scholarly journals A functional family of fluorescent nucleotide analogues to investigate actin dynamics and energetics

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jessica Colombo ◽  
Adrien Antkowiak ◽  
Konstantin Kogan ◽  
Tommi Kotila ◽  
Jenna Elliott ◽  
...  
Keyword(s):  
Single Molecule ◽  
Actin Polymerization ◽  
Eukaryotic Cell ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Actin Assembly ◽  
Nucleotide Exchange ◽  
Nucleotide Analogues ◽  
Highly Sensitive ◽  
Fluorescent Nucleotides

AbstractActin polymerization provides force for vital processes of the eukaryotic cell, but our understanding of actin dynamics and energetics remains limited due to the lack of high-quality probes. Most current probes affect dynamics of actin or its interactions with actin-binding proteins (ABPs), and cannot track the bound nucleotide. Here, we identify a family of highly sensitive fluorescent nucleotide analogues structurally compatible with actin. We demonstrate that these fluorescent nucleotides bind to actin, maintain functional interactions with a number of essential ABPs, are hydrolyzed within actin filaments, and provide energy to power actin-based processes. These probes also enable monitoring actin assembly and nucleotide exchange with single-molecule microscopy and fluorescence anisotropy kinetics, therefore providing robust and highly versatile tools to study actin dynamics and functions of ABPs.

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 role of the Arp2/3 complex in shaping the dynamics and structures of branched actomyosin networks

2020 ◽  
Vol 117 (20) ◽  
pp. 10825-10831 ◽  
Author(s):  
James Liman ◽  
Carlos Bueno ◽  
Yossi Eliaz ◽  
Nicholas P. Schafer ◽  
M. Neal Waxham ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Active Networks ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Marginal Stability ◽  
Actin Assembly ◽  
Heterogeneous Distribution ◽  
Energy Consuming ◽  
Far From Equilibrium

Actomyosin networks give cells the ability to move and divide. These networks contract and expand while being driven by active energy-consuming processes such as motor protein walking and actin polymerization. Actin dynamics is also regulated by actin-binding proteins, such as the actin-related protein 2/3 (Arp2/3) complex. This complex generates branched filaments, thereby changing the overall organization of the network. In this work, the spatiotemporal patterns of dynamical actin assembly accompanying the branching-induced reorganization caused by Arp2/3 were studied using a computational model (mechanochemical dynamics of active networks [MEDYAN]); this model simulates actomyosin network dynamics as a result of chemical reactions whose rates are modulated by rapid mechanical equilibration. We show that branched actomyosin networks relax significantly more slowly than do unbranched networks. Also, branched networks undergo rare convulsive movements, “avalanches,” that release strain in the network. These avalanches are associated with the more heterogeneous distribution of mechanically linked filaments displayed by branched networks. These far-from-equilibrium events arising from the marginal stability of growing actomyosin networks provide a possible mechanism of the “cytoquakes” recently seen in experiments.

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 EPLIN regulates actin dynamics by cross-linking and stabilizing filaments

2003 ◽  
Vol 160 (3) ◽  
pp. 399-407 ◽  
Author(s):  
Raymond S. Maul ◽  
Yuhong Song ◽  
Kurt J. Amann ◽  
Sachi C. Gerbin ◽  
Thomas D. Pollard ◽  
...  
Keyword(s):  
Actin Filament ◽  
Actin Polymerization ◽  
Binding Activity ◽  
Stress Fibers ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Membrane Ruffling ◽  
Cross Links ◽  
As Stress ◽  
Kinetics Of

Epithelial protein lost in neoplasm (EPLIN) is a cytoskeleton-associated protein encoded by a gene that is down-regulated in transformed cells. EPLIN increases the number and size of actin stress fibers and inhibits membrane ruffling induced by Rac. EPLIN has at least two actin binding sites. Purified recombinant EPLIN inhibits actin filament depolymerization and cross-links filaments in bundles. EPLIN does not affect the kinetics of spontaneous actin polymerization or elongation at the barbed end, but inhibits branching nucleation of actin filaments by Arp2/3 complex. Side binding activity may stabilize filaments and account for the inhibition of nucleation mediated by Arp2/3 complex. We propose that EPLIN promotes the formation of stable actin filament structures such as stress fibers at the expense of more dynamic actin filament structures such as membrane ruffles. Reduced expression of EPLIN may contribute to the motility of invasive tumor cells.

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 The integral membrane protein, ponticulin, acts as a monomer in nucleating actin assembly.

1993 ◽  
Vol 120 (4) ◽  
pp. 909-922 ◽  
Author(s):  
C P Chia ◽  
A Shariff ◽  
S A Savage ◽  
E J Luna
Keyword(s):  
Membrane Protein ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Lipid Vesicles ◽  
Integral Membrane Protein ◽  
Actin Binding ◽  
Actin Assembly ◽  
Nucleation Activity

Ponticulin, an F-actin binding transmembrane glycoprotein in Dictyostelium plasma membranes, was isolated by detergent extraction from cytoskeletons and purified to homogeneity. Ponticulin is an abundant membrane protein, averaging approximately 10(6) copies/cell, with an estimated surface density of approximately 300 per microns2. Ponticulin solubilized in octylglucoside exhibited hydrodynamic properties consistent with a ponticulin monomer in a spherical or slightly ellipsoidal detergent micelle with a total molecular mass of 56 +/- 6 kD. Purified ponticulin nucleated actin polymerization when reconstituted into Dictyostelium lipid vesicles, but not when a number of commercially available lipids and lipid mixtures were substituted for the endogenous lipid. The specific activity was consistent with that expected for a protein comprising 0.7 +/- 0.4%, by mass, of the plasma membrane protein. Ponticulin in octylglucoside micelles bound F-actin but did not nucleate actin assembly. Thus, ponticulin-mediated nucleation activity was sensitive to the lipid environment, a result frequently observed with transmembrane proteins. At most concentrations of Dictyostelium lipid, nucleation activity increased linearly with increasing amounts of ponticulin, suggesting that the nucleating species is a ponticulin monomer. Consistent with previous observations of lateral interactions between actin filaments and Dictyostelium plasma membranes, both ends of ponticulin-nucleated actin filaments appeared to be free for monomer assembly and disassembly. Our results indicate that ponticulin is a major membrane protein in Dictyostelium and that, in the proper lipid matrix, it is sufficient for lateral nucleation of actin assembly. To date, ponticulin is the only integral membrane protein known to directly nucleate actin polymerization.

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 Single-molecule nanopore sensing of actin dynamics and drug binding

2019 ◽  
Author(s):  
Xiaoyi Wang ◽  
Mark D. Wilkinson ◽  
Xiaoyan Lin ◽  
Ren Ren ◽  
Keith Willison ◽  
...  
Keyword(s):  
Protein Folding ◽  
Single Molecule ◽  
Eukaryotic Cell ◽  
Molecular State ◽  
Drug Binding ◽  
Actin Dynamics ◽  
Label Free ◽  
Single Molecule Level ◽  
Native Proteins ◽  
Voltage Dependent

AbstractActin is a key protein in the dynamic processes within the eukaryotic cell. To date, methods exploring the molecular state of actin are limited to insights gained from structural approaches, providing a snapshot of protein folding, or methods that require chemical modifications compromising actin monomer thermostability. Nanopore sensing permits label-free investigation of native proteins and is ideally suited to study proteins such as actin that require specialised buffers and cofactors. Using nanopores we determined the state of actin at the macromolecular level (filamentous or globular) and in its monomeric form bound to inhibitors. We revealed urea-dependent and voltage-dependent transitional states and observed unfolding process within which sub-populations of transient actin oligomers are visible. We detected, in real-time, drug-binding and filament-growth events at the single-molecule level. This enabled us to calculate binding stoichiometries and to propose a model for protein dynamics using unmodified, native actin molecules, demostrating the promise of nanopores sensing for in-depth understanding of protein folding landscapes and for drug discovery.

scholarly journals A direct proof that sole actin dynamics drive membrane deformations

2018 ◽  
Author(s):  
Camille Simon ◽  
Rémy Kusters ◽  
Valentina Caorsi ◽  
Antoine Allard ◽  
Majdouline Abou-Ghali ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Actin Polymerization ◽  
Cell Function ◽  
Turgor Pressure ◽  
Direct Proof ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Associated Proteins ◽  
Low Tension ◽  
Membrane Deformations

AbstractCell membrane deformations are crucial for proper cell function. Specialized protein assemblies initiate inward or outward membrane deformations that turn into, for example, filopodia or endocytic intermediates. Actin dynamics and actin-binding proteins are involved in this process, although their detailed role remains controversial. We show here that a dynamic, branched actin network is sufficient, in absence of any membrane-associated proteins, to initiate both inward and outward membrane deformation. With actin polymerization triggered at the membrane of liposomes, we produce inward filopodia-like structures at low tension, while outward endocytosis-like structures are robustly generated regardless of tension. Our results are reminiscent of endocytosis in mammalian cells, where actin polymerization forces are required when membrane tension is increased, and in yeast, where they are always required to overcome the opposing turgor pressure. By combining experimental observations with physical modeling, we propose a mechanism for actin-driven endocytosis under high tension or high pressure conditions.

scholarly journals Burkholderia pseudomalleitype III secreted protein BipC: role in actin modulation and translocation activities required for the bacterial intracellular lifecycle

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2532 ◽  
Author(s):  
Wen Tyng Kang ◽  
Kumutha Malar Vellasamy ◽  
Lakshminarayanan Rajamani ◽  
Roger W. Beuerman ◽  
Jamuna Vadivelu
Keyword(s):  
Actin Polymerization ◽  
Cell Interaction ◽  
Binding Activity ◽  
Host Cells ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Virulence Determinants ◽  
Secretion Systems ◽  
Number Of Patients

Melioidosis, an infection caused by the facultative intracellular pathogenBurkholderia pseudomallei, has been classified as an emerging disease with the number of patients steadily increasing at an alarming rate.B. pseudomalleipossess various virulence determinants that allow them to invade the host and evade the host immune response, such as the type III secretion systems (TTSS). The products of this specialized secretion system are particularly important for theB. pseudomalleiinfection. Lacking in one or more components of the TTSS demonstrated different degrees of defects in the intracellular lifecycle ofB. pseudomallei. Further understanding the functional roles of proteins involved inB. pseudomalleiTTSS will enable us to dissect the enigma ofB. pseudomallei-host cell interaction. In this study, BipC (a translocator), which was previously reported to be involved in the pathogenesis ofB. pseudomallei, was further characterized using the bioinformatics and molecular approaches. ThebipCgene, coding for a putative invasive protein, was first PCR amplified fromB. pseudomalleiK96243genomic DNA and cloned into an expression vector for overexpression inEscherichia coli. The soluble protein was subsequently purified and assayed for actin polymerization and depolymerization. BipC was verified to subvert the host actin dynamics as demonstrated by the capability to polymerize actinin vitro. Homology modeling was also attempted to predict the structure of BipC. Overall, our findings identified that the protein encoded by thebipCgene plays a role as an effector involved in the actin binding activity to facilitate internalization ofB. pseudomalleiinto the host cells.

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