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provide the means by which expression ofregulatory genes with crucial involvement inconferring rhombomere identity is broughtinto tight register with the segmental pattern.

Classical studies of cell aggregates in vitrohave shown that ‘sorting-out’ behaviour canbe mediated by differential adhesion, in par-ticular by the expression of different types ofadhesion molecule or of different amounts ofthe same adhesion molecule13. Despite exten-sive searches, no adhesion molecules withdifferential expression between adjacentrhombomeres have been found, whilemajor-league adhesion molecules such asNCAM and N-cadherin are expressed ratheruniformly throughout the presegmentalhindbrain. So it has been thought that rhom-bomeres might form through the localizedloss of adhesion in an otherwise uniformadhesive system, rather than through differ-ential adhesion between adjacent cell assem-blies. This would be consistent both with thefinding that Eph activation can cause loss ofadhesion14 and with the appearance of largeintercellular spaces early in boundaryformation9. The demonstration that Eph–ephrin interaction can prevent cellintermixing5 is also consistent with this idea.To drive cell sorting in the hindbrain, whichprobably occurs to a limited extent duringnormal boundary sharpening (but, as Xu etal. show, can also occur over a significantlylonger range), it would have to be supposedthat some differential affinity is created

whereby receptor-expressing cells minimizetheir contact with ligand-expressing cells,and vice versa.

These two papers4,5 have significantlyadvanced our understanding of the cellularmechanism of hindbrain segmentation, butignorance persists with regard to the geneticmechanism of regulation of the segmentalpattern. Despite the conserved role of Hoxgenes in specifying segmental identity inboth flies and vertebrates, the segmentationgenes upstream of Hox in Drosophila appearnot to be used during segmentation of thevertebrate hindbrain, suggesting convergentevolution of the mechanisms that controlsegmentation and that couple Hox geneexpression to the segmental array. In contrastto Drosophila, where the genetic regulation ofsegmentation is well understood, there arefew candidate regulators of a segmental pre-pattern in the hindbrain. The best knownexample is the zinc-finger gene Krox-20,which is expressed in two stripes in the neu-ral plate that become r3 and r5 and whoseproduct directly regulates expression ofEphA4. In Krox20-null mutant animals, r3and r5 are deleted and a partially fused r2–r4–r6 territory develops15 — a phenotypeconsistent with Krox-20 being responsiblefor generating single-compartment periodic-ity from cues established by upstream genes.Such cues may include the bZIP transcrip-tion factor encoded by the Kreisler gene,which is expressed in r5 and r6, territories

that are either lost or incorrectly specified inkreisler-null mutant mice and zebrafish16. Amore complete understanding of hindbrainsegmentation will await the discovery of fur-ther transcription factors with a two-seg-ment-repeat pattern, particularly ones thatcould regulate ephrin expression in r2, r4and r6. hAndrew Lumsden is in the Department of Developmental Neurobiology, UMDS, Guy’s Hospital, London SE1 9RT, UK.e-mail: andrew.lumsden@kcl.ac.uk

1. Lumsden, A. & Keynes, R. Nature 337, 424–428 (1989).

2. Champagnat, J. & Fortin, G. Trends Neurosci. 20, 119–124

(1997).

3. Fraser, S. E., Keynes, R. & Lumsden, A. Nature 344, 431–435

(1990).

4. Xu, Q., Mellitzer, G., Robinson, V. & Wilkinson, D. Nature 399,

267–271 (1999).

5. Mellitzer, G., Xu, Q. & Wilkinson, D. Nature 400, 77–81 (1999).

6. Lumsden, A. & Krumlauf, R. Science 274, 1109–1115 (1996).

7. Guthrie, S. & Lumsden, A. Development 112, 221–229 (1991).

8. Wizenmann, A. & Lumsden, A. Mol. Cell. Neurosci. 9, 448–459

(1997).

9. Heyman, I., Kent, A. & Lumsden, A. Dev. Dyn. 198, 241–253

(1993).

10. Xu, Q. & Wilkinson, D. J. Mol. Med. 75, 576–586 (1997).

11. O’Leary, D. D. M. & Wilkinson, D. Curr. Opin. Neurobiol. 9, 65–

73 (1999).

12. Martinez, S., Geijo, E., Sanchez-Vives, M., Puelles, L. & Gallego,

R. Development 116, 1069–1076 (1992).

13. Steinberg, M. & Takeichi, M. Proc. Natl Acad. Sci. USA 91, 206–

209 (1994).

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319 (1996).

15. Schneider-Maunoury, S. et al. Cell 75, 1199–1214 (1993).

16. Moens, C. B., Yan, Y.-L., Appel, B., Force, A. & Kimmel, C. B.

Development 122, 3981–3990 (1996).

Connections count in cell migration

Kristiina Vuori and Erkki Ruoslahti

New results show that cell spreading and migration are regulated by a functional interaction between the tyrosine kinase Src and members of the integrin family of cell-adhesion receptors.

ells change their shape and migrate bycontinuously moulding and remould-ing their actin cytoskeleton. To be able

to propel themselves forward, cells must beable to transmit force from the actincytoskeleton to a ligand anchored to anextracellular-matrix (ECM) substrate. Thisforce transmission typically occurs throughcell-surface-located ligand receptors knownas integrins, so named to emphasize theirrole in connecting ECM substrates with thecell’s interior. Transmission of the forcethrough integrins is highly regulated. Duringcell movement, it is accomplished in a man-

ner that allows cell–substrate linkages at thefront of the cell to provide stable traction,while loosening the linkages at the rear toallow the cell to move along. On page 200 ofthis issue1, Felsenfeld et al. provide newinsight into how force transmission throughintegrins is regulated by showing that thetyrosine kinase Src controls integrin interac-tions with the force-generating cytoskeleton.

Felsenfeld et al. have shown previouslythat binding of integrins to a ligand in theECM induces the coupling of integrins to theactin cytoskeleton2. They showed that beadscoated with integrin ligands and placed on

C

top of a cell migrated toward the trailing endof the cell. The same force that pulls the beadsbackwards can presumably pull cellular com-ponents forward when the integrins arelinked to an immobile extracellular substrate,thus leading to the force transmissionrequired for migration.

The strength of the integrin–cytoskeletalbonds is modulated by cells in response tothe rigidity of the substrate — a type of ‘out-side-in’ signalling3,4. When on a rigid sub-strate, cells make stronger connectionsbetween the substrate and the cytoskeleton,and pull harder on these adhesion sites, thanwhen they are on a pliable substrate. Theforce generated by the cell is proportional tothe rigidity of the substrate. Thus, cells canmigrate from a looser to a more rigid matrix,as integrin–cytoskeletal connections will bestronger where the cell touches the firmersubstrate. This process, mechanotaxis, mayprovide guidance to cell migration in vivo.

Direct measurement of integrin-medi-ated traction forces in migrating cells showsthat these forces oscillate in magnitude5. Thismeans that the integrin–cytoskeletal interac-tions may be regulated by factors in additionto the nature of the substrate, providing apossible mechanism for controlling cell loco-

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motion by means of intracellular stimuli —that is, by a type of ‘inside-out’ signalling thatregulates intracellular interactions ofintegrins.

Two pieces of information led Felsenfeldet al. to investigate the possibility that thetyrosine kinase Src could regulate force gen-eration through integrins. First, the tyrosinephosphatase inhibitor phenyl arsine oxideblocks the strengthening of integrin–cytoskeletal linkages on rigid substrates,indicating that perhaps this process is regu-lated by tyrosine phosphorylation3,4. Also,fibroblasts derived from mice lacking Srcshow delayed spreading and cytoskeletalorganization on a fibronectin substrate(fibronectin is an integrin ligand), suggestingthat Src may regulate integrin function6.

Felsenfeld et al. also find that spreading offibroblasts from Src-deficient mice is com-promised, and that this effect is seen on twodifferent integrin ligands, fibronectin andvitronectin1. They find that this is becauseSrc selectively regulates cell spreadingthrough the integrin receptor for vitronectin,even on fibronectin. They show first that cellspreading on fibronectin depends on the vit-ronectin receptor. Unfortunately, they donot fully identify the receptors that mediatecell attachment, but integrin α5β1 is likely tobe the main fibronectin receptor, andintegrin αvβ3 and/or αvβ5 the vitronectinreceptor, in these fibroblasts.

Felsenfeld et al. then more directly studythe putative function of Src in regulatingintegrin function by placing ligand-coatedbeads on the upper surface of spread fibrob-lasts and holding them there with a laser trap.Beads coated with ligands for the fibronectinor vitronectin receptors bind to the cellsequally well, regardless of Src expression, sothe binding of integrins to extracellular lig-ands is not affected by Src activity. Instead,Src affects the interactions of integrins withthe cytoskeleton. Felsenfeld et al. have shownpreviously that ligand-coated beads that bindto integrins escape from a laser trap as theintegrins are pulled away from the leadingedge of the cell by cytoskeleton moving in aretrograde direction4. Moving a previouslytrapped bead back to the original positionrequires a greater force than does moving anuntouched bead, indicating that the cell hasreinforced its integrin–cytoskeletal linkagesin response to the force imposed on it by thelaser trap (Fig. 1).

Experiments with beads binding to thefibronectin receptor show equal reinforce-ment of fibronectin-receptor–cytoskeletalbonds in wild-type and Src-deficient cells1. Incontrast, the vitronectin receptor shows littleor no reinforcement in Src-expressingfibroblasts, but is strongly reinforced in Src-deficient fibroblasts. There are two possibleinterpretations of these results: either Src isnormally a selective inhibitor of sustained

force generation through the vitronectinreceptor, or Src may promote the turnover oflinks between the integrin and the cytoskele-ton. Because sustained reinforcement ofintegrin–cytoskeletal linkages is inverselycorrelated to cell spreading and motility4,these findings provide a mechanistic expla-nation for the defects in spreading cytoskele-tal dynamics observed in Src-deficient cells6.

Felsenfeld et al.’s latest results may help usto understand the osteopetrotic phenotypeof Src-deficient mice7. Others have estab-lished a crucial role for the vitronectin recep-tor in osteoclast function (see ref. 8 for areview). Thus, it is possible that reinforcedvitronectin-receptor–cytoskeletal connec-tions in Src-deficient mice prevent osteo-clasts from spreading on the bone, restrictingtheir ability to resorb bone. This gives a clueas to what abnormalities to look for in theosteoclasts of these mice. Conversely,increased Src activity may result in increasedcell motility; in this regard, an activatingmutation in the src gene has been identifiedin advanced human colon cancer and shownto promote tumorigenicity and metastasis9.

We don’t yet know how Src regulatesintegrin–cytoskeletal interactions. Felsenfeldet al.’s results provide one hint: they showthat Src localizes together with the vitronec-tin receptor in the cell. Perhaps, therefore,the sites for Src action are at or near the cyto-plasmic membrane. These sites couldinclude focal adhesions — specialized mem-brane-attachment plaques at which integrinscouple the ECM to actin filaments. Supportfor idea this comes from the finding thatspreading of Src-deficient fibroblasts and thefunction of osteoclasts in Src-deficientmutant mice can be restored by kinase-defective Src6,10, indicating that intrinsickinase activity may be expendable for thesefunctions of Src. Instead, localization of Srcto focal adhesions appeared to beimportant6,10. Felsenfeld et al.1 show thatkinase-defective Src co-localizes with the vit-ronectin receptor; it would be interesting tosee whether this mutant can also rescue thereinforcement defect of Src-deficient cells. Ifso, it may be that Src functions in a kinase-independent manner, perhaps by mediatingthe formation of signalling-protein com-plexes that regulate integrin–cytoskeletalinteractions.

Focal-adhesion proteins such as focal-adhesion kinase (FAK) and the docking pro-teins p130Cas (Cas) and paxillin interact withSrc in response to integrin-mediated cellattachment and are phosphorylated by Src.Tyrosine phosphorylation of focal-adhesionproteins is compromised in Src-deficientcells, and can be restored by re-expressingkinase-defective Src (see ref. 11 for a review).Moreover, these proteins are required in cellmigration, indicating that they could mediatethe effects of Src on integrin–cytoskeletalinteractions12–14. However, FAK, Cas and pax-illin appear to reside in pathways that are

Figure 1 Summary of the studies by Felsenfeld et al.1. Beads coated with an extracellular-matrix (ECM)-anchored ligand (yellow) are held in a laser trap on the upper surface of fibroblasts (red tube). Binding of the beads to cell-surface integrins (‘ligand binding’) results in integrin aggregation and induces linkage of the integrin to the actin cytoskeleton (‘integrin aggregation’). Molecular motors (purple) generate a force on the actin cytoskeleton and the ligand-coated bead is pulled away from the laser trap. Upon retrapping of the rearward-moving bead with the laser (curved red lines), the cell reinforces its integrin–cytoskeletal linkages in response to the trap force (‘reinforcement’). Experiments with fibronectin-coated beads show that reinforcement of the linkages between the fibronectin integrin receptor and the cytoskeleton is similar for wild-type and Src-deficient cells. In contrast, the vitronectin receptor shows little or no reinforcement in Src-expressing fibroblasts. On Src-deficient fibroblasts, however, vitronectin-coated beads become reinforced and cannot be retrapped, indicating that the vitronectin receptors have strengthened or increased their number of stable links to the cytoskeleton in Src-deficient cells. These results show that Src is a selective inhibitor of sustained force generation through the vitronectin receptor.

Actin

Reinforcement Integrinaggregation

Ligandbinding

Integrin

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affected by multiple integrin subclasses, so itis unclear how the specificity for the vitronec-tin receptor in Src action would come about.

Small GTPases of the Rho family repre-sent another group of candidates for mediat-ing the effects of Src on integrin–cytoskeletalinteractions. Rho-like GTPases link extracel-lular growth signals and various intracellularstimuli to the assembly and organization ofthe cytoskeleton. Interestingly, a large bodyof evidence indicates that Rho activity is reg-ulated by tyrosine phosphorylation (see ref.15 for a review). As dominant alleles encod-ing FAK and Rho-like GTPases are available,it will be possible to discover whether they

can reverse the effects of loss of Src oncytoskeletal functions. Clearly, finding outwhich events are downstream of Src in thepathway uncovered by Felsenfeld et al. is anexciting challenge for the future. hKristiina Vuori and Erkki Ruoslahti are at the Cancer Research Center, the Burnham Institute, La Jolla, California 92037, USA.e-mails: kvuori@burnham-inst.org ruoslahti@burnham-inst.org

1. Felsenfeld, D. P., Schwartzberg, P. L., Venegas, A., Tse, R. & Sheetz, M. P. Nature Cell Biol. 1, 200–206 (1999).

2. Felsenfeld, D. P., Choquet, D. & Sheetz, M. P. Nature 383, 438–440 (1996).

3. Pelham, R. J. Jr & Wang, Y. Proc. Natl Acad. Sci. USA 94, 13661–13665 (1997).

4. Choquet, D., Felsenfeld, D. P. & Sheetz, M. P. Cell 88, 39–48 (1997).

5. Galbraith, C. G. & Sheetz, M. P. Proc. Natl Acad. Sci. USA 94,

9114–9118 (1997).

6. Kaplan, K. B., Swedlow, J. R., Morgan, D. O. & Varmus, H. E.

Genes Dev. 9, 1505–1517 (1995).

7. Soriano, P., Montgomery, C., Geske, R. & Bradley, A. Cell 64,

693–702 (1991).

8. Rodan, S. B. & Rodan, G. A. J. Endocrinol. 154, S47–S56 (1997).

9. Irby, R. B. et al. Nature Genet. 21, 187–190 (1999).

10. Schwartzberg, P. L. et al. Genes Dev. 11, 2835–2844 (1997).

11. Thomas, S. M. & Brugge, J. S. Annu. Rev. Cell Dev. Biol. 13, 513–

609 (1997).

12. Ilic, D. et al. Nature 377, 539–544 (1995).

13. Cary, L. A., Han, D. C., Polte, T. R., Hanks, S. K. & Guan, J. L. J.

Cell Biol. 140, 211–221 (1998).

14. Klemke, R. L. et al. J. Cell Biol. 140, 961–972 (1998).

15. Schoenwaelder, S. M. & Burridge, K. Curr. Opin. Cell Biol. 11,

274–286 (1999).

Coupling ATP hydrolysis to mechanical work

Alex E. Knight and Justin E. Molloy

Kinesin is a molecular motor that works as a cellular ‘porter’. Its job is to walk along microtubules, carrying membrane-bound bags of chemicals. To do this it must convert the chemical energy of ATP into mechanical work, and ATP hydrolysis may be very tightly coupled to the number and length of steps taken by kinesin.

inesin is a highly processive, microtu-bule-based motor protein that movesalong microtubules in the direction of

their plus (more quickly growing) ends. Thecoordinated activity of kinesin’s two motordomains ensures that at least one of them istightly bound to the microtubule at all times.Kinesin can therefore ‘walk’ along a microtu-bule, taking a hundred or so steps (andhydrolysing a comparable number of ATPmolecules) before falling off. This behaviourenables kinesin to transport vesicles insideyour neurons. You can move your finger toturn this page (not yet we hope!) because bil-lions of slavish kinesin molecules have car-ried vesicles of neurotransmitter all the wayfrom your spine to the nerve endings thatwork the muscles of your fingers.

Visscher et al.1 have now developed a newsingle-molecule mechanical technique withwhich to study processive enzymes. They callit a “molecular force clamp”. Their apparatusenables the load on a single kinesin moleculeto be varied while its stepping motion isobserved. The resolution of the device is suchthat both the duration and the amplitude ofthe individual 8-nanometre steps thatkinesin takes can be measured over a widerange of both chemical and mechanical con-ditions. This has enabled them to answer sev-eral key questions about the basic

mechanism by which the molecular motorkinesin converts the chemical energy of ATPinto movement and force.

The development of in vitro motilityassays has made it possible to observe theactivity of individual kinesin molecules. Inone such assay, glass microspheres are coatedwith kinesin (on average one molecule permicrosphere) and are viewed by light micro-scopy as they walk along a microtubule2. Thevelocity of the microsphere is measured byhigh-resolution tracking of its position and,at low ATP concentrations, the individualsteps taken by the kinesin molecule can beresolved. Hence, on a nanometre scale,movement is not smooth, but shows discretesteps. To improve resolution, ‘optical tweez-ers’ can be used both to restrict brownianmotion and also to apply calibrated loads tothe microsphere. Optical tweezers are a formof nanotechnology: a laser beam, tightlyfocused by a microscope objective lens, gen-erates sufficient photon pressure to captureand manipulate micron-sized particles. Bymonitoring the position of the microspherewith nanometre precision, researchers canmeasure the movements and forces pro-duced by single motor molecules over a widerange of chemical and mechanicalconditions2. Optical tweezers have been usedto study a variety of molecular motors,

K

including actomyosin, RNA polymerase andthe bacterial flagellar motor.

Until now, a problem with many of thesemechanical studies has been the presence of‘series elasticity’ between the motor and themicrosphere. This is an unknown link thatstretches under load, with the result that themicrosphere position no longer faithfullyreports the movements and forces producedby the attached motor molecule. Visscher etal.1 realised that if the load applied to themicrosphere is made constant, the extensionof this compliant link would also be constant,so that true movements and forces couldthen be measured. As the force applied by anoptical tweezer is proportional to the dis-tance between the microsphere and thefocus, they used feedback to keep this dis-tance fixed. This device therefore maintains aconstant load and is termed a ‘force clamp’. Astable force clamp, capable of operating overlong distances, is much more difficult to pro-duce than the position clamps used in acto-myosin studies3. To achieve their goal,Visscher et al. used a computer system to takeinformation from a position sensor and cal-culate the required control signal for theoptical tweezer in just a few microseconds.Simultaneously, the same computer was usedto store, display and analyse all of the data.

There are many unanswered questionsabout exactly how kinesin and other molecu-lar motors work. The question that interestsVisscher et al. is the mechanism by whichATP breakdown is coupled to the mechanicalsteps that kinesin makes. To act as a cellular‘porter’, kinesin must have at least one motordomain (or ‘head’) bound to the microtubuleat any time, and take steps that are of a sizecommensurate with the microtubule lattice.Now, if one ATP is consumed for each steptaken, the efficiency will be high when kinesinoperates at high load, but very low when theload is small. Can kinesin keep efficiency highby altering the coupling between its biochem-ical and mechanical cycles (the number ofATP molecules consumed per step)? Or mustit always break down one ATP for every step