Converging Populations of F-Actin Promote Breakage of Associated Microtubules to Spatially Regulate Microtubule Turnover in Migrating Cells

Gupton, Stephanie L.; Salmon, W. D.; Waterman‐Storer, Clare M.

doi:10.1016/s0960-9822(02)01276-9

Cited by 105 publications

(100 citation statements)

References 37 publications

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“…In addition, we performed a kymographic analysis of mCherry-actin transfected LLC-PK1 cells. Figure 3C shows a kymograph from the cell shown in Figure 3B (and Supplemental Movie 3B) with an actin retrograde flow velocity of 6.9 Ϯ 0.6 nm/s (n ϭ 4 cells) to be on the time scales of seconds which compares well with the values reported by Gupton et al (2002). To further test the role of actomyosin contractility, we inhibited myosin II ATPase activity with blebbistatin.…”

Section: Actomyosin Contractilitysupporting

confidence: 79%

“…Because actomyosin contractility has been reported to buckle and break MTs (Waterman-Storer and Salmon, 1997;Gupton et al, 2002;Brangwynne et al, 2006), we directly imaged actin dynamics to determine its contribution to MT bending in LLC-PK1 cells. As shown in Figure 3A (and Supplemental Movies 3A1 and 3A2), we found that F-actin is nearly stationary, whereas MTs are simultaneously observed to deform.…”

Section: Actomyosin Contractilitymentioning

confidence: 99%

“…It has been shown that acto-myosin contractility can cause MTs to buckle and promote breaking in motile cells (WatermanStorer and Salmon, 1997;Gupton et al, 2002). In particular MTs are observed to deform and break in central cell regions where actin retrograde and anterograde flow powered by myosin motors forms a convergence zone (Gupton et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

“…In particular MTs are observed to deform and break in central cell regions where actin retrograde and anterograde flow powered by myosin motors forms a convergence zone (Gupton et al, 2002). It also has been argued that during cell migration this mechanism plays a major role in spatially modulating MT turnover by regulating MT breakage (Gupton et al, 2002). For this mechanism to work, MTs have to interact with the F-actin bundles, and it has been suggested that MTs and F-actin bind to one another in migrating newt lung epithelial cells .…”

Section: Introductionmentioning

confidence: 99%

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Anterograde Microtubule Transport Drives Microtubule Bending in LLC-PK1 Epithelial Cells

Bicek

Tüzel

Demtchouk

et al. 2009

MBoC

103

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Microtubules (MTs) have been proposed to act mechanically as compressive struts that resist both actomyosin contractile forces and their own polymerization forces to mechanically stabilize cell shape. To identify the origin of MT bending, we directly observed MT bending and F-actin transport dynamics in the periphery of LLC-PK1 epithelial cells. We found that F-actin is nearly stationary in these cells even as MTs are deformed, demonstrating that MT bending is not driven by actomyosin contractility. Furthermore, the inhibition of myosin II activity through the use of blebbistatin results in microtubules that are still dynamically bending. In addition, as determined by fluorescent speckle microscopy, MT polymerization rarely results, if ever, in bending. We suppressed dynamic instability using nocodazole, and we observed no qualitative change in the MT bending dynamics. Bending most often results from anterograde transport of proximal portions of the MT toward a nearly stationary distal tip. Interestingly, we found that in an in vitro kinesin-MT gliding assay, MTs buckle in a similar manner. To make quantitative comparisons, we measured curvature distributions of observed MTs and found that the in vivo and in vitro curvature distributions agree quantitatively. In addition, the measured MT curvature distribution is not Gaussian, as expected for a thermally driven semiflexible polymer, indicating that thermal forces play a minor role in MT bending. We conclude that many of the known mechanisms of MT deformation, such as polymerization and acto-myosin contractility, play an inconsequential role in mediating MT bending in LLC-PK1 cells and that MT-based molecular motors likely generate most of the strain energy stored in the MT lattice. The results argue against models in which MTs play a major mechanical role in LLC-PK1 cells and instead favor a model in which mechanical forces control the spatial distribution of the MT array.

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Section: Actomyosin Contractilitysupporting

confidence: 79%

Section: Actomyosin Contractilitymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Anterograde Microtubule Transport Drives Microtubule Bending in LLC-PK1 Epithelial Cells

Bicek

Tüzel

Demtchouk

et al. 2009

MBoC

103

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show abstract

“…Our assay of total cellular microtubule polymer is not sensitive enough to detect such differences. In addition, Rac1 activation also increases microtubule turnover and minus end depolymerization caused by increased retrograde flow and microtubule breakage, effectively lowering the total amount of microtubule polymer (7,49). This might also explain why the amount of microtubule polymer in the presence of excess Op18 was only slightly increased in Rac1(Q61L)-expressing cells as compared with control cells.…”

Section: Pak-dependent Phosphorylation Downstream Of Rac1 Canmentioning

confidence: 99%

Regulation of Microtubule Destabilizing Activity of Op18/Stathmin Downstream of Rac1

Wittmann

Bokoch

Waterman‐Storer

2004

Journal of Biological Chemistry

Self Cite

218

183

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In the leading edge of migrating cells, a subset of microtubules exhibits net growth in a Rac1-and p21-activated kinase-dependent manner. Here, we explore the possibility of whether phosphorylation and inactivation of the microtubule-destabilizing protein Op18/ stathmin could be a mechanism regulating microtubule dynamics downstream of Rac1 and p21-activated kinases. We find that, in vitro, Pak1 phosphorylates Op18/ stathmin specifically at serine 16 and inactivates its catastrophe promoting activity in biochemical and time lapse microscopy microtubule assembly assays. Furthermore, phosphorylation of either serine 16 or 63 is sufficient to inhibit Op18/stathmin in vitro. In cells, the microtubule-destabilizing effect of an excess of Op18/ stathmin can be partially overcome by expression of constitutively active Rac1(Q61L), which is dependent on Pak activity, suggesting that the microtubule cytoskeleton can be regulated through inactivation of Op18/ stathmin downstream of Rac1 and Pak in vivo. However, in vivo, Pak1 activity alone is not sufficient to phosphorylate Op18, indicating that additional pathways downstream of Rac1 are required for Op18 regulation.

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High‐Content Analysis of Cytoskeleton Functions by Fluorescent Speckle Microscopy

2010

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The development of nanomedical strategies requires precise mechanistic models of cellular pathways. At center stage of many of these pathways is the cytoskeleton, which dynamically organizes cellular architecture and mediates cell morphological functions. In this chapter we review the principles and selected applications of quantitative fluorescent speckle microscopy (qFSM), a combined imaging modality and statistical toolset that delivers high‐resolution measurements of cytoskeleton structure and dynamics in living cells. qFSM has emerged as an invaluable tool in studies aimed at delineating the mechanisms of cytoskeleton regulation by the systematic perturbation of molecular signals upstream of cytoskeleton outputs. Additionally, qFSM can be used as a precise readout of cytoskeleton responses in the search for new drugs and nanomedical strategies that alter cellular behavior by intervening with specific cytoskeleton functions.

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Converging Populations of F-Actin Promote Breakage of Associated Microtubules to Spatially Regulate Microtubule Turnover in Migrating Cells

Abstract: We propose that f-actin movement into the convergence zone plays a major role in spatially modulating MT turnover during cell migration by regulating MT breakage, and thus minus-end dynamics, in central cell regions.

Cited by 105 publications

References 37 publications

Anterograde Microtubule Transport Drives Microtubule Bending in LLC-PK1 Epithelial Cells

Anterograde Microtubule Transport Drives Microtubule Bending in LLC-PK1 Epithelial Cells

Regulation of Microtubule Destabilizing Activity of Op18/Stathmin Downstream of Rac1

High‐Content Analysis of Cytoskeleton Functions by Fluorescent Speckle Microscopy

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