TRENDSin Cell Biology Vol.12 No.2 February 2002http://tcb.trends.com - PDF document

TRENDSin Cell Biology Vol.12 No.2 February 2002http://tcb.trends.com 0962-8924/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S0962-8924(01)02205-X Cell migration is driven by the physical interactions,termed ‘traction forces’, that take place between cells TRENDSin Cell Biology Vol.12 No.2 February 2002http://tcb.trends.com marker particles.Although it should be possible toArecent development in silicone substratausing a curing agent ([15]; Fig. 1c). This generatesmicropatterned molds. Moreover, the micropatternthrough the contact guidance mechanism [16]. LikePolyacrylamide substrataoptical properties [17].The flexibility of the materialThe flexibility of the materialassume that mechanical interactions with suchsubstrata are mediated by the coated ECM orassociated proteins. Deformationis detected by using embedded (a)(b)(c)(e)(d) Fig. 1.Various flexibletraction forces. (a) Motilefish keratocyte on asubstratum. Arrowmigration. Image kindlyprovided by K. Burton. (b) Motile fish keratocytesilicone substratum. Blackmicrobeads; bar, 10 Reproduced, withpermission, from Ref. [14].distortions on amicropatterned siliconesubstratum with regularlyspaced dots. Arrowheadsand magenta dotsunderline the pinchingon the elastomer; bar, m. Reproduced, withpermission, from Ref.[15]. (d) Tail region of achick embryonicfibroblast moving acrossa detection pad of aReproduced, withpermission, from Ref. [20].substratum; bar, 10 Red arrows indicate local TRENDSin Cell Biology Vol.12 No.2 February 2002http://tcb.trends.com Micromachined cantileverss(Fig. 1d). Cells adhere and exert forces onmicrometer-sized pads at one end of the flexiblecantilever, causing displacements that are detectedcan be applied to cells distributed at a high density,with isolated cells. However, the device is difficult toabove. Moreover, the spatial resolution is limited bythe cantilever.How are magnitude and direction of traction forcescalculated?With isolated one-dimensional springs, forces areGenerally, the analysis involves two steps: theOriginally, substrate deformation was determined, substrate deformation was determinedThe coordinates of these markers were then used toconstruct a vectorial map. This painstaking processhas since been replaced with automatic computerprograms based on various forms of the optical flowalgorithm [15,22], which searches for the bestregional matches between a pair of images andgenerates vectors at a specified density. Under a. Under aUsing interpolation algorithms, deformation at agiven location can then be determined with aprecision of 10–100 nm [23].Two different approaches have been used towo different approaches have been used toa prioriassumption of thedistribution of forces other than that forces must beconfined within the boundary of the cell and that netforces and torques equal zero (given the small massand acceleration, the net forces and torques involvedin cell migration are negligible). Because the numberof deformation vectors, superimposed with noise, isgenerally insufficient to provide an unambiguousanswer, a probability-based algorithm that favorsis used to generate a ‘most likely’map of tractionmap of tractionresolution at roughly 2 mm (W.A. Marganski and M. Dembo, unpublished; [27]).etal.al.to force requires the assumption that forces areexerted only at focal adhesions, which significantlyreduces the number of possible answers and possiblyallows a more definitive determination of forces atthese sites. It is unclear, however, whether forces are, whether forces arerequires additional steps of immunofluorescence orimaging with green-fluorescent protein (GFP) and issubject to uncertainties – particularly for small focaladhesions, which might exert stronger forces thanthose of large focal adhesions (see below). Suchuncertainties might lead to systematic errors in thecalculated traction forces.Calculated force or stress distribution can bevisualized as a map of vectorial arrows (Fig. 2a) orrendered as color images after converting the stressmagnitude into different colors ([27]; Fig. 2b). In essence, the latter approach functions as a newform of microscopy and has been referred to as‘traction force microscopy’.Itcan be used to generateWhat has been learned about cellular mechanicalinteractions?[9,11,13,14,20,27,28], growth cone extension [19] and TRENDSin Cell Biology Vol.12 No.2 February 2002http://tcb.trends.com substrata [9].Recent studies with myosin inhibitorsbinding RGD peptideshave indicated further thathave indicated further thatthose in the rear serve as passive anchors [28]. The distribution of forces supports a frontal towingmodel of cell migration, in which the frontal regionsserve as the ‘engine’that tows an adhesive cargothat tows an adhesive cargoAlthough early studies suggested that frontaltraction forces are generally localized near focaladhesions [9,21], subsequent scrutiny indicates thatnot all adhesions produce detectable traction forces.Recent systematic analyses using a combination oftraction force microscopy and GFPimaging has led toimaging has led toThe magnitude of forces decreases as focal adhesionsmature and grow in size. Once the focal adhesionsmature, they seem to maintain a constant stress thatis independent of their size [15]. These observationscorroborate the heterogeneity of size, morphology,,propulsion at nascent focal adhesions provides anelegant, responsive strategy for the cell to coordinatecontractility with migration and adhesion. Together,,where new adhesion sites are forming [31]. In keratocytes, however, this region is located nearcell migration. Although a small propulsiveAlthough a small propulsiveinvolved not only in cell migration but possibly inother functions such as cell–cell communicationand/or mechanosensing (see below).Flexible substrates have also been used as a means to apply mechanical stimulations to adherent cells, totest the ability of cells to sense mechanical changes inthe environment [6,7]. Mechanical forces are exertedby pushing or pulling on the substrate near the cellusing a blunt microneedle [34,35]. Cells have beenfound to reorient towards pulling forces [34],accompanied by an increase in the number and/or sizeof focal adhesions [35,36]. Conversely, pushing move away. . These observations show that cells not only respondto forces exerted through their adhesion sites, but also actively probe mechanical properties of the substratum – a phenomenon termed‘mechanosensing’.What is likely to be learned from future investigations?Clearly, cell–cell and cell–substrate adhesions TRENDS in Cell Biology IntegrinNascent focal adhesionsGeneration of transientpropelling forcesMigration ofMature focal adhesionsAnchorage andstabilization of protrusion New protrusion Relationship between focal adhesions and mechanical forcesduring fibroblast migration. The formation of focal adhesions in thelamellipodium, accompanied by the generation of a pulse of propulsiveforces, drives the forward movement. Cell migration is sustained bypulses of propulsive forces. Mature focal adhesions, such as thoselocated in the tail, play only a passive role in anchoring cells to thesubstrate. Adapted, with permission, from Ref. [27]. (b) (a) Vector plot of traction stress generated by a fish fin fibroblaston a polyacrylamide substratum. Arrowheads indicate direction offorces. (b) Color rendering of the magnitude of traction forces, ‘hot’colors highlight areas of strongest force and ‘cool’ colors indicateregions of weaker force. TRENDSin Cell Biology Vol.12 No.2 February 2002http://tcb.trends.com Traction force microscopy represents a powerful light-microscopy techniques, such as GFPimaging,imaging,developed substrata makes qualitative studies oftraction forces feasible for most laboratories.Although quantitative analyses were initiallyperformed with supercomputers, a combination of hardware/software improvements and availability has enabled personal computers tohandle the task.Many important issues need to be resolved. Forexample, given the differences between nascent focalcomplexes and focal adhesions in mechanical output,it is important to identify the mechanisms thatregulate the production and transduction ofcontractile forces during the maturation of focaladhesions. The process is likely to involve profound changes in protein–protein interactions. An intriguing observation is that stationaryfibroblasts appear to maintain an overall magnitudeof traction force similar to that of migrating cells [15],even though they presumably contain only maturefocal adhesions. During the transition from migratingto stationary state, therefore, a separate processmight cause the traction forces to stay on focaladhesions or to transfer the mechanical load fromnascent focal contacts to existing focal adhesions.Equally important is the mechanism ofmechanosensing. Although focal adhesion kinase,, GTP, proteolysis andjunctions. Although the current methods are designedFinally, there is strong evidence that mechanical, there is strong evidence that mechanicalosteoblast maturation [38] and phagocytosis [39].Although they probably share some common aspects,the specific functions of mechanical forces in theseprocesses are just beginning to be unraveled.Understanding the interplay between extracellularphysical interactions and intracellular chemicalevents is likely to exert a strong impact on manypractical applications, including tissue engineering,stem cell differentiation and treatments ofautoimmune diseases and cancer.AcknowledgementsWe thank Micah DemboThis study was supportedby NIH NRSA grantNIH grant GM-32476 andNASA grant NAG2-1197 to Y-L.W.References1Lauffenburger, D.A. and Horwitz, A.F. (1996) 2Sheetz, M.P. regulation of force on extracellular-Trends Cell Biol.3Elson, E.L. 4Davies, P.F. Annu. Rev.5Gillespie, P.G. and Walker, R.G. (2001) Molecular6Liu, N. 7Brown, T.D. (2000) Techniques for mechanical: a review.J.Biomechanics8Roy, P. 232, 106–1179Harris, A.K. 10Burton, K. and Taylor, D.L. (1997) Traction forces11Burton, K. 12Oliver, T. 13Lee, J. (1994) Traction forces generated by14Oliver, T. (1995) Traction forces in locomoting15Balaban, N.Q. 16Weiss, P. (1958) Cell contact. Int. Rev. Cytol17Pelham, R.J. and Wang, Y-L. (1997) Cellsubstrate flexibility. Proc. Natl. Acad. Sci. U. S. A.18Beningo, K.A. and Wang, Y-L. Flexible19Bridgman, P.C. 20Galbraith, C.B. and Sheetz, M.P. (1997) AProc. Natl. Acad. Sci. 94, 9114–911821Pelham, R.J. and Wang, Y-L. (1999) High22Munevar, S. (2001) Traction force microscopyBiophys. J. 23Marganski, W.A. using correlation based optical flow. 24Dembo, M. and Wang, Y-L. (1999) Stresses at the25Wang, N. Proc. Natl. Acad. Sci. U. S. A. TRENDSin Cell Biology Vol.12 No.2 February 2002 Dept of Cell Biology,Anatomy, University ofOH 45267-0521, USA.http://tcb.trends.com 0962-8924/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S0962-8924(01)02206-126Bray, D. (2001) ,Garland Publishing27Beningo, K.A. 28Munevar, S. 29Zamir, E. . 112, 1655–166930Dembo, M. 31Small, J.V. (1998) Assembling an actin32Oliver, T. 33Galbraith, C. and Sheetz, M.P. (1999) Keratocytes34Lo, C-M. 35Riveline, D. . 153, 1175–118636Wang, H.B. Proc. Natl Acad. Sci. U. S. A. 11295–1130037Oster, G.F. 38Burger, E.H. and Klein-Nulen, J. (1999)invitroAdv. Dent. Res.39Beningo, K.A. and Wang, Y-L. Fc-receptor homeostasis. Any abnormalities in the cell deathAny abnormalities in the cell deathidentifying the molecules involved in cell death andunderstanding the regulation of the death process arecrucial for prevention and management of thesehuman diseases.In normal development and tissue homeostasis,most of the cells die through physiological orprogrammed cell death to remove excessive ordamaged cells [4]. The term ‘apoptosis’was first usedwas first usedmorphological changes were also observed inprogrammed cell death in invertebrates [4]. The apoptotic morphological changes exhibited by the dying cells are followed by phagocytosis byscavenger cells. Abiochemical hallmark of apoptosisis the cleavage of chromosomal DNAintoDNAfragmentation [2]. Apoptosis eliminatesApoptosis eliminatesBecause of the fundamental role of apoptosis indevelopment and tissue homeostasis, a cell-suicideprogram utilizing evolutionarily conserved moleculesis dedicated to the process [5,6]. Elegant genetic andbiochemical work has identified several families ofproteins, such as the Bcl-2 family, that regulate, that regulateAlthough most research efforts have focused on themore upstream death program molecules such as theBcl-2 family proteins and caspases, the key molecules involved in DNAfragmentation and therole of cleavage of chromosomal DNAin apoptosis DNA fragmentation is a hallmark of apoptosis.The tightly controlledactivation of the apoptosis-specific endonucleases provides an effectivemeans to ensure the removal of unwanted DNA and the timely completion ofapoptosis.Over the past several years,crucial progress has been made inidentifying the long-awaited apoptotic endonucleases,and their importancein tissue homeostasis is beginning to unfold.Here,we focus on the mostrecent discoveries about the functions and mechanisms of theseendonucleases in the context of apoptosis.We also discuss consequencesthat defective DNA fragmentation might have for tissue homeostasis anddisease development.Apoptotic DNA fragmentation andJianhua Zhang and Ming Xu