Methods to Measure Perinuclear Actin Dynamics During Nuclear Movement in Migrating Cells

2019 ◽  
pp. 371-385
Author(s):  
Cátia S. Janota ◽  
Francisco J. Calero-Cuenca ◽  
Edgar R. Gomes
Keyword(s):  
Actin Dynamics ◽  
Nuclear Movement ◽  
Migrating Cells

scholarly journals Regulation of leading edge microtubule and actin dynamics downstream of Rac1

2003 ◽  
Vol 161 (5) ◽  
pp. 845-851 ◽  
Author(s):  
Torsten Wittmann ◽  
Gary M. Bokoch ◽  
Clare M. Waterman-Storer
Keyword(s):  
Rho Gtpases ◽  
Actin Polymerization ◽  
Leading Edge ◽  
Time Lapse ◽  
Dominant Negative ◽  
Actin Dynamics ◽  
Retrograde Flow ◽  
Rho Proteins ◽  
Migrating Cells

Actin in migrating cells is regulated by Rho GTPases. However, Rho proteins might also affect microtubules (MTs). Here, we used time-lapse microscopy of PtK1 cells to examine MT regulation downstream of Rac1. In these cells, “pioneer” MTs growing into leading-edge protrusions exhibited a decreased catastrophe frequency and an increased time in growth as compared with MTs further from the leading edge. Constitutively active Rac1(Q61L) promoted pioneer behavior in most MTs, whereas dominant-negative Rac1(T17N) eliminated pioneer MTs, indicating that Rac1 is a regulator of MT dynamics in vivo. Rac1(Q61L) also enhanced MT turnover through stimulation of MT retrograde flow and breakage. Inhibition of p21-activated kinases (Paks), downstream effectors of Rac1, inhibited Rac1(Q61L)-induced MT growth and retrograde flow. In addition, Rac1(Q61L) promoted lamellipodial actin polymerization and Pak-dependent retrograde flow. Together, these results indicate coordinated regulation of the two cytoskeletal systems in the leading edge of migrating cells.

scholarly journals Fascin Regulates Nuclear Movement and Deformation in Migrating Cells

2016 ◽  
Vol 38 (4) ◽  
pp. 371-383 ◽  
Author(s):  
Asier Jayo ◽  
Majid Malboubi ◽  
Susumu Antoku ◽  
Wakam Chang ◽  
Elena Ortiz-Zapater ◽  
...  
Keyword(s):  
Nuclear Movement ◽  
Movement And Deformation ◽  
Migrating Cells

scholarly journals The Piezo channel is used by migrating cells to sense compressive load

2019 ◽  
Author(s):  
Nishit Srivastava ◽  
David Traynor ◽  
Alexandre J. Kabla ◽  
Robert R. Kay
Keyword(s):  
Myosin Ii ◽  
Compressive Load ◽  
Cytosolic Calcium ◽  
Uniaxial Pressure ◽  
Actin Dynamics ◽  
Physical Barriers ◽  
Sense And Respond ◽  
Null Mutants ◽  
Soft Agarose ◽  
Migrating Cells

AbstractMigrating cells face varied mechanical and physical barriers in physiological environments, but how they sense and respond to them remains to be fully understood. We used a custom-built ‘cell squasher’ to apply uniaxial pressure to Dictyostelium cells migrating under soft agarose. Within 10 seconds of application, loads of as little as 100 Pa cause cells to move using blebs instead of pseudopods. Cells lose volume and surface area under pressure and their actin dynamics are perturbed. Myosin-II is recruited to the cortex, potentially increasing contractility and so driving blebbing. The blebbing response depends on extra-cellular calcium, is accompanied by increased cytosolic calcium and largely abrogated in null mutants of the Piezo stretch-operated channel. We propose that migrating cells sense mechanical force through mechano-sensitive channels, leading to an influx of calcium and cortical recruitment of myosin, thus re-directing the motile apparatus to produce blebs rather than pseudopods.

scholarly journals Nuclear Movement Regulated by Cdc42, MRCK, Myosin, and Actin Flow Establishes MTOC Polarization in Migrating Cells

Cell ◽  
2005 ◽  
Vol 121 (3) ◽  
pp. 451-463 ◽  
Author(s):  
Edgar R. Gomes ◽  
Shantanu Jani ◽  
Gregg G. Gundersen
Keyword(s):  
Nuclear Movement ◽  
Migrating Cells

scholarly journals Unbalancing the Phosphatidylinositol-4,5-bisphosphate–Cofilin Interaction Impairs Cell Steering

2009 ◽  
Vol 20 (21) ◽  
pp. 4509-4523 ◽  
Author(s):  
Shirley Leyman ◽  
Mazen Sidani ◽  
Laila Ritsma ◽  
Davy Waterschoot ◽  
Robert Eddy ◽  
...  
Keyword(s):  
Ph Regulation ◽  
Release Kinetics ◽  
Carcinoma Cells ◽  
Actin Dynamics ◽  
Morphological Defects ◽  
Epidermal Growth ◽  
External Stimuli ◽  
Kinetics Of ◽  
Migrating Cells

Cofilin is a key player in actin dynamics during cell migration. Its activity is regulated by (de)phosphorylation, pH, and binding to phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. Here, we here use a human cofilin-1 (D122K) mutant with increased binding affinity for PI(4,5)P2 and slower release from the plasma membrane to study the role of the PI(4,5)P2–cofilin interaction in migrating cells. In fibroblasts in a background of endogenous cofilin, D122K cofilin expression negatively affects cell turning frequency. In carcinoma cells with down-regulated endogenous cofilin, D122K cofilin neither rescues the drastic morphological defects nor restores the effects in cell turning capacity, unlike what has been reported for wild-type cofilin. In cofilin knockdown cells, D122K cofilin expression promotes outgrowth of an existing lamellipod in response to epidermal growth factor (EGF) but does not result in initiation of new lamellipodia. This indicates that, next to phospho- and pH regulation, the normal release kinetics of cofilin from PI(4,5)P2 is crucial as a local activation switch for lamellipodia initiation and as a signal for migrating cells to change direction in response to external stimuli. Our results demonstrate that the PI(4,5)P2 regulatory mechanism, that is governed by EGF-dependent phospholipase C activation, is a determinant for the spatial and temporal control of cofilin activation required for lamellipodia initiation.

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