scholarly journals A role for cofilin and LIM kinase in Listeria-induced phagocytosis

2001 ◽  
Vol 155 (1) ◽  
pp. 101-112 ◽  
Author(s):  
Hélène Bierne ◽  
Edith Gouin ◽  
Pascal Roux ◽  
Pico Caroni ◽  
Helen L. Yin ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Actin Filaments ◽  
Mammalian Cells ◽  
Actin Polymerization ◽  
Essential Feature ◽  
Dominant Negative ◽  
Comet Tail ◽  
Lim Kinase ◽  
Induced Membrane ◽  
Membrane Ruffling

The pathogenic bacterium Listeria monocytogenes is able to invade nonphagocytic cells, an essential feature for its pathogenicity. This induced phagocytosis process requires tightly regulated steps of actin polymerization and depolymerization. Here, we investigated how interactions of the invasion protein InlB with mammalian cells control the cytoskeleton during Listeria internalization. By fluorescence microscopy and transfection experiments, we show that the actin-nucleating Arp2/3 complex, the GTPase Rac, LIM kinase (LIMK), and cofilin are key proteins in InlB-induced phagocytosis. Overexpression of LIMK1, which has been shown to phosphorylate and inactivate cofilin, induces accumulation of F-actin beneath entering particles and inhibits internalization. Conversely, inhibition of LIMK's activity by expressing a dominant negative construct, LIMK1−, or expression of the constitutively active S3A cofilin mutant induces loss of actin filaments at the phagocytic cup and also inhibits phagocytosis. Interestingly, those constructs similarly affect other actin-based phenomenons, such as InlB-induced membrane ruffling or Listeria comet tail formations. Thus, our data provide evidence for a control of phagocytosis by both activation and deactivation of cofilin. We propose a model in which cofilin is involved in the formation and disruption of the phagocytic cup as a result of its local progressive enrichment.

scholarly journals Fascin-mediated propulsion of Listeria monocytogenes independent of frequent nucleation by the Arp2/3 complex

2004 ◽  
Vol 165 (2) ◽  
pp. 233-242 ◽  
Author(s):  
William M. Brieher ◽  
Margaret Coughlin ◽  
Timothy J. Mitchison
Keyword(s):  
Listeria Monocytogenes ◽  
Hollow Cylinder ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Soluble Factor ◽  
Comet Tail ◽  
Structural Differences ◽  
Brain Extracts ◽  
Nucleation Phase ◽  
Elongation Phase

Actin-dependent propulsion of Listeria monocytogenes is thought to require frequent nucleation of actin polymerization by the Arp2/3 complex. We demonstrate that L. monocytogenes motility can be separated into an Arp2/3-dependent nucleation phase and an Arp2/3-independent elongation phase. Elongation-based propulsion requires a unique set of biochemical factors in addition to those required for Arp2/3-dependent motility. We isolated fascin from brain extracts as the only soluble factor required in addition to actin during the elongation phase for this type of movement. The nucleation reaction assembles a comet tail of branched actin filaments directly behind the bacterium. The elongation-based reaction generates a hollow cylinder of parallel bundles that attach along the sides of the bacterium. Bacteria move faster in the elongation reaction than in the presence of Arp2/3, and the rate is limited by the concentration of G-actin. The biochemical and structural differences between the two motility reactions imply that each operates through distinct biochemical and biophysical mechanisms.

scholarly journals Hyperosmotic stress induces Rho/Rho kinase/LIM kinase-mediated cofilin phosphorylation in tubular cells: key role in the osmotically triggered F-actin response

2009 ◽  
Vol 296 (3) ◽  
pp. C463-C475 ◽  
Author(s):  
Ana C. P. Thirone ◽  
Pam Speight ◽  
Matthew Zulys ◽  
Ori D. Rotstein ◽  
Katalin Szászi ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
De Novo ◽  
Rho Kinase ◽  
Small Gtpases ◽  
Hyperosmotic Stress ◽  
Dominant Negative ◽  
Lim Kinase ◽  
Tubular Cells ◽  
Cofilin Phosphorylation ◽  
Underlying Mechanisms

Hyperosmotic stress induces cytoskeleton reorganization and a net increase in cellular F-actin, but the underlying mechanisms are incompletely understood. Whereas de novo F-actin polymerization likely contributes to the actin response, the role of F-actin severing is unknown. To address this problem, we investigated whether hyperosmolarity regulates cofilin, a key actin-severing protein, the activity of which is inhibited by phosphorylation. Since the small GTPases Rho and Rac are sensitive to cell volume changes and can regulate cofilin phosphorylation, we also asked whether they might link osmostress to cofilin. Here we show that hyperosmolarity induced rapid, sustained, and reversible phosphorylation of cofilin in kidney tubular (LLC-PK1 and Madin-Darby canine kidney) cells. Hyperosmolarity-provoked cofilin phosphorylation was mediated by the Rho/Rho kinase (ROCK)/LIM kinase (LIMK) but not the Rac/PAK/LIMK pathway, because 1) dominant negative (DN) Rho and DN-ROCK but not DN-Rac and DN-PAK inhibited cofilin phosphorylation; 2) constitutively active (CA) Rho and CA-ROCK but not CA-Rac and CA-PAK induced cofilin phosphorylation; 3) hyperosmolarity induced LIMK-2 phosphorylation, and 4) inhibition of ROCK by Y-27632 suppressed the hypertonicity-triggered LIMK-2 and cofilin phosphorylation.We thenexamined whether cofilin and its phosphorylation play a role in the hypertonicity-triggered F-actin changes. Downregulation of cofilin by small interfering RNA increased the resting F-actin level and eliminated any further rise upon hypertonic treatment. Inhibition of cofilin phosphorylation by Y-27632 prevented the hyperosmolarity-provoked F-actin increase. Taken together, cofilin is necessary for maintaining the osmotic responsiveness of the cytoskeleton in tubular cells, and the Rho/ROCK/LIMK-mediated cofilin phosphorylation is a key mechanism in the hyperosmotic stress-induced F-actin increase.

scholarly journals Receptor-mediated Drp1 oligomerization on endoplasmic reticulum

2017 ◽  
Vol 216 (12) ◽  
pp. 4123-4139 ◽  
Author(s):  
Wei-Ke Ji ◽  
Rajarshi Chakrabarti ◽  
Xintao Fan ◽  
Lori Schoenfeld ◽  
Stefan Strack ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Actin Filaments ◽  
Mammalian Cells ◽  
Actin Polymerization ◽  
Mitochondrial Division ◽  
Multiple Factors ◽  
Guanosine Triphosphatase

Drp1 is a dynamin guanosine triphosphatase important for mitochondrial and peroxisomal division. Drp1 oligomerization and mitochondrial recruitment are regulated by multiple factors, including interaction with mitochondrial receptors such as Mff, MiD49, MiD51, and Fis. In addition, both endoplasmic reticulum (ER) and actin filaments play positive roles in mitochondrial division, but mechanisms for their roles are poorly defined. Here, we find that a population of Drp1 oligomers is associated with ER in mammalian cells and is distinct from mitochondrial or peroxisomal Drp1 populations. Subpopulations of Mff and Fis1, which are tail-anchored proteins, also localize to ER. Drp1 oligomers assemble on ER, from which they can transfer to mitochondria. Suppression of Mff or inhibition of actin polymerization through the formin INF2 significantly reduces all Drp1 oligomer populations (mitochondrial, peroxisomal, and ER bound) and mitochondrial division, whereas Mff targeting to ER has a stimulatory effect on division. Our results suggest that ER can function as a platform for Drp1 oligomerization, and that ER-associated Drp1 contributes to mitochondrial division.

scholarly journals The Isolated Comet Tail Pseudopodium of Listeria monocytogenes: A Tail of Two Actin Filament Populations, Long and Axial and Short and Random

1997 ◽  
Vol 137 (1) ◽  
pp. 155-167 ◽  
Author(s):  
Antonio S. Sechi ◽  
Jürgen Wehland ◽  
J. Victor Small
Keyword(s):  
Listeria Monocytogenes ◽  
Simple Model ◽  
Actin Filaments ◽  
Adjacent Cell ◽  
Axial Component ◽  
Cell Type ◽  
Macrophage Cell Line ◽  
Comet Tail ◽  
Macrophage Cell ◽  
Host Cytoplasm

Listeria monocytogenes is driven through infected host cytoplasm by a comet tail of actin filaments that serves to project the bacterium out of the cell surface, in pseudopodia, to invade neighboring cells. The characteristics of pseudopodia differ according to the infected cell type. In PtK2 cells, they reach a maximum length of ∼15 μm and can gyrate actively for several minutes before reentering the same or an adjacent cell. In contrast, the pseudopodia of the macrophage cell line DMBM5 can extend to >100 μm in length, with the bacteria at their tips moving at the same speed as when at the head of comet tails in bulk cytoplasm. We have now isolated the pseudopodia from PtK2 cells and macrophages and determined the organization of actin filaments within them. It is shown that they possess a major component of long actin filaments that are more or less splayed out in the region proximal to the bacterium and form a bundle along the remainder of the tail. This axial component of filaments is traversed by variable numbers of short, randomly arranged filaments whose number decays along the length of the pseudopodium. The tapering of the tail is attributed to a grading in length of the long, axial filaments. The exit of a comet tail from bulk cytoplasm into a pseudopodium is associated with a reduction in total F-actin, as judged by phalloidin staining, the shedding of α-actinin, and the accumulation of ezrin. We propose that this transition reflects the loss of a major complement of short, random filaments from the comet, and that these filaments are mainly required to maintain the bundled form of the tail when its borders are not restrained by an enveloping pseudopodium membrane. A simple model is put forward to explain the origin of the axial and randomly oriented filaments in the comet tail.

scholarly journals The Transcription Factor AP-1 Is Required for EGF-induced Activation of Rho-like GTPases, Cytoskeletal Rearrangements, Motility, and In Vitro Invasion of A431 Cells

1998 ◽  
Vol 143 (4) ◽  
pp. 1087-1099 ◽  
Author(s):  
Angeliki Malliri ◽  
Marc Symons ◽  
Robert F. Hennigan ◽  
Adam F.L. Hurlstone ◽  
Richard F. Lamb ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Cortical Actin ◽  
A431 Cells ◽  
Membrane Ruffling ◽  
In Vitro Invasion ◽  
Cytoskeletal Rearrangements ◽  
Lamellipodia Formation

Human squamous cell carcinomas (SCC) frequently express elevated levels of epidermal growth factor receptor (EGFR). EGFR overexpression in SCC-derived cell lines correlates with their ability to invade in an in vitro invasion assay in response to EGF, whereas benign epidermal cells, which express low levels of EGFR, do not invade. EGF-induced invasion of SCC-derived A431 cells is inhibited by sustained expression of the dominant negative mutant of c-Jun, TAM67, suggesting a role for the transcription factor AP-1 (activator protein-1) in regulating invasion. Significantly, we establish that sustained TAM67 expression inhibits growth factor–induced cell motility and the reorganization of the cytoskeleton and cell-shape changes essential for this process: TAM67 expression inhibits EGF-induced membrane ruffling, lamellipodia formation, cortical actin polymerization and cell rounding. Introduction of a dominant negative mutant of Rac and of the Rho inhibitor C3 transferase into A431 cells indicates that EGF-induced membrane ruffling and lamellipodia formation are regulated by Rac, whereas EGF-induced cortical actin polymerization and cell rounding are controlled by Rho. Constitutively activated mutants of Rac or Rho introduced into A431 or A431 cells expressing TAM67 (TA cells) induce equivalent actin cytoskeletal rearrangements, suggesting that the effector pathways downstream of Rac and Rho required for these responses are unimpaired by sustained TAM67 expression. However, EGF-induced translocation of Rac to the cell membrane, which is associated with its activation, is defective in TA cells. Our data establish a novel link between AP-1 activity and EGFR activation of Rac and Rho, which in turn mediate the actin cytoskeletal rearrangements required for cell motility and invasion.

A comparative study of the actin-based motilities of the pathogenic bacteria Listeria monocytogenes, Shigella flexneri and Rickettsia conorii

1999 ◽  
Vol 112 (11) ◽  
pp. 1697-1708 ◽  
Author(s):  
E. Gouin ◽  
H. Gantelet ◽  
C. Egile ◽  
I. Lasa ◽  
H. Ohayon ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Actin Filaments ◽  
Pathogenic Bacteria ◽  
Actin Polymerization ◽  
Shigella Flexneri ◽  
Rickettsia Conorii ◽  
Bacterial Proteins ◽  
Egg Extracts ◽  
Infected Cells ◽  
Syndrome Protein

Listeria monocytogenes, Shigella flexneri, and Rickettsia conorii are three bacterial pathogens that are able to polymerize actin into ‘comet tail’ structures and move within the cytosol of infected cells. The actin-based motilities of L. monocytogenes and S. flexneri are known to require the bacterial proteins ActA and IcsA, respectively, and several mammalian cytoskeleton proteins including the Arp2/3 complex and VASP (vasodilator-stimulated phosphoprotein) for L. monocytogenes and vinculin and N-WASP (the neural Wiskott-Aldrich syndrome protein) for S. flexneri. In contrast, little is known about the motility of R. conorii. In the present study, we have analysed the actin-based motility of this bacterium in comparison to that of L. monocytogenes and S. flexneri. Rickettsia moved at least three times more slowly than Listeria and Shigella in both infected cells and Xenopus laevis egg extracts. Decoration of actin with the S1 subfragment of myosin in infected cells showed that the comet tails of Rickettsia have a structure strikingly different from those of L. monocytogenes or S. flexneri. In Listeria and Shigella tails, actin filaments form a branching network while Rickettsia tails display longer and not cross-linked actin filaments. Immunofluorescence studies revealed that the two host proteins, VASP and (α)-actinin colocalized with actin in the tails of Rickettsia but neither the Arp2/3 complex which we detected in the Shigella actin tails, nor N-WASP, were detected in Rickettsia actin tails. Taken together, these results suggest that R. conorii may use a different mechanism of actin polymerization.

scholarly journals Rapid actin monomer–insensitive depolymerization of Listeria actin comet tails by cofilin, coronin, and Aip1

2006 ◽  
Vol 175 (2) ◽  
pp. 315-324 ◽  
Author(s):  
William M. Brieher ◽  
Hao Yuan Kueh ◽  
Bryan A. Ballif ◽  
Timothy J. Mitchison
Keyword(s):  
Listeria Monocytogenes ◽  
Actin Filaments ◽  
Actin Monomer ◽  
Comet Tail ◽  
Biochemical Basis ◽  
Thymus Extract ◽  
Low Concentrations ◽  
Biochemical Fractionation ◽  
High Concentrations ◽  
Actin Comet Tails

Actin filaments in cells depolymerize rapidly despite the presence of high concentrations of polymerizable G actin. Cofilin is recognized as a key regulator that promotes actin depolymerization. In this study, we show that although pure cofilin can disassemble Listeria monocytogenes actin comet tails, it cannot efficiently disassemble comet tails in the presence of polymerizable actin. Thymus extracts also rapidly disassemble comet tails, and this reaction is more efficient than pure cofilin when normalized to cofilin concentration. By biochemical fractionation, we identify Aip1 and coronin as two proteins present in thymus extract that facilitate the cofilin-mediated disassembly of Listeria comet tails. Together, coronin and Aip1 lower the amount of cofilin required to disassemble the comet tail and permit even low concentrations of cofilin to depolymerize actin in the presence of polymerizable G actin. The cooperative activities of cofilin, coronin, and Aip1 should provide a biochemical basis for understanding how actin filaments can grow in some places in the cell while shrinking in others.

scholarly journals The Host GTPase Arf1 and Its Effectors AP1 and PICK1 Stimulate Actin Polymerization and Exocytosis To Promote Entry of Listeria monocytogenes

2019 ◽  
Vol 88 (2) ◽  
Author(s):  
Susan Saila ◽  
Gaurav Chandra Gyanwali ◽  
Mazhar Hussain ◽  
Antonella Gianfelice ◽  
Keith Ireton
Keyword(s):  
Plasma Membrane ◽  
Listeria Monocytogenes ◽  
Actin Polymerization ◽  
Surface Protein ◽  
Dominant Negative ◽  
Host Cells ◽  
Filamin A ◽  
Content Type ◽  
Bacterial Surface ◽  
Dominant Negative Allele

ABSTRACT Listeria monocytogenes is a foodborne bacterium that causes gastroenteritis, meningitis, or abortion. Listeria induces its internalization (entry) into some human cells through interaction of the bacterial surface protein InlB with its host receptor, the Met tyrosine kinase. InlB and Met promote entry through stimulation of localized actin polymerization and exocytosis. How actin cytoskeletal changes and exocytosis are controlled during entry is not well understood. Here, we demonstrate important roles for the host GTPase Arf1 and its effectors AP1 and PICK1 in actin polymerization and exocytosis during InlB-dependent uptake. Depletion of Arf1 by RNA interference (RNAi) or inhibition of Arf1 activity using a dominant-negative allele impaired InlB-dependent internalization, indicating an important role for Arf1 in this process. InlB stimulated an increase in the GTP-bound form of Arf1, demonstrating that this bacterial protein activates Arf1. RNAi and immunolocalization studies indicated that Arf1 controls exocytosis and actin polymerization during entry by recruiting the effectors AP1 and PICK1 to the plasma membrane. In turn, AP1 and PICK1 promoted plasma membrane translocation of both Filamin A (FlnA) and Exo70, two host proteins previously found to mediate exocytosis during InlB-dependent internalization (M. Bhalla, H. Van Ngo, G. C. Gyanwali, and K. Ireton, Infect Immun 87:e00689-18, 2018, https://doi.org/10.1128/IAI.00689-18). PICK1 mediated recruitment of Exo70 but not FlnA. Collectively, these results indicate that Arf1, AP1, and PICK1 stimulate exocytosis by redistributing FlnA and Exo70 to the plasma membrane. We propose that Arf1, AP1, and PICK1 are key coordinators of actin polymerization and exocytosis during infection of host cells by Listeria.

scholarly journals Phosphorylation of Adducin by Rho-Kinase Plays a Crucial Role in Cell Motility

1999 ◽  
Vol 145 (2) ◽  
pp. 347-361 ◽  
Author(s):  
Yuko Fukata ◽  
Noriko Oshiro ◽  
Nagatoki Kinoshita ◽  
Yoji Kawano ◽  
Yoichiro Matsuoka ◽  
...  
Keyword(s):  
Cell Motility ◽  
Crucial Role ◽  
Plasma Membranes ◽  
Mdck Cells ◽  
Rho Kinase ◽  
Dominant Negative ◽  
Actin Binding ◽  
Dependent Manner ◽  
Induced Membrane ◽  
Membrane Ruffling

Adducin is a membrane skeletal protein that binds to actin filaments (F-actin) and thereby promotes the association of spectrin with F-actin to form a spectrin-actin meshwork beneath plasma membranes such as ruffling membranes. Rho-associated kinase (Rho- kinase), which is activated by the small guanosine triphosphatase Rho, phosphorylates α-adducin and thereby enhances the F-actin–binding activity of α-adducin in vitro. Here we identified the sites of phosphorylation of α-adducin by Rho-kinase as Thr445 and Thr480. We prepared antibody that specifically recognized α-adducin phosphorylated at Thr445, and found by use of this antibody that Rho-kinase phosphorylated α-adducin at Thr445 in COS7 cells in a Rho-dependent manner. Phosphorylated α-adducin accumulated in the membrane ruffling area of Madin-Darby canine kidney (MDCK) epithelial cells and the leading edge of scattering cells during the action of tetradecanoylphorbol-13-acetate (TPA) or hepatocyte growth factor (HGF). The microinjection of Botulinum C3 ADP-ribosyl-transferase, dominant negative Rho-kinase, or α-adducinT445A,T480A (substitution of Thr445 and Thr480 by Ala) inhibited the TPA-induced membrane ruffling in MDCK cells and wound-induced migra- tion in NRK49F cells. α-AdducinT445D,T480D (substi- tution of Thr445 and Thr480 by Asp), but not α-adducinT445A,T480A, counteracted the inhibitory effect of the dominant negative Rho-kinase on the TPA-induced membrane ruffling in MDCK cells. Taken together, these results indicate that Rho-kinase phosphorylates α-adducin downstream of Rho in vivo, and that the phosphorylation of adducin by Rho-kinase plays a crucial role in the regulation of membrane ruffling and cell motility.

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