scholarly journals Profilin connects actin assembly with microtubule dynamics

2016 ◽  
Vol 27 (15) ◽  
pp. 2381-2393 ◽  
Author(s):  
Michaela Nejedla ◽  
Sara Sadi ◽  
Vadym Sulimenko ◽  
Francisca Nunes de Almeida ◽  
Hans Blom ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Actin Dynamics ◽  
Control Element ◽  
Actin Assembly ◽  
Actin Nucleation ◽  
Superresolution Microscopy ◽  
Drug Treatments ◽  
And Migration ◽  
Wasp Family Proteins ◽  
Microscopy Techniques

Profilin controls actin nucleation and assembly processes in eukaryotic cells. Actin nucleation and elongation promoting factors (NEPFs) such as Ena/VASP, formins, and WASP-family proteins recruit profilin:actin for filament formation. Some of these are found to be microtubule associated, making actin polymerization from microtubule-associated platforms possible. Microtubules are implicated in focal adhesion turnover, cell polarity establishment, and migration, illustrating the coupling between actin and microtubule systems. Here we demonstrate that profilin is functionally linked to microtubules with formins and point to formins as major mediators of this association. To reach this conclusion, we combined different fluorescence microscopy techniques, including superresolution microscopy, with siRNA modulation of profilin expression and drug treatments to interfere with actin dynamics. Our studies show that profilin dynamically associates with microtubules and this fraction of profilin contributes to balance actin assembly during homeostatic cell growth and affects micro­tubule dynamics. Hence profilin functions as a regulator of microtubule (+)-end turnover in addition to being an actin control element.

scholarly journals Systematic analysis of the molecular architecture of endocytosis reveals a nanoscale actin nucleation template that drives efficient vesicle formation

2017 ◽  
Author(s):  
Markus Mund ◽  
Johannes Albertus van der Beek ◽  
Joran Deschamps ◽  
Serge Dmitrieff ◽  
Jooske Louise Monster ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Design Principle ◽  
Vesicle Formation ◽  
Molecular Architecture ◽  
Actin Nucleation ◽  
Systematic Analysis ◽  
Superresolution Microscopy ◽  
Membrane Invagination ◽  
Wasp Family Proteins ◽  
Endocytic Vesicles

Clathrin-mediated endocytosis is an essential cellular function in all eukaryotes that is driven by a self-assembled macromolecular machine of over 50 different proteins in tens to hundreds of copies. How these proteins are organized to produce endocytic vesicles with high precision and efficiency is not understood. Here, we developed high-throughput superresolution microscopy to reconstruct the nanoscale structural organization of 23 endocytic proteins from over 100,000 endocytic sites in yeast. We found that proteins assemble by radially-ordered recruitment according to function. WASP family proteins form a circular nano-scale template on the membrane to spatially control actin nucleation during vesicle formation. Mathematical modeling of actin polymerization showed that this WASP nano-template creates sufficient force for membrane invagination and substantially increases the efficiency of endocytosis. Such nanoscale pre-patterning of actin nucleation may represent a general design principle for directional force generation in membrane remodeling processes such as during cell migration and division.

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.

scholarly journals The C-terminus SH3-binding domain of Kv1.3 is required for the actin-mediated immobilization of the channel via cortactin

2015 ◽  
Vol 26 (9) ◽  
pp. 1640-1651 ◽  
Author(s):  
Peter Hajdu ◽  
Geoffrey V. Martin ◽  
Ameet A. Chimote ◽  
Orsolya Szilagyi ◽  
Koichi Takimoto ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Actin Polymerization ◽  
Actin Dynamics ◽  
Actin Network ◽  
Immune Synapse ◽  
Proximity Ligation ◽  
C Terminus ◽  
Kv1.3 Channels ◽  
And Migration ◽  
Channel Polarization

Kv1.3 channels play a pivotal role in the activation and migration of T-lymphocytes. These functions are accompanied by the channels' polarization, which is essential for associated downstream events. However, the mechanisms that govern the membrane movement of Kv1.3 channels remain unclear. F-actin polymerization occurs concomitantly to channel polarization, implicating the actin cytoskeleton in this process. Here we show that cortactin, a factor initiating the actin network, controls the membrane mobilization of Kv1.3 channels. FRAP with EGFP-tagged Kv1.3 channels demonstrates that knocking down cortactin decreases the actin-based immobilization of the channels. Using various deletion and mutation constructs, we show that the SH3 motif of Kv1.3 mediates the channel immobilization. Proximity ligation assays indicate that deletion or mutation of the SH3 motif also disrupts interaction of the channel with cortactin. In T-lymphocytes, the interaction between HS1 (the cortactin homologue) and Kv1.3 occurs at the immune synapse and requires the channel's C-terminal domain. These results show that actin dynamics regulates the membrane motility of Kv1.3 channels. They also provide evidence that the SH3 motif of the channel and cortactin plays key roles in this process.

scholarly journals Changes in Vasodilator-Stimulated Phosphoprotein Phosphorylation, Profilin-1, and Cofilin-1 in Accreta and Protection by DHA

2018 ◽  
Vol 26 (6) ◽  
pp. 757-765 ◽  
Author(s):  
Mehboob Ali ◽  
Lynette K. Rogers ◽  
Kathryn M. Heyob ◽  
Catalin S. Buhimschi ◽  
Irina A. Buhimschi
Keyword(s):  
Actin Polymerization ◽  
Cellular Proliferation ◽  
Placenta Accreta ◽  
Gestational Trophoblastic Disease ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Migration And Invasion ◽  
Aberrant Expression ◽  
Vasp Phosphorylation ◽  
And Migration

Accreta and gestational trophoblastic disease (ie, choriocarcinoma) are placental pathologies characterized by hyperproliferative and invasive trophoblasts. Cellular proliferation, migration, and invasion are heavily controlled by actin-binding protein (ABP)-mediated actin dynamics. The ABP vasodilator-stimulated phosphoprotein (VASP) carries key regulatory role. Profilin-1, cofilin-1, and VASP phosphorylated at Ser157 (pVASP-S157) and Ser239 (pVASP-S239) are ABPs that regulate actin polymerization and stabilization and facilitate cell metastases. Docosahexaenoic acid (DHA) inhibits cancer cell migration and proliferation. We hypothesized that analogous to malignant cells, ABPs regulate these processes in extravillous trophoblasts (EVTs), which exhibit aberrant expression in placenta accreta. Placental–myometrial junction biopsies of histologically confirmed placenta accreta had significantly increased immunostaining levels of cofilin-1, VASP, pVASP-S239, and F-actin. Treatment of choriocarcinoma-derived trophoblast (BeWo) cells with DHA (30 µM) for 24 hours significantly suppressed proliferation, migration, and pVASP-S239 levels and altered protein profiles consistent with increased apoptosis. We concluded that in accreta changes in the ABP expression profile were a response to restore homeostasis by counteracting the hyperproliferative and invasive phenotype of the EVT. The observed association between VASP phosphorylation, apoptosis, and trophoblast proliferation and migration suggest that DHA may offer a therapeutic solution for conditions where EVT is hyperinvasive.

scholarly journals Dictyostelium discoideum plasma membranes contain an actin-nucleating activity that requires ponticulin, an integral membrane glycoprotein.

1990 ◽  
Vol 110 (3) ◽  
pp. 681-692 ◽  
Author(s):  
A Shariff ◽  
E J Luna
Keyword(s):  
Dictyostelium Discoideum ◽  
Actin Polymerization ◽  
Plasma Membranes ◽  
Actin Binding ◽  
Heat Denaturation ◽  
Actin Assembly ◽  
Binding Studies ◽  
Actin Nucleation ◽  
Equilibrium Binding ◽  
Nucleation Activity

In previous equilibrium binding studies, Dictyostelium discoideum plasma membranes have been shown to bind actin and to recruit actin into filaments at the membrane surface. However, little is known about the kinetic pathway(s) through which actin assembles at these, or other, membranes. We have used actin fluorescently labeled with N-(1-pyrenyl)iodoacetamide to examine the kinetics of actin assembly in the presence of D. discoideum plasma membranes. We find that these membranes increase the rate of actin polymerization. The rate of membrane-mediated actin polymerization is linearly dependent on membrane protein concentrations up to 20 micrograms/ml. Nucleation (the association of activated actin monomers into oligomers) appears to be the primary step of polymerization that is accelerated. A sole effect on the initial salt-induced actin conformational change (activation) is ruled out because membranes accelerate the polymerization of pre-activated actin as well as actin activated in the presence of membranes. Elongation of preexisting filaments also is not the major step of polymerization facilitated by membranes since membranes stripped of all peripheral components, including actin, increase the rate of actin assembly to about the same extent as do membranes containing small amounts of endogenous actin. Acceleration of the nucleation step by membranes also is supported by an analysis of the dependence of polymerization lag time on actin concentration. The barbed ends of membrane-induced actin nuclei are not obstructed by the membranes because the barbed end blocking agent, cytochalasin D, reduces the rate of membrane-mediated actin nucleation. Similarly, the pointed ends of the nuclei are not blocked by membranes since the depolymerization rate of gelsolin-capped actin is unchanged in the presence of membranes. These results are consistent with previous observations of lateral interactions between membranes and actin filaments. These results also are consistent with two predictions from a model based on equilibrium binding studies; i.e., that plasma membranes should nucleate actin assembly and that membrane-bound actin nuclei should have both ends free (Schwartz, M. A., and E. J. Luna. 1988. J. Cell Biol. 107:201-209). Integral membrane proteins mediate the actin nucleation activity because activity is eliminated by heat denaturation, treatment with reducing agents, or proteolysis of membranes. Activity also is abolished by solubilization with octylglucoside but is reconstituted upon removal or dilution of the detergent. Ponticulin, the major actin-binding protein in plasma membranes, appears to be necessary for nucleation activity since activity is not reconstituted from detergent extracts depleted of ponticulin.

scholarly journals Arf6 Can Trigger Wave Regulatory Complex-Dependent Actin Assembly Independent of Arno

2020 ◽  
Vol 21 (7) ◽  
pp. 2457 ◽  
Author(s):  
Vikash Singh ◽  
Anthony C. Davidson ◽  
Peter J. Hume ◽  
Vassilis Koronakis
Keyword(s):  
Lipid Bilayers ◽  
Membrane Trafficking ◽  
Actin Polymerization ◽  
Small Gtpase ◽  
Actin Dynamics ◽  
Actin Assembly ◽  
Nucleotide Exchange ◽  
Cortical Actin ◽  
Nucleotide Exchange Factor ◽  
Regulatory Complex

The small GTPase ADP-ribosylation factor 6 (Arf6) anchors at the plasma membrane to orchestrate key functions, such as membrane trafficking and regulating cortical actin cytoskeleton rearrangement. A number of studies have identified key players that interact with Arf6 to regulate actin dynamics in diverse cell processes, yet it is still unknown whether Arf6 can directly signal to the wave regulatory complex to mediate actin assembly. By reconstituting actin dynamics on supported lipid bilayers, we found that Arf6 in co-ordination with Rac1(Ras-related C3 botulinum toxin substrate 1) can directly trigger actin polymerization by recruiting wave regulatory complex components. Interestingly, we demonstrated that Arf6 triggers actin assembly at the membrane directly without recruiting the Arf guanine nucleotide exchange factor (GEF) ARNO (ARF nucleotide-binding site opener), which is able to activate Arf1 to enable WRC-dependent actin assembly. Furthermore, using labelled E. coli, we demonstrated that actin assembly by Arf6 also contributes towards efficient phagocytosis in THP-1 macrophages. Taken together, this study reveals a mechanism for Arf6-driven actin polymerization.

scholarly journals The Diaphanous-related Formin mDia1 Controls Serum Response Factor Activity through its Effects on Actin Polymerization

2002 ◽  
Vol 13 (11) ◽  
pp. 4088-4099 ◽  
Author(s):  
John W. Copeland ◽  
Richard Treisman
Keyword(s):  
Actin Polymerization ◽  
Serum Response Factor ◽  
Signal Pathway ◽  
Small Gtpase ◽  
Actin Dynamics ◽  
Actin Assembly ◽  
Extracellular Signals ◽  
Activation Assay ◽  
The Relationship ◽  
Serum Response

SRF-dependent transcription is regulated by the small GTPase RhoA via its effects on actin dynamics. The diaphanous-related formin (DRF) proteins have been identified as candidate RhoA effectors mediating signaling to SRF. Here we investigate the relationship between SRF activation and actin polymerization by the DRF mDia1. We show that the ability of mDia1 to potentiate SRF activity is strictly correlated with its ability to promote F-actin assembly. Both processes can occur independently of the mDia1 FH1 domain but require sequences in an extended C-terminal region encompassing the conserved FH2 domain. mDia-mediated SRF activation, but not F-actin assembly, can be blocked by a nonpolymerizable actin mutant, placing actin downstream of mDia in the signal pathway. The SRF activation assay was used to identify inactive mDia1 derivatives that inhibit serum- and LPA-induced signaling to SRF. We show that these interfering mutants also block F-actin assembly, whether induced by mDia proteins or extracellular signals. These results identify novel functional elements of mDia1 and show that it regulates SRF activity by inducing depletion of the cellular pool of G-actin.

scholarly journals Three Regions within Acta Promote Arp2/3 Complex-Mediated Actin Nucleation and Listeria monocytogenes Motility

2000 ◽  
Vol 150 (3) ◽  
pp. 527-538 ◽  
Author(s):  
Justin Skoble ◽  
Daniel A. Portnoy ◽  
Matthew D. Welch
Keyword(s):  
Listeria Monocytogenes ◽  
Actin Polymerization ◽  
Sequence Similarity ◽  
Binding Region ◽  
Actin Nucleation ◽  
Homology Sequence ◽  
Infected Cells ◽  
Wasp Family Proteins ◽  
In Cells

The Listeria monocytogenes ActA protein induces actin-based motility by enhancing the actin nucleating activity of the host Arp2/3 complex. Using systematic truncation analysis, we identified a 136-residue NH2-terminal fragment that was fully active in stimulating nucleation in vitro. Further deletion analysis demonstrated that this fragment contains three regions, which are important for nucleation and share functional and/or limited sequence similarity with host WASP family proteins: an acidic stretch, an actin monomer–binding region, and a cofilin homology sequence. To determine the contribution of each region to actin-based motility, we compared the biochemical activities of ActA derivatives with the phenotypes of corresponding mutant bacteria in cells. The acidic stretch functions to increase the efficiency of actin nucleation, the rate and frequency of motility, and the effectiveness of cell–cell spread. The monomer-binding region is required for actin nucleation in vitro, but not for actin polymerization or motility in infected cells, suggesting that redundant mechanisms may exist to recruit monomer in host cytosol. The cofilin homology sequence is critical for stimulating actin nucleation with the Arp2/3 complex in vitro, and is essential for actin polymerization and motility in cells. These data demonstrate that each region contributes to actin-based motility, and that the cofilin homology sequence plays a principal role in activation of the Arp2/3 complex, and is an essential determinant of L. monocytogenes pathogenesis.

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

2004 ◽  
Vol 24 (24) ◽  
pp. 10905-10922 ◽  
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.

scholarly journals ALS-linked PFN1 variants exhibit loss and gain of functions in the context of formin-induced actin polymerization

2021 ◽  
Vol 118 (23) ◽  
pp. e2024605118
Author(s):  
Eric J. Schmidt ◽  
Salome Funes ◽  
Jeanne E. McKeon ◽  
Brittany R. Morgan ◽  
Sivakumar Boopathy ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Actin Polymerization ◽  
Alpha Helix ◽  
Actin Dynamics ◽  
Internal Dynamic ◽  
Actin Assembly ◽  
Loss Of Function ◽  
Dynamics Simulations ◽  
Induced Changes ◽  
Lateral Sclerosis

Profilin-1 (PFN1) plays important roles in modulating actin dynamics through binding both monomeric actin and proteins enriched with polyproline motifs. Mutations in PFN1 have been linked to the neurodegenerative disease amyotrophic lateral sclerosis (ALS). However, whether ALS-linked mutations affect PFN1 function has remained unclear. To address this question, we employed an unbiased proteomics analysis in mammalian cells to identify proteins that differentially interact with mutant and wild-type (WT) PFN1. These studies uncovered differential binding between two ALS-linked PFN1 variants, G118V and M114T, and select formin proteins. Furthermore, both variants augmented formin-mediated actin assembly relative to PFN1 WT. Molecular dynamics simulations revealed mutation-induced changes in the internal dynamic couplings within an alpha helix of PFN1 that directly contacts both actin and polyproline, as well as structural fluctuations within the actin- and polyproline-binding regions of PFN1. These data indicate that ALS-PFN1 variants have the potential for heightened flexibility in the context of the ternary actin–PFN1–polyproline complex during actin assembly. Conversely, PFN1 C71G was more severely destabilized than the other PFN1 variants, resulting in reduced protein expression in both transfected and ALS patient lymphoblast cell lines. Moreover, this variant exhibited loss-of-function phenotypes in the context of actin assembly. Perturbations in actin dynamics and assembly can therefore result from ALS-linked mutations in PFN1. However, ALS-PFN1 variants may dysregulate actin polymerization through different mechanisms that depend upon the solubility and stability of the mutant protein.

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