scholarly journals Coronin-1 Function Is Required for Phagosome Formation

2005 ◽  
Vol 16 (7) ◽  
pp. 3077-3087 ◽  
Author(s):  
Ming Yan ◽  
Richard F. Collins ◽  
Sergio Grinstein ◽  
William S. Trimble
Keyword(s):  
Actin Polymerization ◽  
Coiled Coil ◽  
Dominant Negative ◽  
Actin Dynamics ◽  
Raw 264.7 Cells ◽  
Live Cells ◽  
Raw 264.7 ◽  
Dimerization Domain ◽  
Actin Remodeling ◽  
Downstream Signaling

Coronin-1 is an actin-associated protein whose function in actin dynamics has remained obscure. All coronin proteins have a variable N-terminal domain, followed by WD repeats and a C-terminal coiled-coil dimerization domain. Transfection of coronin-1-GFP into RAW 264.7 cells revealed that coronin rapidly and transiently associates with the phagosome. To determine if coronin is involved in mammalian phagocytosis we used a dominant-negative approach by expressing only the central WD domains. However, this caused cell rounding and dissociation from the substratum, hampering analysis of their phenotype. We therefore developed TAT-fusion constructs of coronin-1 WD domains to acutely introduce the recombinant protein fragment into live cells. We show that although TAT-WD has no effect on binding of opsonized RBCs to RAW 264.7 cells, receptor clustering or several downstream signaling events, lamellipodial extensions, and actin accumulation at the base of the bound particle were diminished. Furthermore, Arp3 accumulation at the phagosome was impaired after TAT-WD treatment. Interestingly, whereas coronin-1 also accumulates at the sites of actin remodeling associated with Salmonella invasion, TAT-WD had no effect on this process. Together, our data demonstrates that coronin-1 is required for an early step in phagosome formation, consistent with a role in actin polymerization.

scholarly journals Syndecan-2 induces filopodia and dendritic spine formation via the neurofibromin–PKA–Ena/VASP pathway

2007 ◽  
Vol 177 (5) ◽  
pp. 829-841 ◽  
Author(s):  
Yi-Ling Lin ◽  
Ya-Ting Lei ◽  
Chen-Jei Hong ◽  
Yi-Ping Hsueh
Keyword(s):  
Hippocampal Neurons ◽  
Actin Polymerization ◽  
Kinase Inhibitors ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Human Embryonic Kidney Cells ◽  
Spine Formation ◽  
Downstream Signaling

Syndecan-2 induced filopodia before spinogenesis; therefore, filopodia formation was used here as a model to study the early downstream signaling of syndecan-2 that leads to spinogenesis. Screening using kinase inhibitors indicated that protein kinase A (PKA) is required for syndecan-2–induced filopodia formation in both human embryonic kidney cells and hippocampal neurons. Because neurofibromin, a syndecan-2–binding partner, activates the cyclic adenosine monophosphate pathway, the role of neurofibromin in syndecan-2–induced filopodia formation was investigated by deletion mutant analysis, RNA interference, and dominant-negative mutant. The results showed that neurofibromin mediates the syndecan-2 signal to PKA. Among actin-associated proteins, Enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) were predicted as PKA effectors downstream of syndecan-2, as Ena/VASP, which is activated by PKA, induces actin polymerization. Indeed, when the activities of Ena/VASP were blocked, syndecan-2 no longer induced filopodia formation. Finally, in addition to filopodia formation, neurofibromin and Ena/VASP contributed to spinogenesis. This study reveals a novel signaling pathway in which syndecan-2 activates PKA via neurofibromin and PKA consequently phosphorylates Ena/VASP, promoting filopodia and spine formation.

scholarly journals Sphingosine Kinase Protects Lipopolysaccharide-Activated Macrophages from Apoptosis

2004 ◽  
Vol 24 (17) ◽  
pp. 7359-7369 ◽  
Author(s):  
Weicheng Wu ◽  
Raymond D. Mosteller ◽  
Daniel Broek
Keyword(s):  
Critical Role ◽  
Sphingosine Kinase ◽  
Dominant Negative ◽  
Raw 264.7 Cells ◽  
Raw 264.7 ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Lps Signaling ◽  
Lps Treatment ◽  
Stimulation Of

ABSTRACT Lipopolysaccharide (LPS) signaling is critical for the innate immune response to gram-negative bacteria. Here, evidence is presented for LPS stimulation of sphingosine kinase (SPK) in the RAW 264.7 murine macrophage cell line and rat primary hepatic macrophages (HMs). LPS treatment of RAW 264.7 cells resulted in a time- and dose-dependent activation of SPK and membrane translocation of SPK1. Further, LPS-induced SPK activation was blocked by SPK1-specific small interfering RNA (siRNA). Overexpression of Toll-like receptor 4 and MD2, the receptor and coreceptor of LPS, in HEK 293 cells activated SPK activity in the absence of LPS treatment. Inhibition of SPK by the pharmacological inhibitor N,N-dimethylsphingosine (DMS) or SPK1-specific siRNA blocked LPS stimulation of extracellular signal-regulated kinase 1/2 and p38 but enhanced LPS-induced c-Jun N-terminal kinase activation. The SPK inhibitor DMS and dominant-negative SPK1 also blocked LPS activation of Elk-1 and NF-κB reporters in RAW 264.7 cells. Inhibition of SPK sensitized RAW 264.7 cells and HMs to LPS-induced apoptosis. These data demonstrate the critical role of SPK1 in LPS signaling in macrophages and suggest that SPK1 is a potential therapeutic target to block hyperimmune responses induced by gram-negative bacteria.

scholarly journals A role for PKC-ε in FcγR-mediated phagocytosis by RAW 264.7 cells

2002 ◽  
Vol 159 (6) ◽  
pp. 939-944 ◽  
Author(s):  
Elaine C. Larsen ◽  
Takehiko Ueyama ◽  
Pamela M. Brannock ◽  
Yasuhito Shirai ◽  
Naoaki Saito ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Variable Region ◽  
Raw 264.7 Cells ◽  
Raw 264.7 ◽  
Dependent Manner ◽  
Actin Assembly ◽  
Fcγ Receptor ◽  
Raw 264.7 Macrophages ◽  
Kinase C ◽  
Pkc Isoforms

Protein kinase C (PKC) plays a prominent role in immune signaling, and the paradigms for isoform selective signaling are beginning to be elucidated. Real-time microscopy was combined with molecular and biochemical approaches to demonstrate a role for PKC-ε in Fcγ receptor (FcγR)–dependent phagocytosis. RAW 264.7 macrophages were transfected with GFP-conjugated PKC isoforms, and GFP movement was followed during phagocytosis of fluorescent IgG–opsonized beads. PKC-ε, but not PKC-δ, concentrated around the beads. PKC-ε accumulation was transient; apparent as a “flash” on target ingestion. Similarly, endogenous PKC-ε was specifically recruited to the nascent phagosomes in a time-dependent manner. Overexpression of PKC-ε, but not PKC-α, PKC-δ, or PKC-γ enhanced bead uptake 1.8-fold. Additionally, the rate of phagocytosis in GFP PKC-ε expressors was twice that of cells expressing GFP PKC-δ. Expression of the regulatory domain (εRD) and the first variable region (εV1) of PKC-ε inhibited uptake, whereas the corresponding PKC-δ region had no effect. Actin polymerization was enhanced on expression of GFP PKC-ε and εRD, but decreased in cells expressing εV1, suggesting that the εRD and εV1 inhibition of phagocytosis is not due to effects on actin polymerization. These results demonstrate a role for PKC-ε in FcγR-mediated phagocytosis that is independent of its effects on actin assembly.

scholarly journals Cortactin and dynamin are required for the clathrin-independent endocytosis of γc cytokine receptor

2004 ◽  
Vol 168 (1) ◽  
pp. 155-163 ◽  
Author(s):  
Nathalie Sauvonnet ◽  
Annick Dujeancourt ◽  
Alice Dautry-Varsat
Keyword(s):  
Rna Interference ◽  
Actin Polymerization ◽  
Dominant Negative ◽  
Cytokine Receptor ◽  
Actin Dynamics ◽  
Dominant Negative Mutant ◽  
Cellular Functions ◽  
Receptor Endocytosis ◽  
The Common ◽  
Negative Mutant

Endocytosis is critical for many cellular functions. We show that endocytosis of the common γc cytokine receptor is clathrin independent by using a dominant-negative mutant of Eps15 or RNA interference to knock down clathrin heavy chain. This pathway is synaptojanin independent and requires the GTPase dynamin. In addition, this process requires actin polymerization. To further characterize the function of dynamin in clathrin-independent endocytosis, in particular its connection with the actin cytoskeleton, we focused on dynamin-binding proteins that interact with F-actin. We compared the involvement of these proteins in the clathrin-dependent and -independent pathways. Thus, we observed that intersectin, syndapin, and mAbp1, which are necessary for the uptake of transferrin (Tf), a marker of the clathrin route, are not required for γc receptor endocytosis. Strikingly, cortactin is needed for both γc and Tf internalizations. These results reveal the ubiquitous action of cortactin in internalization processes and suggest its role as a linker between actin dynamics and clathrin-dependent and -independent endocytosis.

scholarly journals The Role of the Exocyst in Matrix Metalloproteinase Secretion and Actin Dynamics during Tumor Cell Invadopodia Formation

2009 ◽  
Vol 20 (16) ◽  
pp. 3763-3771 ◽  
Author(s):  
Jianglan Liu ◽  
Peng Yue ◽  
Vira V. Artym ◽  
Susette C. Mueller ◽  
Wei Guo
Keyword(s):  
Extracellular Matrix ◽  
Cell Invasion ◽  
Actin Polymerization ◽  
Dominant Negative ◽  
The Other ◽  
Actin Dynamics ◽  
Secretory Vesicles ◽  
Membrane Protrusions ◽  
Invadopodia Formation

Invadopodia are actin-rich membrane protrusions formed by tumor cells that degrade the extracellular matrix for invasion. Invadopodia formation involves membrane protrusions driven by Arp2/3-mediated actin polymerization and secretion of matrix metalloproteinases (MMPs) at the focal degrading sites. The exocyst mediates the tethering of post-Golgi secretory vesicles at the plasma membrane for exocytosis and has recently been implicated in regulating actin dynamics during cell migration. Here, we report that the exocyst plays a pivotal role in invadopodial activity. With RNAi knockdown of the exocyst component Exo70 or Sec8, MDA-MB-231 cells expressing constitutively active c-Src failed to form invadopodia. On the other hand, overexpression of Exo70 promoted invadopodia formation. Disrupting the exocyst function by siEXO70 or siSEC8 treatment or by expression of a dominant negative fragment of Exo70 inhibited the secretion of MMPs. We have also found that the exocyst interacts with the Arp2/3 complex in cells with high invasion potential; blocking the exocyst-Arp2/3 interaction inhibited Arp2/3-mediated actin polymerization and invadopodia formation. Together, our results suggest that the exocyst plays important roles in cell invasion by mediating the secretion of MMPs at focal degrading sites and regulating Arp2/3-mediated actin dynamics.

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 Burkholderia mallei Cluster 1 Type VI Secretion Mutants Exhibit Growth and Actin Polymerization Defects in RAW 264.7 Murine Macrophages

2009 ◽  
Vol 78 (1) ◽  
pp. 88-99 ◽  
Author(s):  
Mary N. Burtnick ◽  
David DeShazer ◽  
Vinod Nair ◽  
Frank C. Gherardini ◽  
Paul J. Brett
Keyword(s):  
Fluorescence Microscopy ◽  
Actin Polymerization ◽  
Severe Disease ◽  
Critical Role ◽  
Raw 264.7 Cells ◽  
Raw 264.7 ◽  
Burkholderia Mallei ◽  
Type Vi Secretion System ◽  
Murine Macrophages ◽  
Type Vi Secretion

ABSTRACT Burkholderia mallei is a facultative intracellular pathogen that causes severe disease in animals and humans. Recent studies have shown that the cluster 1 type VI secretion system (T6SS-1) expressed by this organism is essential for survival in a hamster model of glanders. To better understand the role of T6SS-1 in the pathogenesis of disease, studies were initiated to examine the interactions of B. mallei tssE mutants with RAW 264.7 murine macrophages. Results obtained by utilizing modified gentamicin protection assays indicated that although the tssE mutants were able to survive within RAW 264.7 cells, significant growth defects were observed in comparison to controls. In addition, analysis of infected monolayers by differential interference contrast and fluorescence microscopy demonstrated that the tssE mutants lacked the ability to induce multinucleated giant cell formation. Via the use of fluorescence microscopy, tssE mutants were shown to undergo escape from lysosome-associated membrane protein 1-positive vacuoles. Curiously, however, following entry into the cytosol, the mutants exhibited actin polymerization defects resulting in inefficient intra- and intercellular spread characteristics. Importantly, all mutant phenotypes observed in this study could be restored by complementation. Based upon these findings, it appears that T6SS-1 plays a critical role in growth and actin-based motility following uptake of B. mallei by RAW 264.7 cells.

Invited Review: Focal adhesion and small heat shock proteins in the regulation of actin remodeling and contractility in smooth muscle

2001 ◽  
Vol 91 (2) ◽  
pp. 963-972 ◽  
Author(s):  
William T. Gerthoffer ◽  
Susan J. Gunst
Keyword(s):  
Smooth Muscle ◽  
Heat Shock ◽  
Signaling Pathways ◽  
Focal Adhesion ◽  
Actin Polymerization ◽  
Smooth Muscles ◽  
Actin Dynamics ◽  
Actin Remodeling ◽  
Adhesion Proteins ◽  
Focal Adhesion Proteins

Smooth muscle cells are able to adapt rapidly to chemical and mechanical signals impinging on the cell surface. It has been suggested that dynamic changes in the actin cytoskeleton contribute to the processes of contractile activation and mechanical adaptation in smooth muscle. In this review, evidence for functionally important changes in actin polymerization during smooth muscle contraction is summarized. The functions and regulation of proteins associated with “focal adhesion complexes” (membrane-associated dense plaques) in differentiated smooth muscle, including integrins, focal adhesion kinase (FAK), c-Src, paxillin, and the 27-kDa small heat shock protein (HSP27) are described. Integrins in smooth muscles are key elements of mechanotransduction pathways that communicate with and are regulated by focal adhesion proteins that include FAK, c-Src, and paxillin as well as proteins known to mediate cytoskeletal remodeling. Evidence that functions of FAK and c-Src protein kinases are closely intertwined is discussed as well as evidence that focal adhesion proteins mediate key signal transduction events that regulate actin remodeling and contraction. HSP27 is reviewed as a potentially significant effector protein that may regulate actin dynamics and cross-bridge function in response to activation of p21-activated kinase and the p38 mitogen-activated protein kinase signaling pathway by signaling pathways linked to integrin proteins. These signaling pathways are only part of a large number of yet to be defined pathways that mediate acute adaptive responses of the cytoskeleton in smooth muscle to environmental stimuli.

scholarly journals The Tyrosine Kinase Activity of c-Src Regulates Actin Dynamics and Organization of Podosomes in Osteoclasts

2008 ◽  
Vol 19 (1) ◽  
pp. 394-404 ◽  
Author(s):  
Olivier Destaing ◽  
Archana Sanjay ◽  
Cecile Itzstein ◽  
William C. Horne ◽  
Derek Toomre ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Tyrosine Kinase ◽  
Life Span ◽  
Actin Polymerization ◽  
Fluorescence Recovery After Photobleaching ◽  
Kinase Activity ◽  
Dominant Negative ◽  
Actin Dynamics ◽  
Tyrosine Kinase Activity ◽  
The Core

Podosomes are dynamic actin-rich structures composed of a dense F-actin core surrounded by a cloud of more diffuse F-actin. Src performs one or more unique functions in osteoclasts (OCLs), and podosome belts and bone resorption are impaired in the absence of Src. Using Src−/−OCLs, we investigated the specific functions of Src in the organization and dynamics of podosomes. We found that podosome number and the podosome-associated actin cloud were decreased in Src−/−OCLs. Videomicroscopy and fluorescence recovery after photobleaching analysis revealed that the life span of Src−/−podosomes was increased fourfold and that the rate of actin flux in the core was decreased by 40%. Thus, Src regulates the formation, structure, life span, and rate of actin polymerization in podosomes and in the actin cloud. Rescue of Src−/−OCLs with Src mutants showed that both the kinase activity and either the SH2 or the SH3 binding domain are required for Src to restore normal podosome organization and dynamics. Moreover, inhibition of Src family kinase activities in Src−/−OCLs by Src inhibitors or by expressing dominant-negative SrcK295Minduced the formation of abnormal podosomes. Thus, Src is an essential regulator of podosome structure, dynamics and organization.

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