Analysis of actin dynamics at the leading edge of crawling cells: implications for the shape of keratocyte lamellipodia

Grimm, H P; Verkhovsky, Alexander B.; Mogilner, Alex; Meister, J.‐J.

doi:10.1007/s00249-003-0300-4

Cited by 100 publications

(118 citation statements)

References 36 publications

Supporting

Mentioning

112

Contrasting

Order By: Relevance

“…To maintain their ellipsoidal shape while progressing on surfaces, keratocytes need to protrude more on their main symmetry axis than on their sides (Lee et al, 1993b). This has been generally attributed to the difference in actin polymerization rates (Mogilner and EdelsteinKeshet, 2002;Grimm et al, 2003); however, our finding that keratocyte lamellipodia display a retrograde flow even in their center requires this issue to be reconsidered. We analyzed the relationship between retrograde flow and protrusion rate along the leading edge and estimated the actual polymerization rate as the sum of the two.…”

Section: Flow Dynamics and The Protrusion Ratementioning

confidence: 88%

“…During locomotion, the keratocyte lamellipodium maintains a crescent shape, which was explained by graded radial extension, i.e., a monotonically decreasing protrusion rate from the cell center toward the sides (Lee et al, 1993b). Given the apparent absence of a retrograde flow, efforts toward modeling keratocyte dynamics have relied on the assumption that graded protrusion is entirely regulated by factors controlling actin polymerization (Mogilner and Edelstein-Keshet, 2002;Grimm et al, 2003).In this article, we revisit the question of the existence of retrograde flow and challenge the polymerization-centered models. We exploit fluorescent speckle microscopy (FSM) to probe actin network flow with hundreds of fluorescent marks, producing sensitive and detailed maps of the flow pattern .…”

mentioning

confidence: 99%

See 1 more Smart Citation

Tracking Retrograde Flow in Keratocytes: News from the Front

Vallotton

Danuser

Bohnet

et al. 2005

MBoC

130

111

View full text Add to dashboard Cite

Actin assembly at the leading edge of the cell is believed to drive protrusion, whereas membrane resistance and contractile forces result in retrograde flow of the assembled actin network away from the edge. Thus, cell motion and shape changes are expected to depend on the balance of actin assembly and retrograde flow. This idea, however, has been undermined by the reported absence of flow in one of the most spectacular models of cell locomotion, fish epidermal keratocytes. Here, we use enhanced phase contrast and fluorescent speckle microscopy and particle tracking to analyze the motion of the actin network in keratocyte lamellipodia. We have detected retrograde flow throughout the lamellipodium at velocities of 1-3 m/min and analyzed its organization and relation to the cell motion during both unobstructed, persistent migration and events of cell collision. Freely moving cells exhibited a graded flow velocity increasing toward the sides of the lamellipodium. In colliding cells, the velocity decreased markedly at the site of collision, with striking alteration of flow in other lamellipodium regions. Our findings support the universality of the flow phenomenon and indicate that the maintenance of keratocyte shape during locomotion depends on the regulation of both retrograde flow and actin polymerization. INTRODUCTIONThe lamellipodium of motile cells is formed of a dense network of branched actin filaments (F-actin) polymerizing in the direction of cell movement. Network assembly at the leading edge is thought to generate the forces that push the plasma membrane forward (Theriot and Mitchison, 1991;Mogilner and Oster, 1996;Carlier et al., 2003;Pollard and Borisy, 2003). In most motile cells, the network is simultaneously transported away from the leading edge in a process known as retrograde flow (Cramer, 1997). The flow may be the result of forces of membrane tension counteracting actin polymerization at the edge of the cell (Raucher and Sheetz, 2000) and/or contractile forces of myosin motors (Lin et al., 1996). The balance between forward movement of the cell and backward movement of the actin network is believed to depend on a clutch-like substrate-adhesion mechanism (Smilenov et al., 1999;Jay, 2000). When the actin network is tightly coupled to the substrate (clutch fully engaged), actin polymerization effectively pushes the plasma membrane forward, and myosin-based contraction pulls the cell body in the direction of the leading edge. Conversely, when the clutch is loose, polymerization against the membrane pushes the network backward in concert with contraction pulling the actin network away from the leading edge. Thus, a possible mechanism for the cell to control its shape and net movement is by shifting the balance between protrusion and retrograde flow, supplementing the more widespread notion of spatial variation of actin assembly being the key regulator of cell motility (Pollard et al., 2000). Indeed, several studies report a relationship between retrograde flow and cell motion: the rate of retrograde...

show abstract

Section: Flow Dynamics and The Protrusion Ratementioning

confidence: 88%

mentioning

confidence: 99%

Tracking Retrograde Flow in Keratocytes: News from the Front

Vallotton

Danuser

Bohnet

et al. 2005

MBoC

130

111

View full text Add to dashboard Cite

show abstract

“…Remarkably, even well-prepared fragments of a keratocyte, lacking nuclei and organelles, can, given a transient mechanical stimulus, initiate and sustain such 'gliding' motility (Verkhovsky et al, 1999). Although not all details of the biochemistry are known, this robustness makes the keratocyte-shape an ideal subject of modelling studies (Grimm et al, 2003;Rubinstein et al, 2005). The common aspects of actin-based protrusion suggest that the mechanisms underlying cell motility share some universal properties, and this will be of interest in our paper.…”

Section: Actin Cytoskeletonmentioning

confidence: 89%

“…These include the thermal ratchet model for polymerization-dependent force Oster, 1996a,b, 2003), filament side-branching (Carlsson, 2001(Carlsson, , 2003, and filament-turnover (Mogilner and EdelsteinKeshet, 2002;Grimm et al, 2003). An overarching recent objective has been to start combining diverse aspects of cell motility into a framework of a spatial cell.…”

Section: Discussionmentioning

confidence: 99%

Polarization and Movement of Keratocytes: A Multiscale Modelling Approach

et al. 2006

View full text Add to dashboard Cite

“…Fish keratocytes are characterized by a fast and persistent motion and a simple shape and cytoskeletal organization, which remain nearly constant during migration (Lee et al, 1993;Grimm et al, 2003). The steady-state character of keratocyte migration simplifies quantitative description of the cytoskeletal dynamics and imposes constrains on the theoretical models.…”

Section: Introductionmentioning

confidence: 99%

Comparative Maps of Motion and Assembly of Filamentous Actin and Myosin II in Migrating Cells

Schaub

Bohnet

Laurent

et al. 2007

MBoC

120

View full text Add to dashboard Cite

To understand the mechanism of cell migration, one needs to know how the parts of the motile machinery of the cell are assembled and how they move with respect to each other. Actin and myosin II are thought to be the major structural and force-generating components of this machinery (Mitchison and Cramer, 1996; Parent, 2004). The movement of myosin II along actin filaments is thought to generate contractile force contributing to cell translocation, but the relative motion of the two proteins has not been investigated. We use fluorescence speckle and conventional fluorescence microscopy, image analysis, and computer tracking techniques to generate comparative velocity and assembly maps of actin and myosin II over the entire cell in a simple model system of persistently migrating fish epidermal keratocytes. The results demonstrate contrasting polarized assembly patterns of the two components, indicate force generation at the lamellipodium-cell body transition zone, and suggest a mechanism of anisotropic network contraction via sliding of myosin II assemblies along divergent actin filaments. INTRODUCTIONCrawling cell motion involves a cycle of several distinct processes: protrusion at the front of the cell, attachment to the substratum, and forward translocation of the cell body followed or accompanied by detachment and withdrawal of the rear of the cell. Crawling motion is thought to be dependent on the actin-myosin II cytoskeletal system (Mitchison and Cramer, 1996): protrusion is thought to be driven by the assembly of actin network, which is anchored to the substratum through integrin-containing adhesions; forward translocation of the cell body and contraction of the rear are thought to depend on the interaction of the actin network with the motor protein myosin II. Actin assembly during the protrusion at the leading edge of motile cells has recently received the most attention both in experimental (Pantaloni et al., 2001;Pollard and Borisy, 2003;Ridley et al., 2003) and theoretical studies (for review, see Mogilner, 2006). In contrast, the mechanisms involved in the forward translocation of the cell body remain largely unclear: the exact layout and the mode of action of the contractile actin-myosin II machinery are controversial. Qualitative models proposed in the literature include shortening of small contractile units similar to muscle sarcomeres, myosin II-dependent transport along uniformly polarized actin arrays, and a dynamic network contraction mechanism where contraction results from alignment of actin filaments by myosin II assemblies (Cramer, 1999;Verkhovsky et al., 1999a). More sophisticated biophysical models aim to understand the contractile cytoskeletal machinery in quantitative physical terms. In the past, considering the cytoskeleton as a gel made of cross-linked semiflexible polymers has helped understanding its passive visco-elastic properties. More recently, attempts were made to develop biophysical models taking into account intrinsic activity of the cytoskeleton: polarized assembly of acti...

show abstract

Analysis of actin dynamics at the leading edge of crawling cells: implications for the shape of keratocyte lamellipodia

Cited by 100 publications

References 36 publications

Tracking Retrograde Flow in Keratocytes: News from the Front

Tracking Retrograde Flow in Keratocytes: News from the Front

Polarization and Movement of Keratocytes: A Multiscale Modelling Approach

Comparative Maps of Motion and Assembly of Filamentous Actin and Myosin II in Migrating Cells

Contact Info

Product

Resources

About