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 RacG Regulates Morphology, Phagocytosis, and Chemotaxis

2006 ◽  
Vol 5 (10) ◽  
pp. 1648-1663 ◽  
Author(s):  
Baggavalli P. Somesh ◽  
Georgia Vlahou ◽  
Miho Iijima ◽  
Robert H. Insall ◽  
Peter Devreotes ◽  
...  
Keyword(s):  
Rho Gtpases ◽  
Actin Polymerization ◽  
Kinase Inhibitor ◽  
Dominant Negative ◽  
Dependent Manner ◽  
Free System ◽  
Mild Phenotype ◽  
Particle Uptake ◽  
Phosphoinositide 3 Kinase

ABSTRACTRacG is an unusual member of the complex family of Rho GTPases inDictyostelium. We have generated a knockout (KO) strain, as well as strains that overexpress wild-type (WT), constitutively active (V12), or dominant negative (N17) RacG. The protein is targeted to the plasma membrane, apparently in a nucleotide-dependent manner, and induces the formation of abundant actin-driven filopods. RacG is enriched at the rim of the progressing phagocytic cup, and overexpression of RacG-WT or RacG-V12 induced an increased rate of particle uptake. The positive effect of RacG on phagocytosis was abolished in the presence of 50 μM LY294002, a phosphoinositide 3-kinase inhibitor, indicating that generation of phosphatidylinositol 3,4,5-trisphosphate is required for activation of RacG. RacG-KO cells showed a moderate chemotaxis defect that was stronger in the RacG-V12 and RacG-N17 mutants, in part because of interference with signaling through Rac1. The in vivo effects of RacG-V12 could not be reproduced by a mutant lacking the Rho insert region, indicating that this region is essential for interaction with downstream components. Processes like growth, pinocytosis, exocytosis, cytokinesis, and development were unaffected in Rac-KO cells and in the overexpressor mutants. In a cell-free system, RacG induced actin polymerization upon GTPγS stimulation, and this response could be blocked by an Arp3 antibody. While the mild phenotype of RacG-KO cells indicates some overlap with one or moreDictyosteliumRho GTPases, like Rac1 and RacB, the significant changes found in overexpressors show that RacG plays important roles. We hypothesize that RacG interacts with a subset of effectors, in particular those concerned with shape, motility, and phagocytosis.

Small-Molecule Screen Identifies ROS as Key Regulators of Actin Dynamics in Neutrophils

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4641-4641
Author(s):  
Hidenori Hattori ◽  
Kulandayan K Subramanian ◽  
Hongbo R. Luo
Keyword(s):  
Small Molecule ◽  
Actin Polymerization ◽  
Neutrophil Migration ◽  
Physiological Role ◽  
Leading Edge ◽  
Actin Dynamics ◽  
Functional Screening ◽  
Cellular Processes

Abstract Precise spatial and temporal control of actin polymerization and depolymerization is essential for mediating various cellular processes such as migration, phagocytosis, vesicle trafficking and adhesion. In this study, we used a small-molecule functional screening approach to identify novel regulators of actin dynamics during neutrophil migration. Here we show that NADPH-oxidase dependent Reactive Oxygen Species act as negative regulators of actin polymerization. Neutrophils with pharmacologically inhibited oxidase or isolated from Chronic Granulomatous Disease (CGD) patient and mice displayed enhanced F-actin polymerization, multiple pseudopods formation and impaired chemotaxis. ROS localized to pseudopodia and inhibited actin polymerization by driving actin glutathionylation at the leading edge of migrating cells. Consistent with these in vitro results, adoptively transferred CGD murine neutrophils also showed impaired in vivo recruitment to sites of inflammation. Together, these results present a novel physiological role for ROS in regulation of action polymerization and shed new light on the pathogenesis of CGD.

scholarly journals Analysis of the Actin–Myosin II System in Fish Epidermal Keratocytes: Mechanism of Cell Body Translocation

1997 ◽  
Vol 139 (2) ◽  
pp. 397-415 ◽  
Author(s):  
Tatyana M. Svitkina ◽  
Alexander B. Verkhovsky ◽  
Kyle M. McQuade ◽  
Gary G. Borisy
Keyword(s):  
Cell Body ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Myosin Ii ◽  
Leading Edge ◽  
Time Lapse ◽  
Supramolecular Organization ◽  
Retrograde Flow ◽  
Body Boundary ◽  
Filament Bundles

While the protrusive event of cell locomotion is thought to be driven by actin polymerization, the mechanism of forward translocation of the cell body is unclear. To elucidate the mechanism of cell body translocation, we analyzed the supramolecular organization of the actin–myosin II system and the dynamics of myosin II in fish epidermal keratocytes. In lamellipodia, long actin filaments formed dense networks with numerous free ends in a brushlike manner near the leading edge. Shorter actin filaments often formed T junctions with longer filaments in the brushlike area, suggesting that new filaments could be nucleated at sides of preexisting filaments or linked to them immediately after nucleation. The polarity of actin filaments was almost uniform, with barbed ends forward throughout most of the lamellipodia but mixed in arc-shaped filament bundles at the lamellipodial/cell body boundary. Myosin II formed discrete clusters of bipolar minifilaments in lamellipodia that increased in size and density towards the cell body boundary and colocalized with actin in boundary bundles. Time-lapse observation demonstrated that myosin clusters appeared in the lamellipodia and remained stationary with respect to the substratum in locomoting cells, but they exhibited retrograde flow in cells tethered in epithelioid colonies. Consequently, both in locomoting and stationary cells, myosin clusters approached the cell body boundary, where they became compressed and aligned, resulting in the formation of boundary bundles. In locomoting cells, the compression was associated with forward displacement of myosin features. These data are not consistent with either sarcomeric or polarized transport mechanisms of cell body translocation. We propose that the forward translocation of the cell body and retrograde flow in the lamellipodia are both driven by contraction of an actin–myosin network in the lamellipodial/cell body transition zone.

scholarly journals Junction-based lamellipodia drive endothelial cell rearrangements in vivo via a VE-cadherin/F-actin based oscillatory ratchet mechanism

2017 ◽  
Author(s):  
Ilkka Paatero ◽  
Loïc Sauteur ◽  
Minkyoung Lee ◽  
Anne K. Lagendijk ◽  
Daniel Heutschi ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Cell Elongation ◽  
Actin Polymerization ◽  
Time Lapse ◽  
Cell Junctions ◽  
Actin Dynamics ◽  
Cell Movements ◽  
Cell Behaviors ◽  
Wide Range

AbstractAngiogenesis and vascular remodeling are driven by a wide range of endothelial cell behaviors, such as cell divisions, cell movements, cell shape and polarity changes. To decipher the cellular and molecular mechanism of cell movements, we have analyzed the dynamics of different junctional components during blood vessel anastomosis in vivo. We show that endothelial cell movements are associated with oscillating lamellipodia-like structures, which are orientated in the direction of these movements. These structures emerge from endothelial cell junctions and we thus call them junction-based lamellipodia (JBL). High-resolution time-lapse imaging shows that JBL are formed by F-actin based protrusions at the front end of moving cells. These protrusions also contain diffusely distributed VE-cadherin, whereas the junctional protein ZO-1 (Zona occludens 1) remains at the junction. Subsequently, a new junction is formed at the front of the JBL and the proximal junction is pulled towards the newly established distal junction. JBL function is highly dependent on F-actin dynamics. Inhibition of F-actin polymerization prevents JBL formation, whereas Rac-1 inhibition interferes with JBL oscillations. Both interventions disrupt endothelial junction formation and cell elongation. To examine the role of VE-cadherin (encoded by cdh5 gene) in this process, we generated a targeted mutation in VE-cadherin gene (cdh5ubs25), which prevents VE-cad/F-actin interaction. Although homozygous ve-cadherin mutants form JBL, these JBL are less dynamic and do not promote endothelial cell elongation. Taken together, our observations suggest a novel oscillating ratchet-like mechanism, which is used by endothelial cells to move along or over each other and thus provides the physical means for cell rearrangements.

scholarly journals THz irradiation inhibits cell division by affecting actin dynamics

PLoS ONE ◽  
2021 ◽  
Vol 16 (8) ◽  
pp. e0248381
Author(s):  
Shota Yamazaki ◽  
Yuya Ueno ◽  
Ryosuke Hosoki ◽  
Takanori Saito ◽  
Toshitaka Idehara ◽  
...  
Keyword(s):  
Cell Division ◽  
Actin Polymerization ◽  
Time Lapse ◽  
Actin Dynamics ◽  
Filamentous Actin ◽  
Contractile Ring ◽  
Biological Phenomena ◽  
Underlying Mechanisms

Biological phenomena induced by terahertz (THz) irradiation are described in recent reports, but underlying mechanisms, structural and dynamical change of specific molecules are still unclear. In this paper, we performed time-lapse morphological analysis of human cells and found that THz irradiation halts cell division at cytokinesis. At the end of cytokinesis, the contractile ring, which consists of filamentous actin (F-actin), needs to disappear; however, it remained for 1 hour under THz irradiation. Induction of the functional structures of F-actin was also observed in interphase cells. Similar phenomena were also observed under chemical treatment (jasplakinolide), indicating that THz irradiation assists actin polymerization. We previously reported that THz irradiation enhances the polymerization of purified actin in vitro; our current work shows that it increases cytoplasmic F-actin in vivo. Thus, we identified one of the key biomechanisms affected by THz waves.

scholarly journals THz irradiation inhibits cell division by affecting actin dynamics

2021 ◽  
Author(s):  
shota yamazaki ◽  
Yuya Ueno ◽  
Ryosuke Hosoki ◽  
Takanori Saito ◽  
Toshitaka Idehara ◽  
...  
Keyword(s):  
Cell Division ◽  
Actin Polymerization ◽  
Time Lapse ◽  
Actin Dynamics ◽  
Filamentous Actin ◽  
Contractile Ring ◽  
Biological Phenomena ◽  
Underlying Mechanisms

Biological phenomena induced by terahertz (THz) irradiation are described in recent reports, but underlying mechanisms, structural and dynamical change of specific molecules are still unclear. In this paper, we performed time-lapse morphological analysis of human cells and found that THz irradiation halts cell division at cytokinesis. At the end of cytokinesis, the contractile ring, which consists of filamentous actin (F-actin), needs to disappear; however, it remained for 1 hour under THz irradiation. Induction of the functional structures of F-actin was also observed in interphase cells. Similar phenomena were also observed under chemical treatment (jasplakinolide), indicating that THz irradiation assists actin polymerization. We previously reported that THz irradiation enhances the polymerization of purified actin in vitro; our current work shows that it increases cytoplasmic F-actin in vivo. Thus, we identified one of the key biomechanisms affected by THz waves.

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

2002 ◽  
Vol 158 (1) ◽  
pp. 31-37 ◽  
Author(s):  
Wendy C. Salmon ◽  
Michael C. Adams ◽  
Clare M. Waterman-Storer
Keyword(s):  
Cell Body ◽  
Time Lapse ◽  
Lung Epithelial Cells ◽  
Dual Wavelength ◽  
Convergence Zone ◽  
Retrograde Flow ◽  
Filamentous Actin ◽  
Actin Bundles ◽  
Migrating Cells

Interactions between microtubules (MTs) and filamentous actin (f-actin) are involved in directed cell locomotion, but are poorly understood. To test the hypothesis that MTs and f-actin associate with one another and affect each other's organization and dynamics, we performed time-lapse dual-wavelength spinning-disk confocal fluorescent speckle microscopy (FSM) of MTs and f-actin in migrating newt lung epithelial cells. F-actin exhibited four zones of dynamic behavior: rapid retrograde flow in the lamellipodium, slow retrograde flow in the lamellum, anterograde flow in the cell body, and no movement in the convergence zone between the lamellum and cell body. Speckle analysis showed that MTs moved at the same trajectory and velocity as f-actin in the cell body and lamellum, but not in the lamellipodium or convergence zone. MTs grew along f-actin bundles, and quiescent MT ends moved in association with f-actin bundles. These results show that the movement and organization of f-actin has a profound effect on the dynamic organization of MTs in migrating cells, and suggest that MTs and f-actin bind to one another in vivo.

scholarly journals Rho GTPases as Key Molecular Players within Intestinal Mucosa and GI Diseases

Cells ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 66
Author(s):  
Rashmita Pradhan ◽  
Phuong A. Ngo ◽  
Luz d. C. Martínez-Sánchez ◽  
Markus F. Neurath ◽  
Rocío López-Posadas
Keyword(s):  
Intestinal Mucosa ◽  
Rho Gtpases ◽  
Current Knowledge ◽  
Cell Types ◽  
Effector Proteins ◽  
Special Focus ◽  
Specific Cell ◽  
Rho Proteins

Rho proteins operate as key regulators of the cytoskeleton, cell morphology and trafficking. Acting as molecular switches, the function of Rho GTPases is determined by guanosine triphosphate (GTP)/guanosine diphosphate (GDP) exchange and their lipidation via prenylation, allowing their binding to cellular membranes and the interaction with downstream effector proteins in close proximity to the membrane. A plethora of in vitro studies demonstrate the indispensable function of Rho proteins for cytoskeleton dynamics within different cell types. However, only in the last decades we have got access to genetically modified mouse models to decipher the intricate regulation between members of the Rho family within specific cell types in the complex in vivo situation. Translationally, alterations of the expression and/or function of Rho GTPases have been associated with several pathological conditions, such as inflammation and cancer. In the context of the GI tract, the continuous crosstalk between the host and the intestinal microbiota requires a tight regulation of the complex interaction between cellular components within the intestinal tissue. Recent studies demonstrate that Rho GTPases play important roles for the maintenance of tissue homeostasis in the gut. We will summarize the current knowledge on Rho protein function within individual cell types in the intestinal mucosa in vivo, with special focus on intestinal epithelial cells and T cells.

scholarly journals Rho-Proteins and Downstream Pathways as Potential Targets in Sepsis and Septic Shock: What Have We Learned from Basic Research

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1844
Author(s):  
Maria Luísa da Silveira Hahmeyer ◽  
José Eduardo da Silva-Santos
Keyword(s):  
Septic Shock ◽  
Actin Polymerization ◽  
Current Knowledge ◽  
Cecal Ligation ◽  
Experimental Models ◽  
Rho Proteins ◽  
Growing Cell ◽  
Sepsis And Septic Shock

Sepsis and septic shock are associated with acute and sustained impairment in the function of the cardiovascular system, kidneys, lungs, liver, and brain, among others. Despite the significant advances in prevention and treatment, sepsis and septic shock sepsis remain global health problems with elevated mortality rates. Rho proteins can interact with a considerable number of targets, directly affecting cellular contractility, actin filament assembly and growing, cell motility and migration, cytoskeleton rearrangement, and actin polymerization, physiological functions that are intensively impaired during inflammatory conditions, such as the one that occurs in sepsis. In the last few decades, Rho proteins and their downstream pathways have been investigated in sepsis-associated experimental models. The most frequently used experimental design included the exposure to bacterial lipopolysaccharide (LPS), in both in vitro and in vivo approaches, but experiments using the cecal ligation and puncture (CLP) model of sepsis have also been performed. The findings described in this review indicate that Rho proteins, mainly RhoA and Rac1, are associated with the development of crucial sepsis-associated dysfunction in different systems and cells, including the endothelium, vessels, and heart. Notably, the data found in the literature suggest that either the inhibition or activation of Rho proteins and associated pathways might be desirable in sepsis and septic shock, accordingly with the cellular system evaluated. This review included the main findings, relevance, and limitations of the current knowledge connecting Rho proteins and sepsis-associated experimental models.

scholarly journals Smurf1 regulates tumor cell plasticity and motility through degradation of RhoA leading to localized inhibition of contractility

2006 ◽  
Vol 176 (1) ◽  
pp. 35-42 ◽  
Author(s):  
Erik Sahai ◽  
Raquel Garcia-Medina ◽  
Jacques Pouysségur ◽  
Emmanuel Vial
Keyword(s):  
Cell Migration ◽  
Tumor Cell ◽  
Rho Gtpases ◽  
Rho Kinase ◽  
Cell Movement ◽  
Leading Edge ◽  
Cell Periphery ◽  
Tumor Cell Migration ◽  
Tumor Cell Motility

Rho GTPases participate in various cellular processes, including normal and tumor cell migration. It has been reported that RhoA is targeted for degradation at the leading edge of migrating cells by the E3 ubiquitin ligase Smurf1, and that this is required for the formation of protrusions. We report that Smurf1-dependent RhoA degradation in tumor cells results in the down-regulation of Rho kinase (ROCK) activity and myosin light chain 2 (MLC2) phosphorylation at the cell periphery. The localized inhibition of contractile forces is necessary for the formation of lamellipodia and for tumor cell motility in 2D tissue culture assays. In 3D invasion assays, and in in vivo tumor cell migration, the inhibition of Smurf1 induces a mesenchymal–amoeboid–like transition that is associated with a more invasive phenotype. Our results suggest that Smurf1 is a pivotal regulator of tumor cell movement through its regulation of RhoA signaling.

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