Dual-wavelength fluorescent speckle microscopy reveals coupling of microtubule and actin movements in migrating cells

Salmon, W. D.; Adams, Michael C.; Waterman‐Storer, Clare M.

doi:10.1083/jcb.200203022

Cited by 201 publications

(183 citation statements)

References 24 publications

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“…The results obtained using two types of FSM images and phase contrast images in different cells were all remarkably similar in both flow structure and velocity magnitudes. We measured retrograde flow velocities in the lamellipodia in the range 1-3 m/min, comparable with those reported for other cell types (Salmon et al, 2002;Vallotton et al, 2003). In all cells analyzed, flow maps revealed a flow approximately perpendicular to the edge of the cell.…”

Section: Characterization Of Actin Flowsupporting

confidence: 63%

“…Kymographs indicated anterograde movement of actin in the cell body ( Figure 1I, yellow arrow), converging with the lamellipodial retrograde flow at the lamellipodium/cell body junction, where myosin motors are believed to generate strong contractile forces (Verkhovsky et al, 1999a). The existence of such pronounced convergence zone also has been described in other, less motile cell types (Salmon et al, 2002;Vallotton et al, 2004).The information obtained in kymographs is, however, limited. Kymographs show the movement within the selected region and along the selected direction only.…”

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confidence: 96%

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Tracking Retrograde Flow in Keratocytes: News from the Front

Vallotton

Danuser

Bohnet

et al. 2005

MBoC

130

111

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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...

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Section: Characterization Of Actin Flowsupporting

confidence: 63%

mentioning

confidence: 96%

Tracking Retrograde Flow in Keratocytes: News from the Front

Vallotton

Danuser

Bohnet

et al. 2005

MBoC

130

111

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“…Anterograde flow of actin structures and convergence zone of the retrograde and anterograde flow are also thought to be a characteristic feature of migrating cells (Salmon et al, 2002). However, these features are not inherent to all motile systems.…”

Section: Discussionmentioning

confidence: 99%

“…Our tracking approach based on cross-correlation between small areas of the image produced similar results, further supporting the notion of the generality of the retrograde flow phenomenon. Anterograde flow of actin structures and convergence zone of the retrograde and anterograde flow are also thought to be a characteristic feature of migrating cells (Salmon et al, 2002). However, these features are not inherent to all motile systems.…”

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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

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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...

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“…Despite this dependence on MTs dynamics, nuclear movement itself does not require a structured MTs cytoskeleton, as neurons treated with nocodazole still display actin condensations and nuclear movements. Previous work has shown that in migrating cells MTs and F-actin interact and affect each otherЈs organization and dynamics (Salmon et al, 2002). Microtubule network emanates from the centrosome toward the leading process and nucleus, forming in the later a cage-like structure (Rivas and Hatten, 1995).…”

Section: Discussionmentioning

confidence: 99%

Actomyosin Contraction at the Cell Rear Drives Nuclear Translocation in Migrating Cortical Interneurons

Martini

Valdeolmillos²

2010

Journal of Neuroscience

111

134

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Neuronal migration is a complex process requiring the coordinated interaction of cytoskeletal components and regulated by calcium signaling among other factors. Migratory neurons are polarized cells in which the largest intracellular organelle, the nucleus, has to move repeatedly. Current views support a central role for pulling forces that drive nuclear movement. The participation of actomyosin driven forces acting at the nucleus rear has been suggested, however its precise contribution has not been directly addressed. By analyzing interneurons migrating in cortical slices of mouse brains, we have found that nucleokinesis is associated with a precise pattern of actin dynamics characterized by the initial formation of a cup-like actin structure at the rear nuclear pole. Time-lapse experiments show that progressive actomyosin contraction drives the nucleus forward. Nucleokinesis concludes with the complete contraction of the cup-like structure, resulting in an actin spot at the base of the retracting trailing process. Our results demonstrate that this actin remodeling requires a threshold calcium level provided by low-frequency spontaneous fast intracellular calcium transients. Microtubule stabilization with taxol treatment prevents actin remodeling and nucleokinesis, whereas cells with a collapsed microtubule cytoskeleton induced by nocodazole treatment, display nearly normal actin dynamics and nucleokinesis. In summary, the results presented here demonstrate that actomyosin forces acting at the rear side of the nucleus drives nucleokinesis in tangentially migrating interneurons in a process that requires calcium and a dynamic cytoskeleton of microtubules.

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Dual-wavelength fluorescent speckle microscopy reveals coupling of microtubule and actin movements in migrating cells

Cited by 201 publications

References 24 publications

Tracking Retrograde Flow in Keratocytes: News from the Front

Tracking Retrograde Flow in Keratocytes: News from the Front

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

Actomyosin Contraction at the Cell Rear Drives Nuclear Translocation in Migrating Cortical Interneurons

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