scholarly journals The Actin-Driven Movement and Formation of Acetylcholine Receptor Clusters

2000 ◽  
Vol 150 (6) ◽  
pp. 1321-1334 ◽  
Author(s):  
Zhengshan Dai ◽  
Xiaoyan Luo ◽  
Hongbo Xie ◽  
H. Benjamin Peng
Keyword(s):  
Actin Cytoskeleton ◽  
Acetylcholine Receptor ◽  
Actin Polymerization ◽  
Fluorescent Protein ◽  
Actin Binding ◽  
Heparin Binding ◽  
Actin Assembly ◽  
Achr Cluster ◽  
Green Fluorescent ◽  
Receptor Clusters

A new method was devised to visualize actin polymerization induced by postsynaptic differentiation signals in cultured muscle cells. This entails masking myofibrillar filamentous (F)-actin with jasplakinolide, a cell-permeant F-actin–binding toxin, before synaptogenic stimulation, and then probing new actin assembly with fluorescent phalloidin. With this procedure, actin polymerization associated with newly induced acetylcholine receptor (AChR) clustering by heparin-binding growth-associated molecule–coated beads and by agrin was observed. The beads induced local F-actin assembly that colocalized with AChR clusters at bead–muscle contacts, whereas both the actin cytoskeleton and AChR clusters induced by bath agrin application were diffuse. By expressing a green fluorescent protein–coupled version of cortactin, a protein that binds to active F-actin, the dynamic nature of the actin cytoskeleton associated with new AChR clusters was revealed. In fact, the motive force generated by actin polymerization propelled the entire bead-induced AChR cluster with its attached bead to move in the plane of the membrane. In addition, actin polymerization is also necessary for the formation of both bead and agrin-induced AChR clusters as well as phosphotyrosine accumulation, as shown by their blockage by latrunculin A, a toxin that sequesters globular (G)-actin and prevents F-actin assembly. These results show that actin polymerization induced by synaptogenic signals is necessary for the movement and formation of AChR clusters and implicate a role of F-actin as a postsynaptic scaffold for the assembly of structural and signaling molecules in neuromuscular junction formation.

scholarly journals G Protein β Subunit–null Mutants Are Impaired in Phagocytosis and Chemotaxis Due to Inappropriate Regulation of the Actin Cytoskeleton

1998 ◽  
Vol 141 (7) ◽  
pp. 1529-1537 ◽  
Author(s):  
Barbara Peracino ◽  
Jane Borleis ◽  
Tian Jin ◽  
Monika Westphal ◽  
Jean-Marc Schwartz ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
G Protein ◽  
Actin Polymerization ◽  
Fluorescent Protein ◽  
Constitutive Expression ◽  
Fluid Phase ◽  
Β Subunit ◽  
Wild Type ◽  
Fluid Phase Endocytosis ◽  
Green Fluorescent

Chemotaxis and phagocytosis are basically similar in cells of the immune system and in Dictyostelium amebae. Deletion of the unique G protein β subunit in D. discoideum impaired phagocytosis but had little effect on fluid-phase endocytosis, cytokinesis, or random motility. Constitutive expression of wild-type β subunit restored phagocytosis and normal development. Chemoattractants released by cells or bacteria trigger typical transient actin polymerization responses in wild-type cells. In β subunit–null cells, and in a series of β subunit point mutants, these responses were impaired to a degree that correlated with the defect in phagocytosis. Image analysis of green fluorescent protein–actin transfected cells showed that β subunit– null cells were defective in reshaping the actin network into a phagocytic cup, and eventually a phagosome, in response to particle attachment. Our results indicate that signaling through heterotrimeric G proteins is required for regulating the actin cytoskeleton during phagocytic uptake, as previously shown for chemotaxis. Inhibitors of phospholipase C and intracellular Ca2+ mobilization inhibited phagocytosis, suggesting the possible involvement of these effectors in the process.

scholarly journals Association of cortactin with dynamic actin in lamellipodia and on endosomal vesicles

2000 ◽  
Vol 113 (24) ◽  
pp. 4421-4426 ◽  
Author(s):  
M. Kaksonen ◽  
H.B. Peng ◽  
H. Rauvala
Keyword(s):  
Actin Polymerization ◽  
Fluorescent Protein ◽  
Leading Edge ◽  
Time Lapse ◽  
Actin Binding ◽  
Common Mechanism ◽  
Membrane Protrusions ◽  
Green Fluorescent ◽  
Time Lapse Imaging ◽  
Endosomal Vesicles

We have used fluorescent protein tagging to study the localization and dynamics of the actin-binding protein cortactin in living NIH 3T3 fibroblast cells. Cortactin was localized to active lamellipodia and to small cytoplasmic spots. Time-lapse imaging revealed that these cortactin labeled structures were very dynamic. In the lamellipodia, cortactin labeled structures formed at the leading edge and then moved toward the cell center. Experiments with green fluorescent protein (GFP)-tagged actin showed that cortactin movement was coincident with the actin retrograde flow in the lamellipodia. Cytoplasmic cortactin spots also contained F-actin and were propelled by actin polymerization. Arp3, a component of the arp2/3 complex which is a key regulator of actin polymerization, co-localized with cortactin. Cytoplasmic cortactin-labeled spots were found to be associated with endosomal vesicles. Association was asymmetric and approximately half of the endosomes were associated with cortactin spots. Time-lapse imaging suggested that these cortactin and F-actin-containing spots propelled endosomes. Actin polymerization based propulsion may be a common mechanism for endomembrane trafficking in the same manner as used in the plasma membrane protrusions. As cortactin is known to interact with membrane-associated signaling proteins it could have a role in linking signaling complexes with dynamic actin on endosomes and in lamellipodia.

scholarly journals Rho Family Gtpase Cdc42 Is Essential for the Actin-Based Motility of Shigella in Mammalian Cells

1999 ◽  
Vol 191 (11) ◽  
pp. 1905-1920 ◽  
Author(s):  
Toshihiko Suzuki ◽  
Hitomi Mimuro ◽  
Hiroaki Miki ◽  
Tadaomi Takenawa ◽  
Takuya Sasaki ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Actin Polymerization ◽  
Fluorescent Protein ◽  
Host Cells ◽  
Actin Dynamics ◽  
Cloud Formation ◽  
Actin Assembly ◽  
Egg Extracts ◽  
Rho Family ◽  
Green Fluorescent

Shigella, the causative agent of bacillary dysentery, is capable of directing its movement within host cells by exploiting actin dynamics. The VirG protein expressed at one pole of the bacterium can recruit neural Wiskott-Aldrich syndrome protein (N-WASP), a downstream effector of Cdc42. Here, we show that Cdc42 is required for the actin-based motility of Shigella. Microinjection of a dominant active mutant Cdc42, but not Rac1 or RhoA, into Swiss 3T3 cells accelerated Shigella motility. In add-back experiments in Xenopus egg extracts, addition of a guanine nucleotide dissociation inhibitor for the Rho family, RhoGDI, greatly diminished the bacterial motility or actin assembly, which was restored by adding activated Cdc42. In N-WASP–depleted extracts, the bacterial movement almost arrested was restored by adding exogenous N-WASP but not H208D, an N-WASP mutant defective in binding to Cdc42. In pyrene actin assay, Cdc42 enhanced VirG-stimulating actin polymerization by N-WASP–actin-related protein (Arp)2/3 complex. Actually, Cdc42 stimulated actin cloud formation on the surface of bacteria expressing VirG in a solution containing N-WASP, Arp2/3 complex, and G-actin. Immunohistological study of Shigella-infected cells expressing green fluorescent protein–tagged Cdc42 revealed that Cdc42 accumulated by being colocalized with actin cloud at one pole of intracellular bacterium. Furthermore, overexpression of H208D mutant in cells interfered with the actin assembly of infected Shigella and diminished the intra- and intercellular spreading. These results suggest that Cdc42 activity is involved in initiating actin nucleation mediated by VirG–N-WASP–Arp2/3 complex formed on intracellular Shigella.

Stable transformation and actin visualization in callus cultures of dodder (Cuscuta europaea)

Biologia ◽  
2013 ◽  
Vol 68 (4) ◽  
Author(s):  
Renáta Švubová ◽  
Alžbeta Blehová
Keyword(s):  
Actin Cytoskeleton ◽  
Fluorescent Protein ◽  
Callus Cultures ◽  
Visual Selection ◽  
Actin Binding ◽  
Selection Marker ◽  
Nptii Gene ◽  
Cortical Actin ◽  
Cuscuta Europaea ◽  
Green Fluorescent

AbstractAgrobacterium tumefaciens-mediated transformation of callus culture, combined with a visual selection of GFP-tagged fimbrin actin binding domain (FABD2) expression is described for parasitic species (Cuscuta europaea). The conditions for callus induction from 1 cm-long explants from the basal part of 7-day-old dodder seedlings were defined. We obtained light-green calli, which were transformed with A. tumefaciens bacterial strain GV3101 carrying plasmid pCB302 (35S::ABD2:gfp) with neomycin phosphotransferase (nptII) gene. The limitations of selection procedures based on antibiotics were avoided using green fluorescent protein (GFP) detection, as a visual selection marker subcellularly targeted to the actin cytoskeleton. Fluorescence microscopy analyses demonstrated a network of nucleus-associated actin arrays and dense cortical actin arrangements in stably transformed Cuscuta callus cells. RT-PCR analyses confirmed gfp expression in transformed calli 7, 14 and 21 days after transformation. Although the GFP fluorescence associated with the actin cytoskeleton has retained for at least six months without silencing, no shoot regeneration was observed. It can be concluded that, C. europaea callus cells are competent for transformation, but under given conditions, these cells failed to realize their morphogenic and regeneration potentials.

scholarly journals Dynamin Is Required for GnRH Signaling to L-Type Calcium Channels and Activation of ERK

Endocrinology ◽  
2015 ◽  
Vol 157 (2) ◽  
pp. 831-843 ◽  
Author(s):  
Brian S. Edwards ◽  
An K. Dang ◽  
Dilyara A. Murtazina ◽  
Melissa G. Dozier ◽  
Jennifer D. Whitesell ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Actin Cytoskeleton ◽  
Fluorescent Protein ◽  
Leading Edge ◽  
Dominant Negative ◽  
Actin Binding ◽  
Erk Phosphorylation ◽  
Green Fluorescent ◽  
Ca2 Channels

Abstract We have shown that GnRH-mediated engagement of the cytoskeleton induces cell movement and is necessary for ERK activation. It also has previously been established that a dominant negative form of the mechano-GTPase dynamin (K44A) attenuates GnRH activation of ERK. At present, it is not clear at what level these cellular events might be linked. To explore this, we used live cell imaging in the gonadotrope-derived αT3–1 cell line to determine that dynamin-green fluorescent protein accumulated in GnRH-induced lamellipodia and plasma membrane protrusions. Coincident with translocation of dynamin-green fluorescent protein to the plasma membrane, we demonstrated that dynamin colocalizes with the actin cytoskeleton and the actin binding protein, cortactin at the leading edge of the plasma membrane. We next wanted to assess the physiological significance of these findings by inhibiting dynamin GTPase activity using dynasore. We find that dynasore suppresses activation of ERK, but not c-Jun N-terminal kinase, after exposure to GnRH agonist. Furthermore, exposure of αT3–1 cells to dynasore inhibited GnRH-induced cyto-architectural rearrangements. Recently it has been discovered that GnRH induced Ca2+ influx via the L-type Ca2+ channels requires an intact cytoskeleton to mediate ERK phosphorylation. Interestingly, not only does dynasore attenuate GnRH-mediated actin reorganization, it also suppresses Ca2+ influx through L-type Ca2+ channels visualized in living cells using total internal reflection fluorescence microscopy. Collectively, our data suggest that GnRH-induced membrane remodeling events are mediated in part by the association of dynamin and cortactin engaging the actin cytoskeleton, which then regulates Ca2+ influx via L-type channels to facilitate ERK phosphorylation.

scholarly journals Toxofilin, a Novel Actin-binding Protein from Toxoplasma gondii, Sequesters Actin Monomers and Caps Actin Filaments

2000 ◽  
Vol 11 (1) ◽  
pp. 355-368 ◽  
Author(s):  
Olivier Poupel ◽  
Haralabia Boleti ◽  
Sophie Axisa ◽  
Evelyne Couture-Tosi ◽  
Isabelle Tardieux
Keyword(s):  
Green Fluorescent Protein ◽  
Toxoplasma Gondii ◽  
Host Cell ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Fluorescent Protein ◽  
Binding Protein ◽  
Actin Binding ◽  
Actin Binding Protein ◽  
Green Fluorescent

Toxoplasma gondii relies on its actin cytoskeleton to glide and enter its host cell. However, T. gondii tachyzoites are known to display a strikingly low amount of actin filaments, which suggests that sequestration of actin monomers could play a key role in parasite actin dynamics. We isolated a 27-kDa tachyzoite protein on the basis of its ability to bind muscle G-actin and demonstrated that it interacts with parasite G-actin. Cloning and sequence analysis of the gene coding for this protein, which we named Toxofilin, showed that it is a novel actin-binding protein. In in vitro assays, Toxofilin not only bound to G-actin and inhibited actin polymerization as an actin-sequestering protein but also slowed down F-actin disassembly through a filament end capping activity. In addition, when green fluorescent protein-tagged Toxofilin was overexpressed in mammalian nonmuscle cells, the dynamics of actin stress fibers was drastically impaired, whereas green fluorescent protein-Toxofilin copurified with G-actin. Finally, in motile parasites, during gliding or host cell entry, Toxofilin was localized in the entire cytoplasm, including the rear end of the parasite, whereas in intracellular tachyzoites, especially before they exit from the parasitophorous vacuole of their host cell, Toxofilin was found to be restricted to the apical end.

scholarly journals Dual labeling of the fibronectin matrix and actin cytoskeleton with green fluorescent protein variants

2002 ◽  
Vol 115 (6) ◽  
pp. 1221-1229 ◽  
Author(s):  
Tomoo Ohashi ◽  
Daniel P. Kiehart ◽  
Harold P. Erickson
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Filament ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Actin Binding ◽  
Matrix Assembly ◽  
Actin Binding Domain ◽  
Filament Bundles ◽  
Green Fluorescent ◽  
Protein Variants

We have prepared 3T3 cells doubly labeled to visualize simultaneously the extracellular fibronectin (FN) matrix and intracellular actin cytoskeleton in living cell cultures. We used FN-yellow fluorescent protein (FN-yfp) for the FN matrix, and the actin-binding domain of moesin fused to cyan fluorescent protein (cfp-Moe) to stain actin. Actin filament bundles were clearly seen in the protruding lamellae of the cells. FN matrix assembly appeared to be initiated as small spots of FN at the ends of actin filament bundles. The spots then elongated along the actin filament bundle toward the cell center to form FN fibrils. The end of the fibril towards the cell edge appeared immobile, and probably attached to the substrate, whereas the end toward the cell center frequently showed movements, suggesting attachment to the cell. Combining our data with the observations of Pankov et al. we suggest that fibrils grow by stretching this mobile end toward the cell center while adding new FN molecules at the end and along the entire lenght. When the cell culture was treated with cytochalasin to disrupt the actin cytoskeleton, some fibrils contracted substantially, suggesting that the segment attached primarily to the cell surface is stretched.

scholarly journals A Balance of Capping Protein and Profilin Functions Is Required to Regulate Actin Polymerization in Drosophila Bristle

2003 ◽  
Vol 14 (1) ◽  
pp. 118-128 ◽  
Author(s):  
Roberta Hopmann ◽  
Kathryn G. Miller
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Filament ◽  
Actin Polymerization ◽  
Genetic Interaction ◽  
Actin Binding ◽  
Actin Assembly ◽  
Loss Of Function ◽  
Capping Protein ◽  
Filament Dynamics

Profilin is a well-characterized protein known to be important for regulating actin filament assembly. Relatively few studies have addressed how profilin interacts with other actin-binding proteins in vivo to regulate assembly of complex actin structures. To investigate the function of profilin in the context of a differentiating cell, we have studied an instructive genetic interaction between mutations in profilin (chickadee) and capping protein (cpb). Capping protein is the principal protein in cells that caps actin filament barbed ends. When its function is reduced in the Drosophila bristle, F-actin levels increase and the actin cytoskeleton becomes disorganized, causing abnormal bristle morphology. chickadee mutations suppress the abnormal bristle phenotype and associated abnormalities of the actin cytoskeleton seen in cpb mutants. Furthermore, overexpression of profilin in the bristle mimics many features of thecpb loss-of-function phenotype. The interaction betweencpb and chickadee suggests that profilin promotes actin assembly in the bristle and that a balance between capping protein and profilin activities is important for the proper regulation of F-actin levels. Furthermore, this balance of activities affects the association of actin structures with the membrane, suggesting a link between actin filament dynamics and localization of actin structures within the cell.

scholarly journals Regulation of the Cortical Actin Cytoskeleton in Budding Yeast by Twinfilin, a Ubiquitous Actin Monomer-sequestering Protein

1998 ◽  
Vol 142 (3) ◽  
pp. 723-733 ◽  
Author(s):  
Bruce L. Goode ◽  
David G. Drubin ◽  
Pekka Lappalainen
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Filament ◽  
Fluorescent Protein ◽  
Budding Yeast ◽  
Yeast Cells ◽  
Actin Binding ◽  
Actin Monomer ◽  
Nucleotide Exchange ◽  
Cortical Actin ◽  
Green Fluorescent

Here we describe the identification of a novel 37-kD actin monomer binding protein in budding yeast. This protein, which we named twinfilin, is composed of two cofilin-like regions. In our sequence database searches we also identified human, mouse, and Caenorhabditis elegans homologues of yeast twinfilin, suggesting that twinfilins form an evolutionarily conserved family of actin-binding proteins. Purified recombinant twinfilin prevents actin filament assembly by forming a 1:1 complex with actin monomers, and inhibits the nucleotide exchange reaction of actin monomers. Despite the sequence homology with the actin filament depolymerizing cofilin/actin-depolymerizing factor (ADF) proteins, our data suggests that twinfilin does not induce actin filament depolymerization. In yeast cells, a green fluorescent protein (GFP)–twinfilin fusion protein localizes primarily to cytoplasm, but also to cortical actin patches. Overexpression of the twinfilin gene (TWF1) results in depolarization of the cortical actin patches. A twf1 null mutation appears to result in increased assembly of cortical actin structures and is synthetically lethal with the yeast cofilin mutant cof1-22, shown previously to cause pronounced reduction in turnover of cortical actin filaments. Taken together, these results demonstrate that twinfilin is a novel, highly conserved actin monomer-sequestering protein involved in regulation of the cortical actin cytoskeleton.

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