Dictyostelium LIS1 Is a Centrosomal Protein Required for Microtubule/Cell Cortex Interactions, Nucleus/Centrosome Linkage, and Actin Dynamics

The widespread LIS1-proteins were originally identified as the target for sporadic mutations causing lissencephaly in humans. Dictyostelium LIS1 (DdLIS1) is a microtubule-associated protein exhibiting 53% identity to human LIS1. It colocalizes with dynein at isolated, microtubule-free centrosomes, suggesting that both are integral centrosomal components. Replacement of the DdLIS1 gene by the hypomorphic D327H allele or overexpression of an MBP-DdLIS1 fusion disrupted various dynein-associated functions. Microtubules lost contact with the cell cortex and were dragged behind an unusually motile centrosome. Previously, this phenotype was observed in cells overexpressing fragments of dynein or the XMAP215-homologue DdCP224. DdLIS1 was coprecipitated with DdCP224, suggesting that both act together in dynein-mediated cortical attachment of microtubules. Furthermore, DdLIS1-D327H mutants showed Golgi dispersal and reduced centrosome/nucleus association. Defects in DdLIS1 function also altered actin dynamics characterized by traveling waves of actin polymerization correlated with a reduced F-actin content. DdLIS1 could be involved in actin dynamics through Rho-GTPases, because DdLIS1 interacted directly with Rac1A in vitro. Our results show that DdLIS1 is required for maintenance of the microtubule cytoskeleton, Golgi apparatus and nucleus/centrosome association, and they suggest that LIS1-dependent alterations of actin dynamics could also contribute to defects in neuronal migration in lissencephaly patients.

Download Full-text

Primary granule exocytosis in human neutrophils is regulated by Rac-dependent actin remodeling

AJP Cell Physiology ◽

10.1152/ajpcell.00239.2008 ◽

2008 ◽

Vol 295 (5) ◽

pp. C1354-C1365 ◽

Cited By ~ 59

Author(s):

Troy Mitchell ◽

Andrea Lo ◽

Michael R. Logan ◽

Paige Lacy ◽

Gary Eitzen

Keyword(s):

Actin Polymerization ◽

Microscopic Analysis ◽

Formyl Peptide Receptor ◽

Human Neutrophils ◽

Cell Cortex ◽

Secretory Cells ◽

Actin Remodeling ◽

Granule Exocytosis ◽

Primary Granule

The actin cytoskeleton regulates exocytosis in all secretory cells. In neutrophils, Rac2 GTPase has been shown to control primary (azurophilic) granule exocytosis. In this report, we propose that Rac2 is required for actin cytoskeletal remodeling to promote primary granule exocytosis. Treatment of neutrophils with low doses (≤10 μM) of the actin-depolymerizing drugs latrunculin B (Lat B) or cytochalasin B (CB) enhanced both formyl peptide receptor- and Ca2+ionophore-stimulated exocytosis. Higher concentrations of CB or Lat B, or stabilization of F-actin with jasplakinolide (JP), inhibited primary granule exocytosis measured as myeloperoxidase release but did not affect secondary granule exocytosis determined by lactoferrin release. These results suggest an obligatory role for F-actin disassembly before primary granule exocytosis. However, lysates from secretagogue-stimulated neutrophils showed enhanced actin polymerization activity in vitro. Microscopic analysis showed that resting neutrophils contain significant cortical F-actin, which was redistributed to sites of primary granule translocation when stimulated. Exocytosis and actin remodeling was highly polarized when cells were primed with CB; however, polarization was reduced by Lat B preincubation, and both polarization and exocytosis were blocked when F-actin was stabilized with JP. Treatment of cells with the small molecule Rac inhibitor NSC23766 also inhibited actin remodeling and primary granule exocytosis induced by Lat B/fMLF or CB/fMLF, but not by Ca2+ionophore. Therefore, we propose a role for F-actin depolymerization at the cell cortex coupled with Rac-dependent F-actin polymerization in the cell cytoplasm to promote primary granule exocytosis.

Download Full-text

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

Blood ◽

10.1182/blood.v112.11.4641.4641 ◽

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.

Download Full-text

Mammalian Adenylyl Cyclase-associated Protein 1 (CAP1) Regulates Cofilin Function, the Actin Cytoskeleton, and Cell Adhesion

Journal of Biological Chemistry ◽

10.1074/jbc.m113.484535 ◽

2013 ◽

Vol 288 (29) ◽

pp. 20966-20977 ◽

Cited By ~ 51

Author(s):

Haitao Zhang ◽

Pooja Ghai ◽

Huhehasi Wu ◽

Changhui Wang ◽

Jeffrey Field ◽

...

Keyword(s):

Cell Adhesion ◽

Actin Cytoskeleton ◽

Hela Cells ◽

Actin Polymerization ◽

Actin Dynamics ◽

Ras Signaling ◽

Filamentous Actin ◽

Camp Pathway

CAP (adenylyl cyclase-associated protein) was first identified in yeast as a protein that regulates both the actin cytoskeleton and the Ras/cAMP pathway. Although the role in Ras signaling does not extend beyond yeast, evidence supports that CAP regulates the actin cytoskeleton in all eukaryotes including mammals. In vitro actin polymerization assays show that both mammalian and yeast CAP homologues facilitate cofilin-driven actin filament turnover. We generated HeLa cells with stable CAP1 knockdown using RNA interference. Depletion of CAP1 led to larger cell size and remarkably developed lamellipodia as well as accumulation of filamentous actin (F-actin). Moreover, we found that CAP1 depletion also led to changes in cofilin phosphorylation and localization as well as activation of focal adhesion kinase (FAK) and enhanced cell spreading. CAP1 forms complexes with the adhesion molecules FAK and Talin, which likely underlie the cell adhesion phenotypes through inside-out activation of integrin signaling. CAP1-depleted HeLa cells also had substantially elevated cell motility as well as invasion through Matrigel. In summary, in addition to generating in vitro and in vivo evidence further establishing the role of mammalian CAP1 in actin dynamics, we identified a novel cellular function for CAP1 in regulating cell adhesion.

Download Full-text

Arp2/3- and Cofilin-coordinated Actin Dynamics Is Required for Insulin-mediated GLUT4 Translocation to the Surface of Muscle Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e10-04-0316 ◽

2010 ◽

Vol 21 (20) ◽

pp. 3529-3539 ◽

Cited By ~ 53

Author(s):

Tim Ting Chiu ◽

Nish Patel ◽

Alisa E. Shaw ◽

James R. Bamburg ◽

Amira Klip

Keyword(s):

Actin Polymerization ◽

Molecular Mechanisms ◽

Muscle Cells ◽

Actin Dynamics ◽

Cell Cortex ◽

Glut4 Translocation ◽

Filamentous Actin ◽

Cortical Actin ◽

Rac Gtpase ◽

Skeletal Myoblasts

GLUT4 vesicles are actively recruited to the muscle cell surface upon insulin stimulation. Key to this process is Rac-dependent reorganization of filamentous actin beneath the plasma membrane, but the underlying molecular mechanisms have yet to be elucidated. Using L6 rat skeletal myoblasts stably expressing myc-tagged GLUT4, we found that Arp2/3, acting downstream of Rac GTPase, is responsible for the cortical actin polymerization evoked by insulin. siRNA-mediated silencing of either Arp3 or p34 subunits of the Arp2/3 complex abrogated actin remodeling and impaired GLUT4 translocation. Insulin also led to dephosphorylation of the actin-severing protein cofilin on Ser-3, mediated by the phosphatase slingshot. Cofilin dephosphorylation was prevented by strategies depolymerizing remodeled actin (latrunculin B or p34 silencing), suggesting that accumulation of polymerized actin drives severing to enact a dynamic actin cycling. Cofilin knockdown via siRNA caused overwhelming actin polymerization that subsequently inhibited GLUT4 translocation. This inhibition was relieved by reexpressing Xenopus wild-type cofilin-GFP but not the S3E-cofilin-GFP mutant that emulates permanent phosphorylation. Transferrin recycling was not affected by depleting Arp2/3 or cofilin. These results suggest that cofilin dephosphorylation is required for GLUT4 translocation. We propose that Arp2/3 and cofilin coordinate a dynamic cycle of actin branching and severing at the cell cortex, essential for insulin-mediated GLUT4 translocation in muscle cells.

Download Full-text

N-WASP inhibitor wiskostatin nonselectively perturbs membrane transport by decreasing cellular ATP levels

AJP Cell Physiology ◽

10.1152/ajpcell.00426.2006 ◽

2007 ◽

Vol 292 (4) ◽

pp. C1562-C1566 ◽

Cited By ~ 32

Author(s):

Christopher J. Guerriero ◽

Ora A. Weisz

Keyword(s):

Membrane Trafficking ◽

Actin Polymerization ◽

Cell Function ◽

Actin Dynamics ◽

Cellular Functions ◽

Intact Cells ◽

Atp Levels ◽

Dependent Inhibition

Wiskott-Aldrich syndrome protein (WASP) and WAVE stimulate actin-related protein (Arp)2/3-mediated actin polymerization, leading to diverse downstream effects, including the formation and remodeling of cell surface protrusions, modulation of cell migration, and intracytoplasmic propulsion of organelles and pathogens. Selective inhibitors of individual Arp2/3 activators would enable more exact dissection of WASP- and WAVE-dependent cellular pathways and are potential therapeutic targets for viral pathogenesis. Wiskostatin is a recently described chemical inhibitor that selectively inhibits neuronal WASP (N-WASP)-mediated actin polymerization in vitro. A growing number of recent studies have utilized this drug in vivo to uncover novel cellular functions for N-WASP; however, the selectivity of wiskostatin in intact cells has not been carefully explored. In our studies with this drug, we observed rapid and dose-dependent inhibition of N-WASP-dependent membrane trafficking steps. Additionally, however, we found that addition of wiskostatin inhibited numerous other cellular functions that are not believed to be N-WASP dependent. Further studies revealed that wiskostatin treatment caused a rapid, profound, and irreversible decrease in cellular ATP levels, consistent with its global effects on cell function. Our data caution against the use of this drug as a selective perturbant of N-WASP-dependent actin dynamics in vivo.

Download Full-text

Burkholderia pseudomalleitype III secreted protein BipC: role in actin modulation and translocation activities required for the bacterial intracellular lifecycle

PeerJ ◽

10.7717/peerj.2532 ◽

2016 ◽

Vol 4 ◽

pp. e2532 ◽

Cited By ~ 5

Author(s):

Wen Tyng Kang ◽

Kumutha Malar Vellasamy ◽

Lakshminarayanan Rajamani ◽

Roger W. Beuerman ◽

Jamuna Vadivelu

Keyword(s):

Actin Polymerization ◽

Cell Interaction ◽

Binding Activity ◽

Host Cells ◽

Actin Dynamics ◽

Actin Binding ◽

Virulence Determinants ◽

Secretion Systems ◽

Number Of Patients

Melioidosis, an infection caused by the facultative intracellular pathogenBurkholderia pseudomallei, has been classified as an emerging disease with the number of patients steadily increasing at an alarming rate.B. pseudomalleipossess various virulence determinants that allow them to invade the host and evade the host immune response, such as the type III secretion systems (TTSS). The products of this specialized secretion system are particularly important for theB. pseudomalleiinfection. Lacking in one or more components of the TTSS demonstrated different degrees of defects in the intracellular lifecycle ofB. pseudomallei. Further understanding the functional roles of proteins involved inB. pseudomalleiTTSS will enable us to dissect the enigma ofB. pseudomallei-host cell interaction. In this study, BipC (a translocator), which was previously reported to be involved in the pathogenesis ofB. pseudomallei, was further characterized using the bioinformatics and molecular approaches. ThebipCgene, coding for a putative invasive protein, was first PCR amplified fromB. pseudomalleiK96243genomic DNA and cloned into an expression vector for overexpression inEscherichia coli. The soluble protein was subsequently purified and assayed for actin polymerization and depolymerization. BipC was verified to subvert the host actin dynamics as demonstrated by the capability to polymerize actinin vitro. Homology modeling was also attempted to predict the structure of BipC. Overall, our findings identified that the protein encoded by thebipCgene plays a role as an effector involved in the actin binding activity to facilitate internalization ofB. pseudomalleiinto the host cells.

Download Full-text

Disease-associated missense mutations in the EVH1 domain disrupt intrinsic WASp function causing dysregulated actin dynamics and impaired dendritic cell migration

Blood ◽

10.1182/blood-2012-01-403857 ◽

2013 ◽

Vol 121 (1) ◽

pp. 72-84 ◽

Cited By ~ 11

Author(s):

Austen J. J. Worth ◽

Joao Metelo ◽

Gerben Bouma ◽

Dale Moulding ◽

Marco Fritzsche ◽

...

Keyword(s):

Actin Polymerization ◽

Actin Dynamics ◽

Missense Mutations ◽

Loss Of Function ◽

Interacting Protein ◽

Mutant Proteins ◽

Dendritic Cell Migration ◽

Biologic Function

Abstract Wiskott Aldrich syndrome (WAS), an X-linked immunodeficiency, results from loss-of-function mutations in the human hematopoietic cytoskeletal regulator gene WAS. Many missense mutations in the Ena Vasp homology1 (EVH1) domain preserve low-level WAS protein (WASp) expression and confer a milder clinical phenotype. Although disrupted binding to WASp-interacting protein (WIP) leads to enhanced WASp degradation in vivo, the intrinsic function of EVH1-mutated WASp is poorly understood. In the present study, we show that, despite mediating enhanced actin polymerization compared with wild-type WASp in vitro, EVH1 missense mutated proteins did not support full biologic function in cells, even when levels were restored by forced overexpression. Podosome assembly was aberrant and associated with dysregulated lamellipodia formation and impaired persistence of migration. At sites of residual podosome-associated actin polymerization, localization of EVH1-mutated proteins was preserved even after deletion of the entire domain, implying that WIP-WASp complex formation is not absolutely required for WASp localization. However, retention of mutant proteins in podosomes was significantly impaired and associated with reduced levels of WASp tyrosine phosphorylation. Our results indicate that the EVH1 domain is important not only for WASp stability, but also for intrinsic biologic activity in vivo.

Download Full-text

Actin Dynamics Is Controlled by a Casein Kinase II and Phosphatase 2C Interplay on Toxoplasma gondii Toxofilin

Molecular Biology of the Cell ◽

10.1091/mbc.e02-08-0462 ◽

2003 ◽

Vol 14 (5) ◽

pp. 1900-1912 ◽

Cited By ~ 44

Author(s):

Violaine Delorme ◽

Xavier Cayla ◽

Grazyna Faure ◽

Alphonse Garcia ◽

Isabelle Tardieux

Keyword(s):

Toxoplasma Gondii ◽

Actin Polymerization ◽

Casein Kinase Ii ◽

Casein Kinase ◽

Gliding Motility ◽

Actin Dynamics ◽

Central Feature ◽

Parasite Motility

Actin polymerization in Apicomplexa protozoa is central to parasite motility and host cell invasion. Toxofilin has been characterized as a protein that sequesters actin monomers and caps actin filaments in Toxoplasma gondii. Herein, we show that Toxofilin properties in vivo as in vitro depend on its phosphorylation. We identify a novel parasitic type 2C phosphatase that binds the Toxofilin/G-actin complex and a casein kinase II-like activity in the cytosol, both of which modulate the phosphorylation status of Toxofilin serine53. The interplay of these two molecules controls Toxofilin binding of G-actin as well as actin dynamics in vivo. Such functional interactions should play a major role in actin sequestration, a central feature of actin dynamics in Apicomplexa that underlies the spectacular speed and nature of parasite gliding motility.

Download Full-text

TetraThymosinβ Is Required for Actin Dynamics in Caenorhabditis elegans and Acts via Functionally Different Actin-binding Repeats

Molecular Biology of the Cell ◽

10.1091/mbc.e04-03-0225 ◽

2004 ◽

Vol 15 (10) ◽

pp. 4735-4748 ◽

Cited By ~ 28

Author(s):

Marleen Van Troys ◽

Kanako Ono ◽

Daisy Dewitte ◽

Veronique Jonckheere ◽

Natalie De Ruyck ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Actin Filament ◽

Actin Polymerization ◽

Actin Dynamics ◽

Actin Binding ◽

In Vitro Assays ◽

Filamentous Actin ◽

Lethal Mutant ◽

Interaction Sites

Generating specific actin structures via controlled actin polymerization is a prerequisite for eukaryote development and reproduction. We here report on an essential Caenorhabditis elegans protein tetraThymosinβ expressed in developing neurons and crucial during oocyte maturation in adults. TetraThymosinβ has four repeats, each related to the actin monomer-sequestering protein thymosinβ 4 and assists in actin filament elongation. For homologues with similar multirepeat structures, a profilin-like mechanism of ushering actin onto filament barbed ends, based on the formation of a 1:1 complex, is proposed to underlie this activity. We, however, demonstrate that tetraThymosinβ binds multiple actin monomers via different repeats and in addition also interacts with filamentous actin. All repeats need to be functional for attaining full activity in various in vitro assays. The activities on actin are thus a direct consequence of the repeated structure. In containing both G- and F-actin interaction sites, tetraThymosinβ may be reminiscent of nonhomologous multimodular actin regulatory proteins implicated in actin filament dynamics. A mutation that suppresses expression of tetraThymosinβ is homozygous lethal. Mutant organisms develop into adults but display a dumpy phenotype and fail to reproduce as their oocytes lack essential actin structures. This strongly suggests that the activity of tetraThymosinβ is of crucial importance at specific developmental stages requiring actin polymerization.

Download Full-text

Abi2-Deficient Mice Exhibit Defective Cell Migration, Aberrant Dendritic Spine Morphogenesis, and Deficits in Learning and Memory

Molecular and Cellular Biology ◽

10.1128/mcb.24.24.10905-10922.2004 ◽

2004 ◽

Vol 24 (24) ◽

pp. 10905-10922 ◽

Cited By ~ 80

Author(s):

Matthew Grove ◽

Galina Demyanenko ◽

Asier Echarri ◽

Patricia A. Zipfel ◽

Marisol E. Quiroz ◽

...

Keyword(s):

Cell Migration ◽

Dendritic Spine ◽

Actin Polymerization ◽

Adherens Junctions ◽

Actin Dynamics ◽

Long Term Memory ◽

Cell Morphogenesis ◽

And Migration

ABSTRACT The Abl-interactor (Abi) family of adaptor proteins has been linked to signaling pathways involving the Abl tyrosine kinases and the Rac GTPase. Abi proteins localize to sites of actin polymerization in protrusive membrane structures and regulate actin dynamics in vitro. Here we demonstrate that Abi2 modulates cell morphogenesis and migration in vivo. Homozygous deletion of murine abi2 produced abnormal phenotypes in the eye and brain, the tissues with the highest Abi2 expression. In the absence of Abi2, secondary lens fiber orientation and migration were defective in the eye, without detectable defects in proliferation, differentiation, or apoptosis. These phenotypes were consistent with the localization of Abi2 at adherens junctions in the developing lens and at nascent epithelial cell adherens junctions in vitro. Downregulation of Abi expression by RNA interference impaired adherens junction formation and correlated with downregulation of the Wave actin-nucleation promoting factor. Loss of Abi2 also resulted in cell migration defects in the neocortex and hippocampus, abnormal dendritic spine morphology and density, and severe deficits in short- and long-term memory. These findings support a role for Abi2 in the regulation of cytoskeletal dynamics at adherens junctions and dendritic spines, which is critical for intercellular connectivity, cell morphogenesis, and cognitive functions.

Download Full-text