.

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U. S. A. 95, 11199Ñ11204 24 Takei, K. et al. (1998) Generation of coated intermediates of clathrin-mediated endocytosis on protein-free liposomes. Cell 94, 131Ñ141 25 Matsuoka, K. et al. (1998) Coat assembly directs v-SNARE concentration into synthetic COPII vesicles. Mol. Cell 2, 703Ñ708 26 Faundez, F. et al. (1998) A function for the AP3 coat complex in synaptic vesicle formation from endosomes. Cell 93, 423Ñ432 27 Owen, D.J. and Evans, P.R. (1998) A structural explanation for the recognition of tyrosine-based endocytotic signals. Science 282, 1327Ñ1332 28 Blagoveschenskaya, A.D. et al. (1999)

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A complex web of signal-dependent trafficking underlies the triorganellar distribution of P-selectin in neuroendocrine PC12 cells. J. Cell Biol. 145, 1419Ñ1433 Chen, C.S. et al. (1997) Changes in the spectral properties of a plasma membrane lipid analog during the first seconds of endocytosis in living cells. Biophys. J. 72, 37Ñ50 Mukherjee, S. et al. (1999) Endocytic sorting of lipid analogues differing solely in the chemistry of their hydrophobic tails. J. Cell Biol. 144, 1271Ñ1284 Simons, K. and Ikonen, E. (1997) Functional rafts in cell membranes. Nature 387, 569Ñ572 Kuehn, M.J. et al. (1998) COPIIÐcargo interactions direct protein sorting into

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ER-derived transport vesicles. Nature 391, 187Ñ190 Grote, E. et al. (1995) A targeting signal in VAMP regulating transport to synaptic vesicles. Cell 81, 581Ñ589 Salem, N. et al. (1998) A v-SNARE participates in synaptic vesicle formation mediated by the AP3 adaptor complex. Nat. Neurosci. 1, 551Ñ556 Springer, S. and Schekman, R. (1998) Nucleation of COPII vesicular coat complex by endoplasmic reticulum to Golgi vesicle SNAREs. Science 281, 698Ñ700 Springer, S. et al. (1999) A primer on vesicle budding. Cell 97, 145Ñ148 Collins, J. and Mayblin, B. (1997) Introducing Derrida, Totem Books

Cell adhesion: old and new questions Richard O. Hynes Metazoans clearly need cell adhesion to hold themselves together, but adhesion does much more than that. Adhesion receptors make transmembrane connections, linking extracellular matrix and adjacent cells to the intracellular cytoskeleton, and they also serve as signal transducers. In this article, I briefly summarize our present understanding of the molecular basis and biological consequences of cell adhesion and discuss how our current knowledge sheds light on questions of specificity of cell adhesion. I offer some thoughts and speculations about the evolution of cell-adhesion molecules and processes, consider their inter-relationships with other forms of cellÑcell communication and discuss unresolved questions ripe for investigation as we enter the postgenomic era. ven a cursory consideration of metazoan anatomy and development forces the realization that the associations of cells in epithelia, their attachment to basement membranes and the migrations of cells and projections of neurons all require selective adhesion of cells to one another and to extracellular matrices (ECMs). Recognition of this requirement led to a spirited debate between proponents of a large number of highly selective adhesion receptors, and advocates of models in which quantitative differences in adhesive strength, without necessarily a large spectrum of individual specificities, were invoked to explain differential cell adhesion. Similarly, the phenomenon of induction, in which one tissue influences the developmental fate of adjacent tissues, clearly relies on cell–cell interactions, and experimental embryologists attempted to define whether induction relies on diffusible signals or on cell–cell or cell–matrix contacts. Neither the issue of specificity of cell adhesion nor the question of the mechanistic bases of induction could be resolved without molecular biology. Now, with the benefit of a couple of decades of molecular analysis, we can see that there is some truth to all of the earlier models. The specificity of cell adhesion comes from combinatorial expression and interactions among a large, but not unlimited, number of adhesion receptors, and induction relies on diffusible ligands binding to receptors, on cell–cell contacts and on cell–matrix adhesion. The distinctions among these three mechanisms are not actually that great – adhesion receptors signal much like receptors for growth factors and should be considered in parallel with them.

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Before considering the biological functions of cell adhesion, we need to define the players. Figure I in Box 1 diagrams the structures of representative cell–cell adhesion receptors. Fortunately, many adhesion receptors fall into a relatively small number of families, the major ones being shown in Fig. I. Other families of adhesion receptors, such as syndecans and other membranebound proteoglycans, the disintegrin family and others are less well understood at this time. In addition to their roles in binding cells to their neighbours (Fig. I) or to ECM (Fig. 1), engagement of cell-adhesion receptors has major effects on many aspects of cell behaviour – cell shape and polarization, cytoskeletal organization, cell motility, proliferation, survival and differentiation. How do they accomplish all these functions?

Cytoskeletal connections Crucial to the effects of adhesion receptors on intracellular organization and cell motility is the fact that their cytoplasmic domains connect to the cytoskeleton. Figure 1a shows how integrins bind to linker proteins, which in turn make direct and indirect connections to F-actin filaments, thus establishing a mechanical link between the fibrils of the ECM and the filaments of the cytoskeleton9,10. The connection of classic cadherins to the actin cytoskeleton that occurs at cell–cell junctions is analogous, although the molecules involved are different (Fig. 2a)1,11,12. Although integrins appear to be the major receptors for ECM, they are not the only ones. One well-studied example, of considerable interest because of its involvement in

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Richard O. Hynes [email protected] Howard Hughes Medical Institute and Center for Cancer Research, Dept of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

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