scholarly journals Rac1 and Rac2 differentially regulate actin free barbed end formation downstream of the fMLP receptor

2007 ◽  
Vol 179 (2) ◽  
pp. 239-245 ◽  
Author(s):  
Chun Xiang Sun ◽  
Marco A.O. Magalhães ◽  
Michael Glogauer
Keyword(s):  
Actin Cytoskeleton ◽  
De Novo ◽  
Leading Edge ◽  
Small Gtpases ◽  
Actin Dynamics ◽  
Membrane Protrusion ◽  
Actin Assembly ◽  
Unique Combination ◽  
High Affinity ◽  
Fmlp Receptor

Actin assembly at the leading edge of migrating cells depends on the availability of high-affinity free barbed ends (FBE) that drive actin filament elongation and subsequent membrane protrusion. We investigated the specific mechanisms through which the Rac1 and Rac2 small guanosine triphosphatases (GTPases) generate free barbed ends in neutrophils. Using neutrophils lacking either Rac1 or Rac2 and a neutrophil permeabilization model that maintains receptor signaling to the actin cytoskeleton, we assessed the mechanisms through which these two small GTPases mediate FBE generation downstream of the formyl-methionyl-leucyl-phenylalanine receptor. We demonstrate here that uncapping of existing barbed ends is mediated through Rac1, whereas cofilin- and ARP2/3-mediated FBE generation are regulated through Rac2. This unique combination of experimental tools has allowed us to identify the relative roles of uncapping (15%), cofilin severing (10%), and ARP2/3 de novo nucleation (75%) in FBE generation and the respective roles played by Rac1 and Rac2 in mediating actin dynamics.

scholarly journals Capping Protein Terminates but Does Not Initiate Chemoattractant-induced Actin Assembly in Dictyostelium

1997 ◽  
Vol 139 (5) ◽  
pp. 1243-1253 ◽  
Author(s):  
R.J. Eddy ◽  
J. Han ◽  
J.S. Condeelis
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Polymerization ◽  
De Novo ◽  
Leading Edge ◽  
Actin Assembly ◽  
Directed Movement ◽  
Capping Protein ◽  
End Capping

The first step in the directed movement of cells toward a chemotactic source involves the extension of pseudopods initiated by the focal nucleation and polymerization of actin at the leading edge of the cell. We have previously isolated a chemoattractant-regulated barbed-end capping activity from Dictyostelium that is uniquely associated with capping protein, also known as cap32/34. Although uncapping of barbed ends by capping protein has been proposed as a mechanism for the generation of free barbed ends after stimulation, in vitro and in situ analysis of the association of capping protein with the actin cytoskeleton after stimulation reveals that capping protein enters, but does not exit, the cytoskeleton during the initiation of actin polymerization. Increased association of capping protein with regions of the cell containing free barbed ends as visualized by exogenous rhodamine-labeled G-actin is also observed after stimulation. An approximate threefold increase in the number of filaments with free barbed ends is accompanied by increases in absolute filament number, whereas the average filament length remains constant. Therefore, a mechanism in which preexisting filaments are uncapped by capping protein, in response to stimulation leading to the generation of free barbed ends and filament elongation, is not supported. A model for actin assembly after stimulation, whereby free barbed ends are generated by either filament severing or de novo nucleation is proposed. In this model, exposure of free barbed ends results in actin assembly, followed by entry of free capping protein into the actin cytoskeleton, which acts to terminate, not initiate, the actin polymerization transient.

Osmotic stress-induced remodeling of the cortical cytoskeleton

2002 ◽  
Vol 283 (3) ◽  
pp. C850-C865 ◽  
Author(s):  
Caterina Di Ciano ◽  
Zilin Nie ◽  
Katalin Szászi ◽  
Alison Lewis ◽  
Takehito Uruno ◽  
...  
Keyword(s):  
Osmotic Stress ◽  
De Novo ◽  
Small Gtpases ◽  
Dominant Negative ◽  
Actin Binding ◽  
Cell Cortex ◽  
Actin Assembly ◽  
Signaling Mechanism ◽  
Actin Binding Domain ◽  
Cytoskeleton Remodeling

Osmotic stress is known to affect the cytoskeleton; however, this adaptive response has remained poorly characterized, and the underlying signaling pathways are unexplored. Here we show that hypertonicity induces submembranous de novo F-actin assembly concomitant with the peripheral translocation and colocalization of cortactin and the actin-related protein 2/3 (Arp2/3) complex, which are key components of the actin nucleation machinery. Additionally, hyperosmolarity promotes the association of cortactin with Arp2/3 as revealed by coimmunoprecipitation. Using various truncation or phosphorylation-incompetent mutants, we show that cortactin translocation requires the Arp2/3- or the F-actin binding domain, but the process is independent of the shrinkage-induced tyrosine phosphorylation of cortactin. Looking for an alternative signaling mechanism, we found that hypertonicity stimulates Rac and Cdc42. This appears to be a key event in the osmotically triggered cytoskeletal reorganization, because 1) constitutively active small GTPases translocate cortactin, 2) Rac and cortactin colocalize at the periphery of hypertonically challenged cells, and 3) dominant-negative Rac and Cdc42 inhibit the hypertonicity-provoked cortactin and Arp3 translocation. The Rho family-dependent cytoskeleton remodeling may be an important osmoprotective response that reinforces the cell cortex.

scholarly journals A new form of actin assembly around the Shigella-containing vacuole regulates its intracellular niche

2020 ◽  
Author(s):  
Sonja Kühn ◽  
John Bergqvist ◽  
Laura Barrio ◽  
Stephanie Lebreton ◽  
Chiara Zurzolo ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Polymerization ◽  
De Novo ◽  
Shigella Flexneri ◽  
Host Cells ◽  
Host Recognition ◽  
Actin Assembly ◽  
Actin Structure ◽  
New Form ◽  
Structure And Functions

SUMMARYThe enteroinvasive bacterium Shigella flexneri forces its uptake into non-phagocytic host cells through the translocation of T3SS effectors that subvert the actin cytoskeleton. Here, we report de novo actin polymerization after cellular entry around the bacterial containing vacuole (BCV) leading to the formation of a dynamic actin cocoon. This cocoon is thicker than any described cellular actin structure and functions as a gatekeeper for the cytosolic access of the pathogen. Host Cdc42, Toca-1, N-WASP, WIP, the Arp2/3 complex, cortactin, coronin, and cofilin are recruited to the actin cocoon. They are subverted by T3SS effectors, such as IpgD, IpgB1, and IcsB. IcsB immobilizes components of the actin polymerization machinery at the BCV. This represents a novel microbial subversion strategy through localized entrapment of host actin regulators causing massive actin assembly. We propose that the cocoon protects Shigella’s niche from canonical maturation or host recognition.

scholarly journals Rab40/Cullin5 complex regulates EPLIN and actin cytoskeleton dynamics during cell migration and invasion

2021 ◽  
Author(s):  
Erik S Linklater ◽  
Emily Duncan ◽  
Ke Jun Han ◽  
Algirdas Kaupinis ◽  
Mindaugas Valius ◽  
...  
Keyword(s):  
Cell Migration ◽  
Actin Cytoskeleton ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Leading Edge ◽  
Stress Fibers ◽  
Actin Dynamics ◽  
Migration And Invasion ◽  
Matrix Remodeling ◽  
Cell Migration And Invasion

Rab40b is a SOCS box containing protein that regulates the secretion of MMPs to facilitate extracellular matrix remodeling during cell migration. Here we show that Rab40b interacts with Cullin5 via the Rab40b SOCS domain. We demonstrate that loss of Rab40b/Cullin5 binding decreases cell motility and invasive potential, and show that defective cell migration and invasion stem from alteration to the actin cytoskeleton, leading to decreased invadopodia formation, decreased actin dynamics at the leading edge, and an increase in stress fibers. We also show that these stress fibers anchor at less dynamic, more stable focal adhesions. Mechanistically, changes in the cytoskeleton and focal adhesion dynamics are mediated in part by EPLIN, which we demonstrate to be a binding partner of Rab40b and a target for Rab40b/Cullin5 dependent localized ubiquitylation and degradation. Thus, we propose a model where the Rab40b/Cullin5 dependent ubiquitylation regulates EPLIN localization to promote cell migration and invasion by altering focal adhesion and cytoskeletal dynamics.

scholarly journals EGF stimulates an increase in actin nucleation and filament number at the leading edge of the lamellipod in mammary adenocarcinoma cells

1998 ◽  
Vol 111 (2) ◽  
pp. 199-211 ◽  
Author(s):  
A.Y. Chan ◽  
S. Raft ◽  
M. Bailly ◽  
J.B. Wyckoff ◽  
J.E. Segall ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
De Novo ◽  
Leading Edge ◽  
Mammary Adenocarcinoma ◽  
Actin Dynamics ◽  
Actin Nucleation ◽  
Nucleation Sites ◽  
Nucleation Activity ◽  
Pointed End ◽  
Stimulation Of

Stimulation of metastatic MTLn3 cells with EGF causes the rapid extension of lamellipods, which contain a zone of F-actin at the leading edge. In order to establish the mechanism for accumulation of F-actin at the leading edge and its relationship to lamellipod extension in response to EGF, we have studied the kinetics and location of EGF-induced actin nucleation activity in MTLn3 cells and characterized the actin dynamics at the leading edge by measuring the changes at the pointed and barbed ends of actin filaments upon EGF stimulation of MTLn3 cells. The major result of this study is that stimulation of MTLn3 cells with EGF causes a transient increase in actin nucleation activity resulting from the appearance of free barbed ends very close to the leading edge of extending lamellipods. In addition, cytochalasin D causes a significant decrease in the total F-actin content in EGF-stimulated cells, indicating that both actin polymerization and depolymerization are stimulated by EGF. Pointed end incorporation of rhodamine-labeled actin by the EGF stimulated cells is 2.12+/−0.47 times higher than that of control cells. Since EGF stimulation causes an increase in both barbed and pointed end incorporation of rhodamine-labeled actin in the same location, the EGF-stimulated nucleation sites are more likely due either to severing of pre-existing filaments or de novo nucleation of filaments at the leading edge thereby creating new barbed and pointed ends. The timing and location of EGF-induced actin nucleation activity in MTLn3 cells can account for the observed accumulation of F-actin at the leading edge and demonstrate that this F-actin rich zone is the primary actin polymerization zone after stimulation.

IgE alone-induced actin assembly modifies calcium signaling and degranulation in RBL-2H3 mast cells

2004 ◽  
Vol 286 (2) ◽  
pp. C256-C263 ◽  
Author(s):  
Tatsuya Oka ◽  
Masatoshi Hori ◽  
Akane Tanaka ◽  
Hiroshi Matsuda ◽  
Hideaki Karaki ◽  
...  
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Cell Activation ◽  
De Novo ◽  
Immunoglobulin E ◽  
Cytochalasin D ◽  
Mast Cell Activation ◽  
Actin Dynamics ◽  
Actin Assembly ◽  
Filamentous Actin

In the mast cell signaling pathways, the binding of immunoglobulin E (IgE) to FcϵRI, its high-affinity receptor, is generally thought to be a passive step. In this study, we examined the effect of IgE alone, that is, without antigen stimulation, on the degranulation in mast cells. Monomeric IgE (500–5,000 ng/ml) alone increased cytosolic Ca2+ level ([Ca2+]i) and induced degranulation in rat basophilic leukemia (RBL)-2H3 mast cells. Monomeric IgE (5,000 ng/ml) alone also increased [Ca2+]i and induced degranulation in bone marrow-derived mast cells. Interestingly, monomeric IgE (5–50 ng/ml) alone, in concentrations too low to induce degranulation, increased filamentous actin content in RBL-2H3 mast cells. We next examined whether actin dynamics affect the IgE alone-induced RBL-2H3 mast cell activation pathways. Cytochalasin D inhibited the ability of IgE alone (50 ng/ml) to induce de novo actin assembly. In cytochalasin D-treated cells, IgE (50 ng/ml) alone increased [Ca2+]i and induced degranulation. We have summarized the current findings into two points. First, IgE alone increases [Ca2+]i and induces degranulation in mast cells. Second, IgE, at concentrations too low to increase either [Ca2+]i or degranulation, significantly induces actin assembly, which serves as a negative feedback control in the mast cell Ca2+ signaling and degranulation.

scholarly journals Actin dynamics in living mammalian cells

1998 ◽  
Vol 111 (12) ◽  
pp. 1649-1658 ◽  
Author(s):  
C. Ballestrem ◽  
B. Wehrle-Haller ◽  
B.A. Imhof
Keyword(s):  
Actin Cytoskeleton ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Leading Edge ◽  
Time Lapse ◽  
Living Cells ◽  
Actin Dynamics ◽  
Mammalian Cell Lines ◽  
Cytoskeletal Dynamics ◽  
Green Fluorescent

The actin cytoskeleton maintains the cellular architecture and mediates cell movements. To explore actin cytoskeletal dynamics, the enhanced green fluorescent protein (EGFP) was fused to human β-actin. The fusion protein was incorporated into actin fibers which became depolymerized upon cytochalasin B treatment. This functional EGFP-actin construct enabled observation of the actin cytoskeleton in living cells by time lapse fluorescence microscopy. Stable expression of the construct was obtained in mammalian cell lines of different tissue origins. In stationary cells, actin rich, ring-like structured ‘actin clouds’ were observed in addition to stress fibers. These ruffle-like structures were found to be involved in the reorganization of the actin cytoskeleton. In migratory cells, EGFP-actin was found in the advancing lamellipodium. Immobile actin spots developed in the lamellipodium and thin actin fibers formed parallel to the leading edge. Thus EGFP-actin expressed in living cells unveiled structures involved in the dynamics of the actin cytoskeleton.

scholarly journals Modulation of Prion Formation, Aggregation, and Toxicity by the Actin Cytoskeleton in Yeast

2006 ◽  
Vol 26 (2) ◽  
pp. 617-629 ◽  
Author(s):  
Elena E. Ganusova ◽  
Laura N. Ozolins ◽  
Srishti Bhagat ◽  
Gary P. Newnam ◽  
Renee D. Wegrzyn ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Prion Protein ◽  
De Novo ◽  
Prion Diseases ◽  
Actin Assembly ◽  
Yeast Prion ◽  
Cortical Actin ◽  
Gene Coding ◽  
Cortical Cytoskeleton ◽  
Yeast Deletion

ABSTRACT Self-perpetuating protein aggregates transmit prion diseases in mammals and heritable traits in yeast. De novo prion formation can be induced by transient overproduction of the corresponding prion-forming protein or its prion domain. Here, we demonstrate that the yeast prion protein Sup35 interacts with various proteins of the actin cortical cytoskeleton that are involved in endocytosis. Sup35-derived aggregates, generated in the process of prion induction, are associated with the components of the endocytic/vacuolar pathway. Mutational alterations of the cortical actin cytoskeleton decrease aggregation of overproduced Sup35 and de novo prion induction and increase prion-related toxicity in yeast. Deletion of the gene coding for the actin assembly protein Sla2 is lethal in cells containing the prion isoforms of both Sup35 and Rnq1 proteins simultaneously. Our data are consistent with a model in which cytoskeletal structures provide a scaffold for generation of large aggregates, resembling mammalian aggresomes. These aggregates promote prion formation. Moreover, it appears that the actin cytoskeleton also plays a certain role in counteracting the toxicity of the overproduced potentially aggregating proteins.

scholarly journals α-Actinin-4/FSGS1 is required for Arp2/3-dependent actin assembly at the adherens junction

2012 ◽  
Vol 196 (1) ◽  
pp. 115-130 ◽  
Author(s):  
Vivian W. Tang ◽  
William M. Brieher
Keyword(s):  
De Novo ◽  
Adherens Junction ◽  
Dominant Negative ◽  
Cell Junctions ◽  
Actin Dynamics ◽  
Actin Assembly ◽  
Vitro Assay ◽  
Apical Junctions ◽  
Cell Cell

We have developed an in vitro assay to study actin assembly at cadherin-enriched cell junctions. Using this assay, we demonstrate that cadherin-enriched junctions can polymerize new actin filaments but cannot capture preexisting filaments, suggesting a mechanism involving de novo synthesis. In agreement with this hypothesis, inhibition of Arp2/3-dependent nucleation abolished actin assembly at cell–cell junctions. Reconstitution biochemistry using the in vitro actin assembly assay identified α-actinin-4/focal segmental glomerulosclerosis 1 (FSGS1) as an essential factor. α-Actinin-4 specifically localized to sites of actin incorporation on purified membranes and at apical junctions in Madin–Darby canine kidney cells. Knockdown of α-actinin-4 decreased total junctional actin and inhibited actin assembly at the apical junction. Furthermore, a point mutation of α-actinin-4 (K255E) associated with FSGS failed to support actin assembly and acted as a dominant negative to disrupt actin dynamics at junctional complexes. These findings demonstrate that α-actinin-4 plays an important role in coupling actin nucleation to assembly at cadherin-based cell–cell adhesive contacts.

scholarly journals A pharmacological cocktail for arresting actin dynamics in living cells

2011 ◽  
Vol 22 (21) ◽  
pp. 3986-3994 ◽  
Author(s):  
Grace E. Peng ◽  
Sarah R. Wilson ◽  
Orion D. Weiner
Keyword(s):  
Leading Edge ◽  
Actin Dynamics ◽  
Actin Assembly ◽  
Actin Reorganization ◽  
Cellular Processes ◽  
Wide Range ◽  
Morphological Polarity ◽  
Nih 3T3 ◽  
External Stimuli

The actin cytoskeleton is regulated by factors that influence polymer assembly, disassembly, and network rearrangement. Drugs that inhibit these events have been used to test the role of actin dynamics in a wide range of cellular processes. Previous methods of arresting actin rearrangements take minutes to act and work well in some contexts, but can lead to significant actin reorganization in cells with rapid actin dynamics, such as neutrophils. In this paper, we report a pharmacological cocktail that not only arrests actin dynamics but also preserves the structure of the existing actin network in neutrophil-like HL-60 cells, human fibrosarcoma HT1080 cells, and mouse NIH 3T3 fibroblast cells. Our cocktail induces an arrest of actin dynamics that initiates within seconds and persists for longer than 10 min, during which time cells maintain their responsivity to external stimuli. With this cocktail, we demonstrate that actin dynamics, and not simply morphological polarity or actin accumulation at the leading edge, are required for the spatial persistence of Rac activation in HL-60 cells. Our drug combination preserves the structure of the existing cytoskeleton while blocking actin assembly, disassembly, and rearrangement, and should prove useful for investigating the role of actin dynamics in a wide range of cellular signaling contexts.

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