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Cell Motility and Mechanicsin Three-DimensionalCollagen MatricesFrederick Grinnell1 and W. Matthew Petroll2

Departments of 1Cell Biology and 2Ophthalmology, University of Texas SouthwesternMedical Center, Dallas, Texas 75390; email: frederick.grinnell@utsouthwestern.edu,matthew.petroll@utsouthwestern.edu

Annu. Rev. Cell Dev. Biol. 2010. 26:335–61

First published online as a Review in Advance onMay 3, 2010

The Annual Review of Cell and DevelopmentalBiology is online at cellbio.annualreviews.org

This article’s doi:10.1146/annurev.cellbio.042308.113318

Copyright c© 2010 by Annual Reviews.All rights reserved

1081-0706/10/1110-0335$20.00

Key Words

biomechanics, extracellular matrix remodeling, Rho GTPases,tensional homeostasis, tissue engineering, wound healing

Abstract

Fibrous connective tissues provide mechanical support and frameworksfor other tissues of the body and play an integral role in normal tis-sue physiology and pathology. Three-dimensional collagen matricesexhibit mechanical and structural features that resemble fibrous con-nective tissue and have become an important model system to study cellbehavior in a tissue-like environment. This review focuses on motileand mechanical interactions between cells—especially fibroblasts—andcollagen matrices. We describe several matrix contraction models, theinteractions between fibroblasts and collagen fibrils at global and sub-cellular levels, unique features of mechanical feedback between cells andthe matrix, and the impact of the cell-matrix tension state on cell mor-phology and mechanical behavior. We develop a conceptual frameworkto explain the balance between cell migration and collagen translocationincluding the concept of promigratory and procontractile growth factorenvironments. Finally, we review the significance of these concepts forthe physiology of wound repair.

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Contents

BEYOND REDUCTIONISM . . . . . . . . 336REGULATION OF MATRIX

CONTRACTION . . . . . . . . . . . . . . . . 337MATRIX MECHANICS . . . . . . . . . . . . . 341CELL MIGRATION. . . . . . . . . . . . . . . . . 343HIGH-RESOLUTION IMAGING

OF LOCAL MATRIXREMODELING . . . . . . . . . . . . . . . . . . 347

REGULATION OF LOCALMATRIX REMODELING BYSMALL G PROTEINS ANDMECHANICAL STIMULI. . . . . . . . 348

WOUND REPAIR ANDMATRIX CONTRACTION . . . . . . 350

CONCLUSIONS AND FUTUREDIRECTIONS . . . . . . . . . . . . . . . . . . . . 352

BEYOND REDUCTIONISM

Snatched from a life of obscurity and installed incontemporary glass and plastic palaces, cells are indanger of becoming Pygmalion’s proteges. Housedin more traditional residences constructed of waterand collagen instead of plastic or glass, do cells leadprimitive, less cultured lives?

Tom Elsdale and Jonathan Bard: “Collagensubstrata for studies on cell behavior” (Elsdale& Bard 1972)

In their 1924 review “Behavior of cells intissue cultures,” Lewis & Lewis summarizedthe contemporary field of tissue culture andits goal to reveal “new facts concerning thebehavior, structure, and physiology of theliving body cells” (Lewis & Lewis 1924).Diverse normal and malignant cell typeshad been observed to migrate out of tissuefragments onto the surface of glass coverslips,thereby making the cells accessible to inves-tigation. Cell pseudopodia were believed toprovide the mechanism for migration. Thenew methodology for establishing and growingtissue cells on glass coverslips rapidly replaced

the techniques of cell culture in 3D matricesprepared with clotted lymph and plasma thathad been introduced 15 years earlier.

When cells are cultured on glass coverslips,the physical framework with which the cellsinteract becomes invisible (literally and oftenfiguratively as well). Recognizing the impor-tance of hierarchical organization for biologicalfunction and structure, developmental biologistPaul Weiss continued to emphasize the need tolearn about cells not only as individuals (a re-ductionist approach) but also in the context ofthe interdependent relationship between cellsand their physical framework (a systems-basedapproach) (Weiss 1959). Figure 1a shows oneof the experiments used by Weiss to illustratesuch interdependence. Stretch (arrows) appliedat the corners of a plasma clot resulted in ori-entation of the fibrin. Mesenchymal cells in thestretched region of the clot developed bipolarmorphology parallel to the lines of tension. Inunstrained regions, where the fibrin lacked ten-sion, cells retained a more symmetric, stellateshape. Figure 1b illustrates a more complex ex-ample. In this case, three tissue fragments wereembedded in a fibrin matrix. Cellular activ-ity resulted in the development of tension andthe orientation of the fibrin between the frag-ments. These tension lines became tracks forsubsequent cell migration—evidence for con-tact guidance.

Acid-solubilized collagen, when coated oncoverslips and polymerized by ammonia vapor,forms an amorphous gel that can be used asa substrate to study cell-collagen interactions(Bornstein 1958). However, adjusting the col-lagen solution to physiological ionic strengthand pH first, and then initiating polymerizationby raising the temperature, produces hydratedcollagen lattices in which cells can be cultured(Elsdale & Bard 1972). Using their new tech-nique, Elsdale and Bard showed that fibroblastsexhibited features of shape, motility andgrowth in 3D collagen matrices not observedin routine 2D culture. Moreover, they made adistinction between cell behavior in restrainedmatrices (i.e., attached to culture dishes) versusfloating matrices (i.e., in culture medium).

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Under floating conditions there occurred acell-dependent “gradual collapse of the latticeto a dense, opaque body less than one-tenththe original size” (Elsdale & Bard 1972).

Subsequently, Michalopoulos and Pitot(1975) cultured liver parenchymal cells on thesurfaces of restrained and floating collagen ma-trices. They reported that cells on floating ma-trices caused the matrix to decrease in diameter,which they described as contraction. Also, cellson floating but not restrained matrices exhib-ited morphologic and biochemical character-istics of differentiated liver parenchymal cells,including formation of structures resemblingbile canaliculi (Michalopoulos & Pitot 1975).Similarly, Emerman & Pitelka (1977) observedthat mammary epithelial cells cultured on thesurfaces of floating collagen matrices causedmatrix contraction and retained morphologi-cal and biochemical characteristics of differen-tiated mammary epithelium including the de-velopment of alveolar-like structures within thematrices.

Although others had noticed the phe-nomenon of collagen matrix contraction,Eugene Bell made the contraction process itselfa problem for investigation. He suggested thatunderstanding how fibroblasts contract a col-lagen matrix into a tissue-like structure mightprovide insights into wound contraction (Bellet al. 1979). Moreover, he used contracted col-lagen matrices containing fibroblasts as the der-mal equivalent component of in vitro skin grafts(Bell et al. 1981), which became a foundingtechnology of the tissue engineering companyOrganogenesis.

Type I collagen is the major protein com-ponent of fibrous connective tissues, and fi-broblasts are the cell type primarily responsi-ble for collagen biosynthesis and remodeling.Because fibrous connective tissues provide me-chanical support and frameworks for the othertissues of the body, collagen matrix contractioncan influence diverse aspects of normal tissuephysiology and pathology. This review will fo-cus on motile and mechanical interactions be-tween cells—especially fibroblasts—and colla-gen matrices, the relationship between matrix

a

b

Figure 1Effect of regional tension on cell shape and migration. (a) Stretch (arrows)applied at the corners of a plasma clot results in orientation of the fibrin.Mesenchymal cells in the stretched region developed bipolar morphologyparallel to the lines of tension. In unstrained regions, cells retained a moresymmetric, stellate shape. (b) Cell migration along tension lines connectingthree tissue fragments in a fibrin clot. Reprinted from Weiss (1959) withpermission of the American Physical Society.

contraction and cell migration, and the signifi-cance of matrix contraction for cell physiology.Several recent reviews provide more generaldiscussions regarding the use of collagen andother 3D matrices to model the morphogene-sis of normal and malignant cells (e.g., Davis &Senger 2008, Griffith & Swartz 2006, Xu et al.2009, Yamada & Cukierman 2007).

REGULATION OF MATRIXCONTRACTION

Many cell types including fibroblasts, smoothmuscle cells, epithelial cells, and endothelial

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Cell traction(specific): rearwardforce transmitted to asubstrate during cellextension, analogousto a tire on the road

Tractional force(generic): forcetransmitted to asubstrate regardless ofmechanism

Stiffness: a measureof the resistanceoffered by an elasticbody to deformation(bending, stretching,or compression)

cells contract collagen matrices (Supplemen-tal Table 1; follow the Supplemental Mate-rial link from the Annual Reviews home page athttp://www.annualreviews.org). Therefore,matrix contraction is a highly conserved fea-ture of cell motile behavior similar to cell adhe-sion, spreading, and migration. Given the po-tential of matrix contraction to influence tissuestructure, understanding the basic mechanismsand regulation of collagen matrix contractionhas become an important design considerationfor tissue engineering (Brown et al. 2005, Clarket al. 2007, Lutolf & Hubbell 2005).

Although we use the word fibroblast in ageneric sense, it should be recognized that fi-broblasts represent a diverse cell population(Rinn et al. 2008, Sorrell & Caplan 2009). Con-siderable variation in matrix contraction by dif-ferent types of fibroblasts has been reported.For instance, papillary fibroblasts contract col-lagen matrices better than reticular fibroblasts(Schafer et al. 1989). Wound fibroblasts con-tract matrices better than dermal fibroblasts(Germain et al. 1994). Early passage fibro-blasts contract matrices better than establishedcell lines, which contract matrices better thanSV40-transformed cells (Steinberg et al. 1980).

The term collagen matrix contraction canbe used to refer to the final organization of thematrix or to the cellular mechanisms responsi-ble for achieving that organization. Early find-ings suggested that matrix contraction couldresult from cell traction (rearward force ex-erted during cell extension) or cell contraction(inward force exerted by cell shortening) (Bellet al. 1979, Bellows et al. 1982, Stopak & Harris1982). The expression “tractional force” is of-ten used in a generic sense to refer to any forceexerted on the matrix by cells regardless of themechanism. We will follow that trend but indi-cate the mechanism where appropriate.

Matrix contraction was initially found to de-pend on serum stimulation (Steinberg et al.1980); however, a wide range of physiologi-cally relevant growth factors have been shownto substitute for serum, many of which activatedifferent signaling pathways (SupplementalTable 2; follow the Supplemental Material

link from the Annual Reviews home page athttp://www.annualreviews.org). In the ab-sence of added growth factors, matrix contrac-tion becomes dependent on autocrine or (in thecase of mixed cell cultures) paracrine cell secre-tion. Also, collagen matrix contraction requiresintegrin-dependent adhesive interactions, andmany different collagen-binding integrins canfunction in matrix contraction including α1β1(Racine-Samson et al. 1997), α2β1 (Klein et al.1991, Schiro et al. 1991), α3β1 (Morales et al.2007), α11β1 (Tiger et al. 2001), and αvβ3(Cooke et al. 2000). No role in matrix con-traction has yet been described for collagen-binding tyrosine kinase receptors (Vogel et al.2006).

Given the multiple cell types, mechanismsfor exerting force, receptors, and signalingpathways involved in collagen matrix contrac-tion, rather than a singular view of the pro-cess, an overall conceptual framework can bedescribed best. Cells interacting with collagenmatrices form a system. Global factors such aswhether the matrix is floating or restrained aswell as local factors such as matrix stiffness in-fluence the pattern and mechanism of contrac-tion. Some of the most important variables thatwe will mention are low versus high cell-matrixtension states, cell tractional force relative tocollagen matrix stiffness, and promigratory ver-sus procontractile growth factor environments.

Figure 2 shows three interrelated modelsthat have been used to study the signaling,regulatory, and motor mechanisms involved incollagen matrix contraction (Grinnell 2000).Floating matrix contraction occurs when cell-containing matrices are released from the cul-ture surfaces soon after matrix polymeriza-tion. During and after contraction, the cellsexhibit few actin stress fibers and focal adhe-sions (low cell-matrix tension state). Restrainedmatrix contraction occurs when cells are in-cubated with matrices that remain attached tothe culture surfaces. During contraction, ma-trix height decreases, cells and collagen be-come aligned parallel to the culture surface,and prominent actin stress fibers and focal ad-hesions form (high cell-matrix tension state).

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Supplemental Material

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LPA:lysophosphatidic acid

PDGF: platelet-derived growth factor

Stress-relaxation occurs upon release of re-strained matrices that previously have devel-oped tension. During stress-relaxation, actinstress fibers disappear, fibronectin matrix andfibronexus junctions are disrupted, and cellsand collagen lose their aligned orientation. Asa practical matter, floating matrix contractionand stress-relaxation are easily quantified bymeasuring the change in matrix diameter.

Serum can stimulate fibroblast contractionof collagen matrices in all three of the mod-els shown in Figure 2. Research using re-strained matrices attached to a force moni-tor showed that serum and the serum lipidgrowth factor lysophosphatidic acid (LPA)were far more effective than platelet-derivedgrowth factor (PDGF) in stimulating contrac-tile force generation by fibroblasts in restrainedcollagen matrices (Kolodney & Elson 1993).Subsequent work demonstrated that PDGFand LPA stimulate collagen matrix contrac-tion by different mechanisms (Grinnell 2000).Figure 3 summarizes key steps in the sig-naling pathways involved (Rhee & Grinnell2006). Three points are worth special emphasis.First, cell-matrix tension state influences whichgrowth factors stimulate collagen matrix con-traction. Both PDGF and LPA stimulate float-ing matrix contraction; only LPA stimulatesstress-relaxation. Second, although LPA stim-ulates collagen matrix contraction at both lowand high cell-matrix tension states, differentsignaling pathways and motor mechanisms areinvolved. LPA-stimulated floating contraction(low cell-matrix tension state) involves p21-activating kinase 1 (PAK1) in cooperation withthe Rho effector mDia1; myosin II is not re-quired. LPA-stimulated stress-relaxation (highcell-matrix tension state) depends on Rho ki-nase and myosin II. Third, although stimu-lation of floating matrix contraction by bothPDGF and LPA involves PAK1 signaling, dif-ferent Rho effectors cooperate with PAK1: Rhokinase in the case of PDGF and mDia1 in thecase of LPA. In general, stimulation of matrixcontraction by serum and by LPA exhibit sim-ilar features.

Fibroblasts polymerizedin collagen matrix

Floating matrix contraction

(dermal equivalent)

ReleaseRestrained matrix

contraction(tension develops)

Culture

12 mm

4–6 mmStress-relaxation

(tension dissipated)

Release

Figure 2Models of collagen matrix contraction. Floating matrix contraction occurswhen cell-containing matrices are released from culture surfaces soon aftermatrix polymerization. Restrained matrix contraction occurs when cells areincubated with matrices that remain attached to the culture surfaces.Stress-relaxation occurs upon release of restrained matrices that previouslyhave developed tension. Adapted from Grinnell (2000).

The images of cell morphology in Figure 4help explain the basis for the alternative reg-ulatory and motor mechanisms described inFigure 3. In newly polymerized collagen ma-trices, early passage human fibroblasts constitu-tively form thin, highly branched pseudopodialextensions, giving the cells a dendritic appear-ance. PDGF stimulates further protrusion ofthe dendritic extensions by a Rac- and PAK1-dependent mechanism. LPA or serum stim-ulates their retraction by a Rho/Rho kinase/myosin II–dependent mechanism (Rhee &Grinnell 2007). These cellular responses rep-resent exaggerated versions of the cell ruf-fling (PDGF) and contraction (serum or LPA)that fibroblasts exhibit on conventional 2D cul-ture surfaces (Hall 1998). In restrained collagenmatrices, cell morphology matures as tensiondevelops. In PDGF-containing medium, den-dritic extensions merge; in serum- and LPA-containing media, retracted extensions repro-trude. In both cases, stress fibers and focal

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PDGF

PI3 kinase Gαi

RhoRho (basal) Rho

Rho kinaseMyosin IImDia1PAK1

Cell rufflingtraction

Floating matrixcontraction

Cell shorteningcontraction

Stress-relaxation

Rho kinaseMyosin II

LPA

a Low cell-matrix tension state b High cell-matrix tension state

LPA

Figure 3Growth factor regulation of collagen matrix contraction by dermal fibroblasts. Cell-matrix tension stateinfluences which growth factors stimulate collagen matrix contraction and the signaling pathways and motormechanisms involved. Gαi, G protein subunit αi; LPA, lysophosphatidic acid; PGDF, platelet-derivedgrowth factor; PI3 kinase, phosphatidylinositol 3-kinase. Adapted from Rhee & Grinnell (2006).

adhesions eventually develop (Tamariz &Grinnell 2002). Therefore, the cells themselvesare phenotypically different under the differentexperimental conditions shown in Figure 3.

In retrospect, it has become clear thatfloating matrix contraction describes outcome,not mechanism, and depends on the tractiongenerated by cell ruffling—hand-over-handcycles of local pseudopodial protrusion andretraction (Glaser et al. 1987). More thanone motor mechanism appears capable ofgenerating the mechanical force necessaryfor collagen translocation during ruffling. Forinstance, following Rac-dependent protrusion,collagen movement toward cells can occurduring Rho/Rho kinase/myosin II–dependentcell retraction (Meshel et al. 2005). Alterna-tively, corneal fibroblasts in 3D matrices can

pull collagen inward during cell protrusion(Petroll et al. 2008a, Roy et al. 1997). Incontrast to floating matrices, cell contraction(shortening) rather than cell ruffling providesthe mechanism for stress-relaxation (Grinnell2003, Tomasek et al. 2002).

Disrupting the cells’ actin cytoskeletonblocks all mechanisms of collagen matrix con-traction, but the effect of disrupting micro-tubules depends on the cell-matrix tensionstate. On one hand, if fibroblasts already areunder tension, then disrupting microtubules in-creases force generation (Brown et al. 1996,Kolodney & Wysolmerski 1992) and promotesstress-relaxation (Lee et al. 2003). On the otherhand, disrupting microtubules of cells in float-ing collagen matrices inhibits contraction (Bellet al. 1979, Lee et al. 2003). At a low cell-matrix

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Constitutive

Serum, LPA (1 h) Restrained matrix(after tension develops)

PDGF (4 h)

Figure 4Retraction and protrusion of dermal fibroblast dendritic extensions in response to platelet-derived growthfactor (PDGF), lysophosphatidic acid (LPA), and serum. Cells in basal medium (no added growth factors)form dendritic extensions in collagen matrices after 1–2 h (constitutive). Addition of PDGF results in furtherprotrusion of dendritic extensions over 4 h, whereas addition of LPA or serum causes retraction within 1 h.If the matrices are restrained, then cells in PDGF merge their dendritic extensions, cells in LPA/serumreprotrude their retracted extensions, and tension eventually develops (appearance of actin stress fibers) inboth cases. Adapted from Grinnell et al. (2003) and Tamariz & Grinnell (2002).

Biphasic: describesmaterial consisting ofboth solid and fluidfractions, such ascollagen fibrils andmedia

Nonaffine: describesmaterial in whichdeformation is notcontinuous throughoutthe volume; that is,local strain may besignificantly differentthan global strain

tension state, microtubules are required for for-mation and stabilization of dendritic extensionsand not solely for cell polarization (Rhee et al.2007).

MATRIX MECHANICS

What accounts for the differences in fibroblastmorphology in Figure 4 compared with thebehavior of cells in “glass and plastic palaces?”On 2D surfaces, cell motile activity generallycan be described as the balance of cellular forcesof protrusion, contraction, and adhesion, inde-pendent of the surface’s mechanical properties

(Lauffenburger & Horwitz 1996, Sheetz et al.1999). Two-dimensional surfaces are generallyhomogeneous, elastic materials. By contrast,collagen matrices are biphasic and viscoelastic,exhibit nonaffine compression (Chandran& Barocas 2006), and can undergo strain-stiffening (Storm et al. 2005). Moreover, cellscan penetrate into and become entangled in 3Dmatrices rather than just interacting with thesurface ( Jiang & Grinnell 2005). In short, thetexture and mechanical properties of 3D colla-gen matrices resemble the connective tissue en-vironment, and matrix structure and mechanicshave increasingly been identified as important

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Tensionalhomeostasis: themaintenance ofconstant mechanicaltension within cells,matrices, or tissues

factors in determining cell behavior (Baro-cas & Tranquillo 1997, Georges & Janmey2005, Pedersen & Swartz 2005, Plant et al.2009, Silver et al. 2002, Wakatsuki et al. 2000).Cells and matrix interact to develop tensionalhomeostasis (Brown et al. 1998, Delvoye et al.1991, Paszek et al. 2005, Petroll et al. 2004b).Biomechanical features of contracted collagenmatrices resemble dermis (Ahlfors & Billiar2007). Application of external forces such ascyclic strain during contraction can influencecell orientation and final mechanical strength(Seliktar et al. 2000).

Based on rheometric measurements, theelastic modulus of newly polymerized 1–2 mg ml−1 collagen matrices, the concentra-tion range frequently utilized for collagen ma-trix contraction experiments, is less than 50 Pa(Barocas et al. 1995, Leung et al. 2007). By con-trast, the stiffness of the plastic polymers andglass used as tissue culture surfaces is >1 GPa.How best to determine the mechanical proper-ties of collagen matrices remains an open ques-tion. Rheometry describes bulk phase materialcharacteristics but provides limited informationabout local mechanical properties. Other meth-ods such as nanoindentation or atomic forcemicroscopy can be used to acquire the latterinformation.

Soft 2D surfaces increasingly are being usedto study how cells exert force on a substrateand how mechanical features of the substrateregulate cell motile function (Discher et al.2005, Munevar et al. 2001, Rehfeldt et al.2007). Increasing substrate stiffness increasesthe formation of actin stress fibers and focaladhesions (Engler et al. 2004, Pelham & Wang1997, Yeung et al. 2005). Overlaying fibroblastswith soft 2D surfaces can cause a switch from2D to more 3D morphology (Beningo et al.2004). However, only fibroblasts interactingwith collagen and other 3D matrices formdendritic extensions. As soon as cells beginto experience tension, dendritic extensionsundergo myosin II–dependent reorganizationinto more lamellar structures (Rhee et al.2007). For fibroblasts on the surfaces ofcollagen matrices, this switch occurs when the

collagen concentration of the matrix is in-creased from 1 mg ml−1 to 4 mg ml−1 (Rheeet al. 2010).

Understanding the dendritic features of fi-broblasts is particularly important because rest-ing connective tissues contain fibroblasts ina dendritic network (Goldsmith et al. 2004,Langevin et al. 2004, Salomon et al. 1988). In-teraction between this network and the sur-rounding collagen extracellular matrix pro-vides a mechanism for homeostatic tissuemechanosensing in response to diverse inputsranging from interstitial fluid flow (Heuchelet al. 1999, Pedersen et al. 2007) to stretch(Langevin et al. 2005) to tissue loss (Grinnellet al. 2003, Tomasek et al. 2002). Moreover,accumulated defects in the matrix as a resultof aging disrupt integration between the fi-broblast network and surrounding collagen ma-trix, thereby interfering with normal mechani-cal homeostasis (Fisher et al. 2008).

Collagen matrix contraction requires thefibril network to be interconnected, which al-lows local force to be propagated over long dis-tances (Barocas & Tranquillo 1997, Sawhney& Howard 2002, Vanni et al. 2003). Duringcontraction, cell-independent noncovalent in-teractions rapidly stabilize reorganized colla-gen fibrils. These interactions occur in the ab-sence of collagen telopeptides; however, thepresence of telopeptides increases contrac-tion and alters collagen fibril packing (Freyet al. 1995, Wolf et al. 2009, Woodley et al.1991). During restrained matrix contraction,the matrix itself develops tension. After re-lease, cell-independent matrix contraction oc-curs (Grinnell & Ho 2002, Marenzana et al.2006).

In addition to biologically polymerizedcollagen matrices and collagen matrices formedby electrospinning (Powell & Boyce 2009),fibroblasts can contract diverse other types ofmatrices ranging from fibrin (Tuan & Grinnell1989) to large-pore (140 μm) collagen–chondroitin sulfate composites (Freyman et al.2002). Increasing collagen matrix stiffness bychemical cross-linking with nonenzymaticglycation inhibits matrix contraction (Howard

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TGFβ: transforminggrowth factor β

MMP: matrixmetalloproteinase

Porosity: the ratio ofthe volume of amaterial’s pores to itstotal volume

et al. 1996). Embedding noncompressibleelements into the matrix such as insolublecollagen fragments (Gentleman et al. 2004),single-walled carbon nanotubes (MacDonaldet al. 2005) or cross-linked hyaluronan (Mehraet al. 2006) slows down the rate of contraction.However, hyaluronan by itself has little effecton matrix contraction even under conditionsshown to alter matrix mechanics (Kreger& Voytik-Harbin 2009). Also, fibronectinprobably is not required for collagen matrixcontraction (Tomasek & Akiyama 1992). How-ever, fibronectin was shown to increase matrixcontraction by fibronectin-null fibroblasts(Hocking et al. 2000).

The ability of matrix composition and me-chanics to influence growth factor binding andactivation provides additional mechanisms forindirect regulation of collagen matrix contrac-tion (Macri et al. 2007, Wipff et al. 2007).Stimulation of contraction caused by throm-bospondin and inhibition of contraction causedby decorin have been linked to the ability ofthese matrix components to modify the avail-ability of transforming growth factor β (TGFβ)(Sakai et al. 2003, Zhang et al. 2009). Ma-trix components also can exert indirect ef-fects on contraction by modulating cell sig-naling. Tenascin C inhibits fibronectin-fibrinmatrix contraction by blocking cell signalingdownstream of Rho activation (Midwood &Schwarzbauer 2002).

The role of matrix metalloproteinases(MMPs) in collagen matrix contraction re-mains unclear. Fibroblasts from stomelysin-1null (Bullard et al. 1999) and MMP-9 nullbut not MMP-2 null mice ( Johnson & Galis2004) show reduced matrix contraction. How-ever, matrix contraction occurs in the presenceof serum, which contains high levels of solu-ble proteinase inhibitors, and little gross degra-dation of the matrix occurs during contrac-tion. Rather than soluble metalloproteinases,membrane-tethered MT1-MMP seems themost likely candidate to play a regulatory rolein collagen matrix contraction (Deryugina et al.1998) through its ability to modify the pericel-lular microenvironment (Itoh & Seiki 2006).

CELL MIGRATION

Thus far, our discussion has focused on globalcontraction of collagen matrices. What aboutthe experimental result described in Figure 1b?As already mentioned, Weiss recognized thattension between tissue fragments resulted in lo-cal fibrin alignment and that tension lines inthe fibrin matrix became the tracks along whichcells migrated. Stopak & Harris, working withtissue explants in collagen matrices, showed thatcellular mechanical activity was responsible forgenerating the tension. They emphasized therole of cell mechanics in connective tissue mor-phogenesis (Stopak & Harris 1982). Throughlocal matrix contraction, migrating cells can es-tablish tracks for their own migration (Friedlet al. 1997, Provenzano et al. 2006, Tranquillo1999) or for the migration of other cells(Gaggioli et al. 2007). Similarly, imposing ten-sion externally on polymeric 3D matrices alsocan establish aligned tracks for cell migra-tion (Guido & Tranquillo 1993, Raeber et al.2008).

If cells migrated only on the surfaces ofaligned fibrils in a 3D matrix, then individualfibrils might become singular migration paths(Doyle et al. 2009). But collagen matrices fre-quently contain fibrils that lack a singular ori-entation, and fibroblasts can interact with fib-rils oriented both parallel and perpendicular tothe direction of cell pseudopodia (Petroll et al.2008b). As a result, cell-induced alignment ofcollagen fibrils is not necessarily confined tothe axis of cell movement (Friedl et al. 1998,Petroll et al. 2004a). Moreover, the porosityof 3D matrices establishes the possibility ofdual modes of cell migration—mesenchymaland amoeboid (Friedl & Brocker 2000). In mes-enchymal migration, the cells attach to and pullon the matrix. In amoeboid migration, cells pro-trude through the spaces and push off the ma-trix. With tumor cells, Rho activation favors theamoeboid mode of migration; Rac activationfavors the mesenchymal mode (Sanz-Morenoet al. 2008).

Matrix porosity makes possible multiplemigratory mechanisms but also establishes

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Dermalequivalent

Reembed incell-free matrix

Act

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Restrained nestedmatrix

Floating nestedmatrix

a

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Figure 5Dermal fibroblast migration and collagen translocation in nested collagenmatrices. (a) Nested collagen matrices contain contracted floating collagenmatrices re-embedded in cell-free matrices. (b) Cells with leading dendriticextensions migrate out of the inner matrix and into the outer matrix (phasecontrast). (c) The global environment of nested matrices influences the balancebetween cell migration (actin) and collagen translocation (fluorescent beadstrapped in the collagen). Cell migration occurs preferentially in restrained nestedmatrices. Adapted from Grinnell et al. (2006) and Miron-Mendoza et al. (2008).

potential barriers to migration in the form ofsteric hindrance. Mechanical deformation andproteolysis provide alternative mechanisms ofdealing with steric hindrance (Friedl & Wolf2009, Packard et al. 2009, Wyckoff et al. 2006,Zaman et al. 2006). In the case of proteo-lysis, MT1-MMP likely plays a particularlyimportant role (Rowe et al. 2009, Stratmanet al. 2009). If steric hindrance is minimaland adhesion force is weak, then amoeboidprotrusion/push off can occur without myosinII–dependent contraction (Lammermann &Sixt 2009).

Population studies of fibroblast migra-tion can be accomplished using nested col-lagen matrices—dermal equivalents embed-ded in cell-free collagen matrices (Figure 5a)(Grinnell et al. 2006). In nested collagen matri-ces, fibroblasts translocate from the inner ma-trix into the outer matrix (Figure 5b). Humanfibroblast migration in nested collagen matricesdepends not only on Rho/Rho kinase/myosin IIbut also on metalloproteinase activity. Time-lapse video microscopy of cell migration showsthat in addition to cells migrating out of theinner matrix, collagen fibrils in the outer ma-trix translocate toward the interface betweenthe outer and inner matrices (SupplementalVideo 1; follow the Supplemental Materiallink from the Annual Reviews home page athttp://www.annualreviews.org). By placingfluorescent beads in the other matrix, it be-comes possible to visualize more easily the bal-ance between cell migration and matrix translo-cation (Figure 5c). The global environment ofnested matrices profoundly influences this bal-ance. Cell migration occurs preferentially inrestrained nested matrices where cell tensioncan be propagated through the matrix to theunderlying restraining surface. In floatingnested matrices, in the absence of a fixed bound-ary against which to pull, matrix translocationpredominates (Miron-Mendoza et al. 2008).

Figure 6 offers a conceptual framework forunderstanding the relationship between cellmigration and collagen translocation. If thecollagen matrix resists the cellular tractionalforce, then the cells move. If the matrix

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S1P: sphingosine-1-phosphate

FBS: fetal bovineserum

cannot resist the cellular tractional force,then the matrix moves. As a result, cell-induced collagen translocation can providea potential mechanism for large-scale tis-sue translocation, such as occurs in paired,floating nested matrices (SupplementalFigure 1; follow the Supplemental Materiallink from the Annual Reviews home page athttp://www.annualreviews.org). Althoughtension lines form between paired matricesand become tracks for cell migration, collagentranslocation predominates in the absenceof global restraint. Over a 48 h period, suf-ficient translocation occurs to close a 1 mmstarting gap between the dermal equivalents(Miron-Mendoza et al. 2008).

Human fibroblast migration in nested colla-gen matrices, floating matrix contraction, andstress-relaxation exhibit their own unique pat-tern of growth factor stimulation. PDGF isby far the best agonist for human fibroblastmigration in nested collagen matrices. How-ever, serum, LPA, sphingosine-1-phosphate(S1P), and endothelin-1 (Endo-1)—all Rhoactivators—are the best agonists for stress-relaxation. Because stress-relaxation occurs bycell contraction, and the Rho-activating growthfactors also cause retraction of fibroblast den-dritic extensions, the presence of these growthfactors establishes a procontractile environ-ment for the cells. Serum is procontractile forhuman fibroblasts but, after removal of S1Pand other lipid growth factors, becomes promi-gratory and stimulates cell migration in nestedcollagen matrices almost as well as PDGF. Incontrast to the specificity of cell migration andstress-relaxation, floating collagen matrix con-traction can be stimulated by both promigra-tory and procontractile growth factors ( Jianget al. 2008).

Changing the growth factor environmentcan have profound effects on morphogeneticcell patterns. For instance, when human fibro-blasts are cultured on the surfaces of collagenmatrices, they contract into clusters in the pres-ence of serum or LPA but migrate as individualsin the presence of PDGF or serum from whichlipid growth factors have been removed [fetal

a If the matrix resists cell tractionalforce, then the cell moves.

b If the matrix does not resist celltractional force, then the matrix moves.

Tractionalforce

Tractionalforce

Figure 6Conceptual framework for understanding the relationship between cellmigration and collagen translocation. If the collagen matrix resists the cellulartractional force, then the cells move. If the matrix cannot resist the cellulartractional force, then the matrix moves. Adapted from Miron-Mendoza et al.(2008).

bovine serum (FBS) + charcoal] (Supplemen-tal Figure 2; follow the Supplemental Mate-rial link from the Annual Reviews home pageat http://www.annualreviews.org). Cluster-ing and individual cell migration patterns arecompletely reversible by switching growth fac-tor conditions (Rhee et al. 2009). In the caseof Ras-transformed human fibroblasts, whosemigration in 3D matrices is constitutively acti-vated without adding growth factors (Menezeset al. 2008), cell clustering in serum or LPA-containing medium does not occur.

In the case of corneal keratocytes (rest-ing fibroblasts of the cornea), both PDGFand serum stimulate cell migration, but bydifferent mechanisms. During migration inthe presence of serum, keratocytes assume abipolar morphology and produce more local

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a 10% FBS

b PDGF 50 ng ml–1

40 μm

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collagen compaction as compared to migrationin the presence of PDGF (Figure 7, Kim et al.2009). Moreover, PDGF-stimulated migrationoccurs by repeated protrusion and retractionof dendritic processes with small tractionalforces localized to the tips (SupplementalVideo 2; follow the Supplemental Materiallink from the Annual Reviews home page athttp://www.annualreviews.org). By contrast,serum-stimulated migration results in largerand more sustained tractional forces at theleading edge and more pronounced ruptureof cell-matrix adhesions at the rear, whichresults in elastic tail recoil and release ofmatrix tension (Supplemental Video 3;follow the Supplemental Material linkfrom the Annual Reviews home page athttp://www.annualreviews.org).

The conceptual framework in Figure 8summarizes the idea that the growth factorenvironment of serum or tissue will contain amixture of promigratory and procontractilegrowth factors and antagonists. As demon-strated above, the same set of growth factorsmay have different promigratory and procon-tractile effects depending on cell type. For in-stance, serum lysophospholipids LPA and S1Pinteract with a family of G-protein coupled

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 7High-resolution imaging of collagen fibrilreorganization induced by migrating cornealkeratocytes using a nested collagen matrix model.Color overlays of maximum intensity projectionsof confocal fluorescent (green) and reflection(red) images showing f-actin and collagen fibrils,respectively. Nested collagen matrices were preparedwith compressed inner collagen matrices rather thandermal equivalents. (a) Cells migrating in 10% fetalbovine serum (FBS) medium developed bipolar mor-phology with occasional stress fibers along the cellbody. Collagen fibrils were compacted and alignedparallel to the long axes of pseudopodia. (b) Cellsmigrating in platelet-derived growth factor (PDGF)exhibited more dendritic morphology. Collagenfibrils remained more randomly aligned around thesecells compared to cells migrating in FBS. Adaptedfrom Kim et al. (2009), copyright c© Associationfor Research in Vision and Ophthalmology.

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receptors that various cell types express dif-ferentially. Depending on receptor expression,S1P potentially can activate cell contraction orcell migration signaling pathways. The S1P1 re-ceptor links to Rac activation and cell migra-tion, whereas the S1P2 receptor links to Rhoactivation, Rac inhibition, and inhibition of cellmigration (Sugimoto et al. 2003).

Ironically, most research on mechanismsof cell migration have been carried out inserum-containing medium. For many celltypes, serum will establish a procontractilerather than promigratory environment, i.e.,Rho rather than Rac activation (Ridley &Hall 1992). Because the presence of a pro-contractile or promigratory environment canalter the balance between the cellular forcesof protrusion, contraction, and adhesion, thegrowth factor environment has the potential toinfluence basic mechanisms and patterns of cellmotility, especially in the tissue-like 3D matrixenvironment, where cell tractional force andmatrix stiffness are closely balanced. IncreasingRho activation will result in an increase incell membrane tension and tensegrity forces(Ingber 1997, Sheetz & Dai 1996) tendingto impede cell spreading in 3D matrices(Grinnell 2003, Rhee et al. 2010). Conversely,increasing Rac activation stimulates formationof dendritic extensions (Grinnell 2003) andincreases random fibroblast migration in 3Dmatrices (Pankov et al. 2005). Cell durotaxis,which has been demonstrated for cells mi-grating in serum-containing media on 2Dsurfaces (Lo et al. 2000) and inside 3D matrices(Hadjipanayi et al. 2009), may turn out to beunique to the procontractile growth factorenvironment.

HIGH-RESOLUTION IMAGINGOF LOCAL MATRIXREMODELING

Imaging collagen matrices that contain micro-spheres has been useful for learning about thelocal and global movements of collagen in re-sponse to cell motile activity (Miron-Mendozaet al. 2008, Roy et al. 1997, Tamariz & Grinnell

S1P PDGF LPA

Extensionand migration

Retractionand contraction

Serum/tissue growth factor environment

Figure 8Conceptual framework for understanding promigratory versus procontractilegrowth factor environments. The growth factor environment of serum or tissuewill contain a mixture of promigratory and procontractile growth factors andantagonists. The same set of growth factors may have different promigratoryand procontractile effects depending on cell type; effects on dermal fibroblastsare depicted here. LPA, lysophosphatidic acid; PDGF, platelet-derived growthfactor; S1P, sphingosine-1-phosphate. Adapted from Jiang et al. (2008).

2002), but such experiments do not providedetailed information on the structural reorga-nization of the matrix. Advances in imagingtechnology combined with live cell labelingtechniques increasingly has made such studiespossible. The three most commonly usedmethods are differential interference contrast(DIC), confocal reflection microscopy, andconfocal microscopy combined with secondharmonic generation imaging.

Bard and Hay introduced DIC imagingto visualize corneal embryonic fibroblastmigration in situ and in collagen matrices(Bard & Hay 1975). This approach alloweddirect visualization of the local collagen fibrilorganization surrounding the cells. Improvedoptics and digital imaging now allow theseprocesses to be studied with high temporaland spatial resolution (Supplemental Video4; follow the Supplemental Material linkfrom the Annual Reviews home page athttp://www.annualreviews.org) (Petroll &Ma 2003). Tracking landmarks on collagenfibrils in the DIC images provides an accuratemeasure of matrix deformation without theneed for embedded microspheres (Vanni et al.2003).

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DIC analysis of green fluorescent protein(GFP)-zyxin expressing fibroblasts spread-ing on or within collagen matrices showsthat new focal adhesions form at the tips ofpseudopodia while existing adhesions movebackward. Simultaneously, adhesions at thebase of pseudopodia move toward those at thetip, resulting in a region of contractile-likeshortening and matrix compression (Sup-plemental Figure 3, Supplemental Video5; follow the Supplemental Material linkfrom the Annual Reviews home page athttp://www.annualreviews.org) (Petroll &Ma 2003). These findings show that pseu-dopodia have distinct mechanical zones atthe leading edge and base (Petroll & Ma2003, Vanni et al. 2003). Similar observationsregarding different mechanical zones havebeen made using traction force microscopyon cells on 2D surfaces (Munevar et al. 2001).However, whereas groups of adjacent focaladhesions form within a cell lamellipodium on2D surfaces, in 3D matrices focal adhesionstend to form and treadmill in a linear patternalong individual collagen fibrils. The alignmentbetween pseudopodial focal adhesions andcollagen fibrils helps account for the contactguidance of cell migration along matrix fibrilsoriginally observed by Weiss (1959) and subse-quently confirmed by many other laboratories.

Taking advantage of refractive index differ-ences within the matrix, Friedl et al. introducedconfocal reflection imaging to generate high-contrast optical section images of matrixfibrils (Friedl et al. 1998). Time-lapse confocalreflection imaging has been particularly usefulin permitting direct observation of the sub-cellular mechanics underlying amoeboid andmesenchymal modes of migration through 3Dmatrices (Wolf et al. 2003). Using this method,the organization of collagen matrix remodelingaround single cells and between adjacent cellscan be visualized at high resolution (Supple-mental Figure 4; follow the SupplementalMaterial link from the Annual Reviews homepage at http://www.annualreviews.org) (Kimet al. 2006, Petroll et al. 2004a). An alternativeto confocal reflection microscopy that permits

both deeper penetration and higher-resolutionimaging of the matrix is second harmonic gen-eration imaging using two-photon microscopy(Friedl 2004, Yeh et al. 2004). With both ofthese techniques, signal intensity depends onthe orientation of the collagen fibrils beingimaged ( Jawerth et al. 2009).

Confocal imaging provides more detailedinformation about 3D collagen matrix reorga-nization than does DIC imaging. Nevertheless,longer acquisition times and potential photo-toxicity can limit the usefulness of this technol-ogy for time-lapse studies of very rapid eventsin living cells. Thus, wide-field fluorescent andDIC imaging are generally used to study rapidchanges in protein organization and matrix de-formation that occur within minutes, whereasconfocal and multiphoton imaging are used toassess more gradual changes in cell-inducedcompaction and alignment of collagen fibrils.

REGULATION OF LOCALMATRIX REMODELING BYSMALL G PROTEINS ANDMECHANICAL STIMULI

As described in previous sections, the Rho-family GTPases play a central role in the regula-tion of cell motile activity in collagen matrices.For instance, Rho activation causes dendriticcell extensions to retract; Rac activation causesthe extensions to protrude further (Figure 4).High-resolution imaging has been used tostudy the effects of Rho and Rac activation oncell motile activity in relationship to collagenremodeling. When corneal fibroblasts on topof or within restrained 3D collagen matrices arestimulated with LPA or serum, local collagencompression accompanies retraction of cellpseudopodia (Petroll et al. 2008b). Conversely,inhibiting Rho kinase results in extension of cellpseudopodia and reversible relaxation of cell-induced tension on the matrix (Figure 9, Sup-plemental Video 6; follow the SupplementalMaterial link from the Annual Reviews homepage at http://www.annualreviews.org)(Vishwanath et al. 2003).

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In contrast to pseudopodial extension causedby blocking Rho kinase, inward tractionalforces are generated as cell pseudopodia ex-tend outward following treatment of cornealfibroblasts in restrained collagen matrices withPDGF (Supplemental Figure 5, Supplemen-tal Video 7; follow the Supplemental Mate-rial link from the Annual Reviews home page athttp://www.annualreviews.org) (Petroll et al.2008a). Subsequent inhibition of Rho kinasestimulates additional spreading and a relaxationof tractional forces. However, even in the pres-ence of Rho kinase inhibition, small inward dis-placements of collagen fibrils at the tips of thecell processes are still observed, which mightprovide a mechanism for cell traction in low-tension environments in which ROCK/myosinII–based contractility is significantly reduced(Figure 3).

Changes in small G proteins can cause rapidvariation in the cellular force exerted on the ma-trix. Changes in matrix organization can causerapid cellular mechanical responses. This pat-tern of bidirectional force modulation—cells tomatrix and matrix to cells—is the basis for ten-sional homeostasis (Brown et al. 1998, Delvoyeet al. 1991, Paszek et al. 2005, Petroll et al.2004b). The impact of transient changes in ex-tracellular matrix tension has been investigatedat the subcellular level by using micropipettesto locally deform the matrix. Reducing effec-tive matrix stiffness by pushing toward the frontof a cell results in rapid cellular shorteningwith corresponding matrix compression alongthe cell body, similar to the shortening pro-duced by releasing one end of a rubber bandthat is under tension (Figure 10, Supplemen-tal Video 8; follow the Supplemental Mate-rial link from the Annual Reviews home pageat http://www.annualreviews.org) (Petroll &Ma 2008). Because shortening is blocked byRho kinase inhibitors, it likely results frommyosin II-based contractile forces Followingthis initial contraction, reprotrusion of pseu-dopodia is observed as fibroblasts attempt to re-establish the original cell-matrix tension state.During this secondary spreading, collagen fib-rils move inward, and there is rapid turnover

a S+ (133 min)

15 μm

b Y-27632 (44 min)

c S+ (69 min)

15 μm

15 μm

Figure 9Reversible effect of inhibiting Rho kinase on corneal fibroblast pseudopodia.High-resolution images show overlays of a green fluorescent protein(GFP)-α-actinin expressing cell ( green) and collagen displacements (red tracks)measured by DIC at different time points. (a) Corneal fibroblasts in restrainedcollagen matrices in serum-containing medium. (b) Forty-four minutes afteradding Rho kinase inhibitor Y-27632, relaxation (additional spreading) of thecell (large arrow) and surrounding collagen matrix was observed (cross marksindicate position just prior to adding Y-27632). GFP-α-actinin was localized tothe tips of the extensions (small arrows). (c) Sixty-nine minutes after reperfusionwith serum-containing medium, cell retraction (large arrow) and recompressionof the matrix (red tracks; cross marks indicate position at the start ofreperfusion) occurred. Adapted from Vishwanath et al. (2003), copyright c©Association for Research in Vision and Ophthalmology.

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c 28 min after needle push

b 1 min after needle push

a Before needle push

15 μm

15 μm

15 μm

Figure 10Corneal fibroblast response to large changes in local matrix tension.(a–c) Differential interference contrast (DIC) images of a corneal fibroblast in arestrained collagen matrix. (a) Cell appearance before insertion of amicroneedle. (b) Cellular contraction (arrows) following a 25-μm needle pushtoward the cell. (c) Subsequent reprotrusion of retracted dendritic extensions(arrows) and accompanying inward collagen movement (red tracks; cross marksindicate start positions at 1 min after needle push). Adapted from Petroll et al.(2004b), copyright c© Association for Research in Vision and Ophthalmology.

of focal adhesions along extending pseudopo-dia. Because matrix tension has been reduced,the ratio of collagen translocation to pseu-dopodial reprotrusion is greater than that ob-served during protrusion of pseudopodia in re-sponse to PDGF stimulation (SupplementalFigure 5; follow the Supplemental Mate-rial link from the Annual Reviews home pageat http://www.annualreviews.org), consistentwith the conceptual framework described inFigure 6. Overall, dynamic feedback betweencellular forces and local ECM stress plays a cen-tral role in regulating fibroblast mechanical be-havior within 3-D matrices.

WOUND REPAIR ANDMATRIX CONTRACTION

Tensional homeostasis applies not only to theiterative relationship between cell and matrixmechanics but also to the iterative relationshipbetween cell physiology and matrix mechanics.As Bell et al. suggested, research on collagenmatrix contraction has turned out to be usefulfor studying wound contraction and repair (Bellet al. 1979). Wound contraction refers to theclosure of full thickness dermal wounds, whichoccurs by large-scale movement of dermal con-nective tissue (Billingham & Russell 1956).Two different mechanisms of wound contrac-tion have been proposed. One centered the cel-lular force for contraction within the newlysynthesized and highly vascularized wound fi-brous connective tissue, i.e., granulation tissue(Abercrombie et al. 1960). Gabbiani et al.showed that isolated granulation tissue itselfwas contractile and contained smooth muscle-like fibroblasts, designated myofibroblasts, thatwere thought to be the cells whose contractionprovided the force (Gabbiani et al. 1972). Theother mechanism localized the force for woundcontraction to cell migration at the wound mar-gins because continuously removing granula-tion tissue from the wound bed during repairdid not decrease the rate of wound closure,nor was wound closure slower in vitamin C-deficient animals lacking normal collagen and

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granulation tissue synthesis (reviewed in Gross1996).

The discovery of α-smooth muscle actinexpression as a biochemical marker for my-ofibroblast differentiation made it possible tocharacterize more precisely the appearanceand function of these cells (Skalli et al. 1986).Myofibroblasts sometimes appear late ingranulation tissue relative to wound closure(Berry et al. 1998, Doillon et al. 1987, Nedelecet al. 2000), and they are not required forfetal wounds to close (Martin 2007). Also, inthe tenascin-C knockout mouse repair occursnormally even though appearance of myofi-broblasts is delayed (Tamaoki et al. 2005).Therefore, at least under some circumstances,wound fibroblasts are sufficient for woundcontraction and closure. However, in mechan-ically restrained wounds that exhibit delayedclosure, the myofibroblast population increasesmarkedly (Hinz et al. 2001, Squier 1981).

Figure 11 shows the interrelationshipsbetween the above findings and collagenmatrix contraction models. At low mechanicalresistance (floating collagen matrices), cellmigration can provide the force for large-scaletissue movement, as observed with pairednested collagen matrices (SupplementalFigure 1; follow the Supplemental Materiallink from the Annual Reviews home page athttp://www.annualreviews.org). As mechan-ical resistance increases (restrained collagenmatrices), a high cell-matrix tension statedevelops and cell contraction becomes themechanism for force generation (Figure 3).In addition to mechanical resistance, thechange from migration to contraction requiresswitching from promigratory to procontractilegrowth factor environments (Figures 3 and8). If differentiation signals such as TGFβ

are present in the growth factor environment,then fibroblasts in restrained collagen matriceswill differentiate into myofibroblasts and exertgreater contractile force (Hinz 2009, Tomaseket al. 2002). The contractile force exerted byfibroblasts and myofibroblasts in combinationwith matrix biosynthesis ultimately resultsin wound closure and dissipation of tissue

Fibroblast

Floating

Restra

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x

Cell type causing wound contraction

Mec

hani

sm o

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Cont

ract

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Myofibroblast

Figure 11Conceptual framework relating collagen matrixcontraction and wound contraction. At lowmechanical resistance (floating collagen matrices),cell migration can provide the force for large-scaletissue movement. As mechanical resistance increases(restrained collagen matrices), cell contractionbecomes the mechanism for force generation. Ifdifferentiation signals are present in the growthfactor environment, then fibroblasts experiencinghigh mechanical resistance differentiate intomyofibroblasts. Adapted from Grinnell (2003).

tension (stress-relaxation). Continued activityof myofibroblasts after wound closure occursunder pathological wound conditions such ascontracture and hypertrophic scar (Abrahamet al. 2007, Tomasek et al. 2002, Wynn 2008).

Myofibroblasts appear in tissue in contextsother than wound repair. For example, thesecells play a central role in fibrotic disease(Abraham et al. 2007, Desmouliere et al. 2003,Wynn 2008) and tumor progression (Butcheret al. 2009, Desmouliere et al. 2004, Weigelt& Bissell 2008). Most evidence suggeststhat fibroblasts differentiate into myofibro-blasts only when they are stimulated withappropriate growth factors when in a highcell-matrix tension state (e.g., in restrainedbut not floating collagen matrices) (Hinz2009, Tomasek et al. 2002). Interstitial fluidflow through collagen matrices containingfibroblasts also provides a myofibroblast-activating signal by increasing matrix tension

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FAK: focal adhesionkinase

PI3 kinase:phosphatidylinositol3-kinase

and autocrine production of TGFβ (Ng et al.2005). However, the ability of endothelin-1 topromote differentiation of fibroblasts into my-ofibroblasts in floating collagen matrices showsthat with some growth factors, high cell-matrixtension may not be required for myofibroblastdifferentiation (Shi-Wen et al. 2004).

Studies on fibroblast proliferation andbiosynthesis in collagen matrices have con-tributed to the mechanoregulated model ofwound repair. Figure 12 summarizes thesefindings. In floating collagen matrices, fibro-blasts are turned off and exhibit little ma-trix biosynthesis or proliferation. In restrainedmatrices, cells are turned on. They prolifer-ate and synthesize and accumulate new ex-tracellular matrix (Eckes et al. 2006, Grinnell2003). After stress-relaxation, cells rapidly be-come quiescent as a result of disrupted signal-ing to cell cycle regulatory proteins (Fringer &Grinnell 2001). The 3D matrix environmentuniquely regulates focal adhesion kinase activa-tion (phospho FAK397) (Cukierman et al. 2001,Wei et al. 2008), and decreased FAK activa-tion probably accounts for cell quiescence infloating collagen matrices (Klein et al. 2009).Also, a subpopulation of fibroblasts in floating

Wounding

Resting fibroblastCell proliferation OFF

Matrix biosynthesis OFF

(Floating collagenmatrix)

Activated fibroblastCell proliferation ON

Matrix biosynthesis ON

(Restrained collagenmatrix)

Low cell-matrix tension High cell-matrix tension

Cell proliferation and biosynthesisCell migration and contraction

(Stress-relaxation)

Figure 12Mechanoregulated model of wound contraction. Inresting tissue, fibroblasts are turned off and exhibitlittle matrix biosynthesis or proliferation. Ingranulation tissue, fibroblasts are activated. Overtime, the combination of cell proliferation,biosynthesis, migration, and contraction returns thetissue from an activated to resting state. Adaptedfrom Grinnell (2003).

collagen matrices becomes apoptotic (Grinnellet al. 1999). Apoptosis results from decreasedphosphatidylinositol 3-kinase (PI3 kinase) sig-naling at least in part as a result of phosphataseand tensin homolog (PTEN) activation (Nhoet al. 2006). Increased expression of cell sur-face tissue transglutaminase may protect hyper-trophic scar cells from undergoing apoptosis infloating collagen matrices (Linge et al. 2005).

In relationship to wound repair, floatingcollagen matrices resemble normal tissue, andrestrained collagen matrices resemble granula-tion tissue. Fibroblasts in normal tissue are in alow cell-matrix tension state—the fibrocytes ofclassical histology. When these cells develophigh cell-matrix tension as part of tissue homeo-stasis or in response to mechanical wounding,they are activated to proliferate and synthesizenew connective tissue matrix. After dissipationof the high cell-matrix tension state throughthe combination of matrix biosynthesis andcontraction, fibroblasts turn off and granula-tion tissue regresses. External pressure therapy(Costa et al. 1999) or closing experimentalwounds by skin flapping releases internaltension and causes granulation tissue to regressrapidly through cell quiescence and apoptosis(Carlson et al. 2002, Darby et al. 2002). Increas-ing tissue tension by external skin splinting(Hinz et al. 2001) or injection of dermal fillers(Wang et al. 2007) increases matrix biosynthe-sis. In addition, increasing tissue tension resultsin decreased myofibroblast apoptosis and for-mation of hypertropic scars (Aarabi et al. 2007).Finally, mice with PTEN-null fibroblasts intheir mammary glands show increased matrixremodeling and formation of epithelial tumors(Trimboli et al. 2009), perhaps because of thefailure of myofibroblasts to undergo apoptosis.

CONCLUSIONS ANDFUTURE DIRECTIONS

Almost one hundred years have passed sincepublication of the Lewis and Lewis article dis-cussed at the beginning of this review. Sincethen, research on cells in 2D culture has pro-vided remarkable insights into cell structure

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and function. Weiss’s hierarchical approach ofstudying cells in 3D matrices lagged behind butnow is beginning to catch up. Advances in imag-ing technology combined with live and fixed celllabeling techniques increasingly makes possiblehigh-resolution analysis of cell-matrix interac-tions at the global and subcellular levels.

In this review we focus on fibroblasts inter-acting with 3D collagen matrices, which exhibitmechanical and structural features that resem-ble fibrous connective tissue. Fibroblasts are thecell type primarily responsible for fibrous con-nective tissue biosynthesis and organization. Fi-broblast tractional force and matrix stiffness areclosely balanced in collagen matrices. Conse-quently, cells show a high degree of plasticityincluding the ability to develop dendritic mor-phology. Fibroblasts in 3D collagen matricesare capable of rapidly protruding and retract-ing cell extensions in response to growth fac-tor stimulation or physical manipulation of thematrix. The small G proteins Rac and Rho areparticularly important regulators of protrusionand retraction. Movement of cell extensions cancause inward (toward the cell body) movementof collagen fibrils not only as the extensions re-tract but also as they protrude.

Cell tractional force can remodel andcompact matrix fibrils into stably reorganizedstructures. Similar to cell adhesion, spreading,and migration, collagen matrix contraction hasturned out to be a highly conserved featureof cell motile behavior exhibited not only byfibroblasts but also by many other cell types.Global factors such as whether the matrix isfloating or restrained as well as local factors suchas matrix stiffness affect the cell-matrix tensionstate and influence cell signaling pathways,cytoskeletal function, and motor mechanismsrequired for contraction. Cells interactingwith collagen matrices form an interactivemechanical system. Tensional homeostasisdescribes this mechanical reciprocity.

The porosity of the matrix makes it pos-sible for cells to penetrate and move throughthe matrix and not just over its surface. Thegeometry of the matrix establishes limited do-mains of cell adhesion along matrix fibrils.

Pseudopodial adhesions in 3D matrices tend toform and treadmill in a linear pattern along in-dividual collagen fibrils, which helps accountfor contact guidance. However, in a matrix lack-ing oriented fibrils, fibroblasts can interact withfibrils oriented both parallel and perpendicularto the direction of cell pseudopodia. At a globallevel, cell migration and collagen translocationcan couple together to produce large-scale tis-sue movement.

We also emphasize the importance of dis-tinguishing promigratory and procontractilegrowth factor environments, which can alterthe balance between the cellular forces of pro-trusion, contraction, and adhesion. These dif-ferences will influence basic mechanisms andpatterns of cell motility, especially in the tissue-like 3D matrix environment, where cell trac-tional force and matrix stiffness are closelybalanced.

Finally, we discuss the relationship betweencollagen matrix contraction models and woundrepair. We suggest that the relative importanceof fibroblasts and myofibroblasts for woundcontraction depends on tissue resistance. In themechanoregulated model of wound repair, cellphysiology links with cell-matrix tension state.

What about the future? For tissue cells on2D surfaces, cell motile activity has been de-scribed in terms of a balance between cellularforces of protrusion, contraction, and adhesion.To successfully model cell mechanical behav-ior in 3D matrices, a new generation of engi-neered biocompatible matrices will be requiredin which matrix porosity, stiffness, and adhesionligand domains are defined and can be varied in-dependently so that their individual effects oncell behavior can be determined.

New types of engineered matrices also areneeded to permit more detailed investigationsof the influence of matrix fibril organizationon cell motility and mechanics. Most currentmethods for preparing 3D fibrillar matricesresult in matrices in which fibrils lack anyparticular organization or become aligned uni-axially. However, tissues exhibit more complexarchitectures such as collagen fibril bundlesand orthogonally arranged collagen sheets.

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Incorporation of these features into 3D matrixmodels could provide unique insights intocell-matrix interactions and might help clarifythe role of proteoglycans and other matrixcomponents in modulating cell behavior in thetissue environment.

Much remains to be learned about thelow cell-matrix tension environment. Relativelylittle is known about the dendritic fibroblast.Fibroblasts and other cell types appear capableof generating myosin II–independent tractionalforces in the low cell-matrix tension environ-

ment. Are other myosins responsible, or does adifferent type of molecular motor produce theseforces?

Lastly, development of new probes andimaging techniques to allow direct assessmentof mechanical signaling events in living cellsshould continue to improve our understandingof how cell motility in 3D matrices is regulated.Application of multiphoton imaging techniquesand novel experimental animal models will in-creasingly allow many of these processes to bestudied in vivo.

DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings thatmight be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

Research in our laboratories has been supported by grants from the National Institutes of Health,GM031321 to F.G. and EY013322 to W.M.P., and from Research to Prevent Blindness, Inc. toW.M.P.

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Annual Reviewof Cell andDevelopmentalBiology

Volume 26, 2010

ContentsEnzymes, Embryos, and Ancestors

John Gerhart � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

Control of Mitotic Spindle LengthGohta Goshima and Jonathan M. Scholey � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �21

Trafficking to the Ciliary Membrane: How to Get Acrossthe Periciliary Diffusion Barrier?Maxence V. Nachury, E. Scott Seeley, and Hua Jin � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �59

Transmembrane Signaling ProteoglycansJohn R. Couchman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �89

Membrane Fusion: Five Lipids, Four SNAREs, Three Chaperones,Two Nucleotides, and a Rab, All Dancing in a Ring on Yeast VacuolesWilliam Wickner � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 115

Tethering Factors as Organizers of Intracellular Vesicular TrafficI-Mei Yu and Frederick M. Hughson � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 137

The Diverse Functions of Oxysterol-Binding ProteinsSumana Raychaudhuri and William A. Prinz � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 157

Ubiquitination in Postsynaptic Function and PlasticityAngela M. Mabb and Michael D. Ehlers � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 179

α-Synuclein: Membrane Interactions and Toxicityin Parkinson’s DiseasePavan K. Auluck, Gabriela Caraveo, and Susan Lindquist � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 211

Novel Research Horizons for Presenilins and γ-Secretases in CellBiology and DiseaseBart De Strooper and Wim Annaert � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 235

Modulation of Host Cell Function by Legionella pneumophilaType IV EffectorsAndree Hubber and Craig R. Roy � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 261

A New Wave of Cellular ImagingDerek Toomre and Joerg Bewersdorf � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 285

Mechanical Integration of Actin and Adhesion Dynamicsin Cell MigrationMargaret L. Gardel, Ian C. Schneider, Yvonne Aratyn-Schaus,

and Clare M. Waterman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 315

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AR425-FM ARI 14 September 2010 18:54

Cell Motility and Mechanics in Three-Dimensional Collagen MatricesFrederick Grinnell and W. Matthew Petroll � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 335

Rolling Cell AdhesionRodger P. McEver and Cheng Zhu � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 363

Assembly of Fibronectin Extracellular MatrixPurva Singh, Cara Carraher, and Jean E. Schwarzbauer � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 397

Interactions Between Nuclei and the Cytoskeleton Are Mediatedby SUN-KASH Nuclear-Envelope BridgesDaniel A. Starr and Heidi N. Fridolfsson � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 421

Plant Nuclear Hormone Receptors: A Role for Small Moleculesin Protein-Protein InteractionsShelley Lumba, Sean R. Cutler, and Peter McCourt � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 445

Mammalian Su(var) Genes in Chromatin ControlBarna D. Fodor, Nicholas Shukeir, Gunter Reuter, and Thomas Jenuwein � � � � � � � � � � � � � � 471

Chromatin Regulatory Mechanisms in PluripotencyJulie A. Lessard and Gerald R. Crabtree � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 503

Presentation Counts: Microenvironmental Regulation of Stem Cells byBiophysical and Material CuesAlbert J. Keung, Sanjay Kumar, and David V. Schaffer � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 533

Paramutation and DevelopmentJay B. Hollick � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 557

Assembling Neural Crest Regulatory Circuits intoa Gene Regulatory NetworkPaola Betancur, Marianne Bronner-Fraser, and Tatjana Sauka-Spengler � � � � � � � � � � � � � � � 581

Regulatory Mechanisms for Specification and Patterning of PlantVascular TissuesAna Cano-Delgado, Ji-Young Lee, and Taku Demura � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 605

Common Factors Regulating Patterning of the Nervousand Vascular SystemsMariana Melani and Brant M. Weinstein � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 639

Stem Cell Models of Cardiac Development and DiseaseKiran Musunuru, Ibrahim J. Domian, and Kenneth R. Chien � � � � � � � � � � � � � � � � � � � � � � � � � � 667

Stochastic Mechanisms of Cell Fate Specification that Yield Randomor Robust OutcomesRobert J. Johnston, Jr. and Claude Desplan � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 689

A Decade of Systems BiologyHan-Yu Chuang, Matan Hofree, and Trey Ideker � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 721

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Editor: Stephen E. Fienberg, Carnegie Mellon UniversityAssociate Editors: Nancy Reid, University of Toronto

Stephen M. Stigler, University of ChicagoThe Annual Review of Statistics and Its Application aims to inform statisticians and quantitative methodologists, as well as all scientists and users of statistics about major methodological advances and the computational tools that allow for their implementation. It will include developments in the field of statistics, including theoretical statistical underpinnings of new methodology, as well as developments in specific application domains such as biostatistics and bioinformatics, economics, machine learning, psychology, sociology, and aspects of the physical sciences.

Complimentary online access to the first volume will be available until January 2015. table of contents:•What Is Statistics? Stephen E. Fienberg•A Systematic Statistical Approach to Evaluating Evidence

from Observational Studies, David Madigan, Paul E. Stang, Jesse A. Berlin, Martijn Schuemie, J. Marc Overhage, Marc A. Suchard, Bill Dumouchel, Abraham G. Hartzema, Patrick B. Ryan

•The Role of Statistics in the Discovery of a Higgs Boson, David A. van Dyk

•Brain Imaging Analysis, F. DuBois Bowman•Statistics and Climate, Peter Guttorp•Climate Simulators and Climate Projections,

Jonathan Rougier, Michael Goldstein•Probabilistic Forecasting, Tilmann Gneiting,

Matthias Katzfuss•Bayesian Computational Tools, Christian P. Robert•Bayesian Computation Via Markov Chain Monte Carlo,

Radu V. Craiu, Jeffrey S. Rosenthal•Build, Compute, Critique, Repeat: Data Analysis with Latent

Variable Models, David M. Blei•Structured Regularizers for High-Dimensional Problems:

Statistical and Computational Issues, Martin J. Wainwright

•High-Dimensional Statistics with a View Toward Applications in Biology, Peter Bühlmann, Markus Kalisch, Lukas Meier

•Next-Generation Statistical Genetics: Modeling, Penalization, and Optimization in High-Dimensional Data, Kenneth Lange, Jeanette C. Papp, Janet S. Sinsheimer, Eric M. Sobel

•Breaking Bad: Two Decades of Life-Course Data Analysis in Criminology, Developmental Psychology, and Beyond, Elena A. Erosheva, Ross L. Matsueda, Donatello Telesca

•Event History Analysis, Niels Keiding•StatisticalEvaluationofForensicDNAProfileEvidence,

Christopher D. Steele, David J. Balding•Using League Table Rankings in Public Policy Formation:

Statistical Issues, Harvey Goldstein•Statistical Ecology, Ruth King•Estimating the Number of Species in Microbial Diversity

Studies, John Bunge, Amy Willis, Fiona Walsh•Dynamic Treatment Regimes, Bibhas Chakraborty,

Susan A. Murphy•Statistics and Related Topics in Single-Molecule Biophysics,

Hong Qian, S.C. Kou•Statistics and Quantitative Risk Management for Banking

and Insurance, Paul Embrechts, Marius Hofert

Access this and all other Annual Reviews journals via your institution at www.annualreviews.org.

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