scholarly journals The actomyosin interface contains an evolutionary conserved core and an ancillary interface involved in specificity

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Julien Robert-Paganin ◽  
Xiao-Ping Xu ◽  
Mark F. Swift ◽  
Daniel Auguin ◽  
James P. Robblee ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Actin Filaments ◽  
Gliding Motility ◽  
Filamentous Actin ◽  
Electron Cryomicroscopy ◽  
Force And Motion ◽  
Rigor State ◽  
Higher Eukaryotes ◽  
Spatial Positioning ◽  
Conserved Core

AbstractPlasmodium falciparum, the causative agent of malaria, moves by an atypical process called gliding motility. Actomyosin interactions are central to gliding motility. However, the details of these interactions remained elusive until now. Here, we report an atomic structure of the divergent Plasmodium falciparum actomyosin system determined by electron cryomicroscopy at the end of the powerstroke (Rigor state). The structure provides insights into the detailed interactions that are required for the parasite to produce the force and motion required for infectivity. Remarkably, the footprint of the myosin motor on filamentous actin is conserved with respect to higher eukaryotes, despite important variability in the Plasmodium falciparum myosin and actin elements that make up the interface. Comparison with other actomyosin complexes reveals a conserved core interface common to all actomyosin complexes, with an ancillary interface involved in defining the spatial positioning of the motor on actin filaments.

scholarly journals Formin-2 drives intracellular polymerisation of actin filaments enabling correct segregation of apicoplasts in Plasmodium falciparum and Toxoplasma gondii

2018 ◽  
Author(s):  
Johannes Felix Stortz ◽  
Mirko Singer ◽  
Jonathan M Wilkes ◽  
Markus Meissner ◽  
Sujaan Das
Keyword(s):  
Plasmodium Falciparum ◽  
Toxoplasma Gondii ◽  
Gliding Motility ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Comparative Approach ◽  
Actin Network ◽  
Filamentous Actin ◽  
Apicomplexan Parasites

AbstractPathogenic obligate-intracellular apicomplexan parasites possess an essential chloroplast-like organelle called the apicoplast that undergoes division and segregation during replication. Parasite actin is essential during intracellular development, implicated in vesicular transport, parasite replication and apicoplast inheritance. However, the inability to visualise live actin dynamics in apicomplexan parasites limited functional characterisation of both filamentous-actin (F-actin) and actin regulatory factors. Apicomplexans possess at least two distinct formins, Formin-1 and Formin-2, predicted to serve as actin-nucleating factors, and previously implicated in regulating gliding motility and host cell invasion. Here, we expressed chromobodies and validated them as F-actin-binding sensors in Plasmodium falciparum and characterised the in vivo dynamics of the F-actin network. The F-actin network could be modulated chemically and disrupted by conditionally deleting the actin-1 gene. In a comparative approach, we demonstrate that Formin-2 is closely associated with apicoplasts and with the F-actin network in P. falciparum and Toxoplasma gondii. Consequently, disruption of Formin-2 resulted not only in an apicoplast segregation defect, but also in complete abrogation of F-actin dynamics in intracellular parasites. Together, our results strongly indicate that Formin-2-mediated filament formation is the common primary mechanism for F-actin nucleation during apicomplexan intracellular growth effecting apicoplast segregation.

Myofibrillar degeneration with diphtheria toxin

2020 ◽  
Vol 45 (4) ◽  
pp. 351-357
Author(s):  
Bilge Özerman Edis ◽  
Muhammet Bektaş ◽  
Rüstem Nurten
Keyword(s):  
Protein Synthesis ◽  
Actin Filaments ◽  
Diphtheria Toxin ◽  
Cardiac Tissue ◽  
Culture Conditions ◽  
Bacterial Toxin ◽  
Filamentous Actin ◽  
Cardiac Damage ◽  
Tissue Samples

AbstractObjectivesCardiac damage in patient with diphtheritic myocarditis is reported as the leading cause of mortality. Diphtheria toxin (DTx) is a well-known bacterial toxin inducing various cytotoxic effects. Mainly, catalytic fragment inhibits protein synthesis, induces cytotoxicity, and depolymerizes actin filaments. In this study, we aimed to demonstrate the extent of myofibrillar damage under DTx treatment to porcine cardiac tissue samples.MethodsTissue samples were incubated with DTx for 1–3 h in culture conditions. To analyze whole toxin (both fragments) distribution, conjugation of DTx with FITC was performed. Measurements were carried out with fluorescence spectrophotometer before and after dialysis. Immunofluorescence microscopy was used to show localization of DTx-FITC (15 nM) on cardiac tissue incubated for 2 h. Ultrastructural characterization of cardiac tissue samples treated with DTx (15 or 150 nM) was performed with transmission electron microscopy.ResultsDTx exerts myofibrillar disorganization. Myofilament degeneration, mitochondrial damage, vacuolization, and abundant lipid droplets were determined with 150 nM of DTx treatment.ConclusionsThis finding is an addition to depolymerization of actin filaments as a result of the DTx-actin interactions in in vitro conditions, indicating that myofilament damage can occur with DTx directly besides protein synthesis inhibition. Ultrastructural results support the importance of filamentous actin degeneration at diphtheritic myocarditis.

scholarly journals Proteins P24 and P41 Function in the Regulation of Terminal-Organelle Development and Gliding Motility in Mycoplasma pneumoniae

2007 ◽  
Vol 189 (20) ◽  
pp. 7442-7449 ◽  
Author(s):  
Benjamin M. Hasselbring ◽  
Duncan C. Krause
Keyword(s):  
Cell Division ◽  
Mycoplasma Pneumoniae ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Gliding Motility ◽  
Atypical Pneumonia ◽  
Reading Frame ◽  
Spatial Positioning ◽  
Time Lapse Imaging ◽  
Gliding Mechanism

ABSTRACT Mycoplasma pneumoniae is a major cause of bronchitis and atypical pneumonia in humans. This cell wall-less bacterium has a complex terminal organelle that functions in cytadherence and gliding motility. The gliding mechanism is unknown but is coordinated with terminal-organelle development during cell division. Disruption of M. pneumoniae open reading frame MPN311 results in loss of protein P41 and downstream gene product P24. P41 localizes to the base of the terminal organelle and is required to anchor the terminal organelle to the cell body, but during cell division, MPN311 insertion mutants also fail to properly regulate nascent terminal-organelle development spatially or gliding activity temporally. We measured gliding velocity and frequency and used fluorescent protein fusions and time-lapse imaging to assess the roles of P41 and P24 individually in terminal-organelle development and gliding function. P41 was necessary for normal gliding velocity and proper spatial positioning of new terminal organelles, while P24 was required for gliding frequency and new terminal-organelle formation at wild-type rates. However, P41 was essential for P24 function, and in the absence of P41, P24 exhibited a dynamic localization pattern. Finally, protein P28 requires P41 for stability, but analysis of a P28− mutant established that the MPN311 mutant phenotype was not a function of loss of P28.

scholarly journals αE-catenin actin-binding domain alters actin filament conformation and regulates binding of nucleation and disassembly factors

2013 ◽  
Vol 24 (23) ◽  
pp. 3710-3720 ◽  
Author(s):  
Scott D. Hansen ◽  
Adam V. Kwiatkowski ◽  
Chung-Yueh Ouyang ◽  
HongJun Liu ◽  
Sabine Pokutta ◽  
...  
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Binding Domain ◽  
Actin Binding ◽  
Filamentous Actin ◽  
Filament Structure ◽  
Actin Binding Domain ◽  
Filament Bundles ◽  
Underlying Mechanisms ◽  
Cell Cell

The actin-binding protein αE-catenin may contribute to transitions between cell migration and cell–cell adhesion that depend on remodeling the actin cytoskeleton, but the underlying mechanisms are unknown. We show that the αE-catenin actin-binding domain (ABD) binds cooperatively to individual actin filaments and that binding is accompanied by a conformational change in the actin protomer that affects filament structure. αE-catenin ABD binding limits barbed-end growth, especially in actin filament bundles. αE-catenin ABD inhibits actin filament branching by the Arp2/3 complex and severing by cofilin, both of which contact regions of the actin protomer that are structurally altered by αE-catenin ABD binding. In epithelial cells, there is little correlation between the distribution of αE-catenin and the Arp2/3 complex at developing cell–cell contacts. Our results indicate that αE-catenin binding to filamentous actin favors assembly of unbranched filament bundles that are protected from severing over more dynamic, branched filament arrays.

scholarly journals Optimized fixation of actin filaments for improved indirect immunofluorescence staining of rickettsiae

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Monika Danchenko ◽  
Lucia Csaderova ◽  
Pierre Edouard Fournier ◽  
Zuzana Sekeyova
Keyword(s):  
Indirect Immunofluorescence ◽  
Actin Filaments ◽  
Immunofluorescence Assay ◽  
Host Cells ◽  
Immunofluorescence Staining ◽  
Sensitive Detection ◽  
Filamentous Actin ◽  
Indirect Immunofluorescence Staining ◽  
Accurate Imaging

Abstract Objective The objective was to investigate fixative solutions: 3.7% formaldehyde, 4% paraformaldehyde, 4% paraformaldehyde in the cytoskeletal buffer and 4% paraformaldehyde in PHEM buffer (containing PIPES, HEPES, EGTA and MgCl2), applicable for immunofluorescence assay. Results Herein we optimized this serological technique, testing four fixative solutions, for the sensitive detection of rickettsial antigens, and preservation of intracellular structures of the host cells, particularly filamentous actin. Rickettsial antigens were presented equally well both with formaldehyde and all paraformaldehyde-based fixations, but only protocol with 4% paraformaldehyde in PHEM buffer allowed accurate imaging of actin filaments, and simultaneously allows monitoring of rickettsiae using actin-based motility during infection inside the host cells.

scholarly journals Unusual Kinetic and Structural Properties Control Rapid Assembly and Turnover of Actin in the Parasite Toxoplasma gondii

2006 ◽  
Vol 17 (2) ◽  
pp. 895-906 ◽  
Author(s):  
Nivedita Sahoo ◽  
Wandy Beatty ◽  
John Heuser ◽  
David Sept ◽  
L. David Sibley
Keyword(s):  
Critical Concentration ◽  
Microscopic Analysis ◽  
Protozoan Parasite ◽  
Gliding Motility ◽  
Host Cells ◽  
Filamentous Actin ◽  
Unusual Behavior ◽  
Electron Microscopic ◽  
Unusual Form ◽  
Kinetics Of

Toxoplasma is a protozoan parasite in the phylum Apicomplexa, which contains a number of medically important parasites that rely on a highly unusual form of motility termed gliding to actively penetrate their host cells. Parasite actin filaments regulate gliding motility, yet paradoxically filamentous actin is rarely detected in these parasites. To investigate the kinetics of this unusual parasite actin, we expressed TgACT1 in baculovirus and purified it to homogeneity. Biochemical analysis showed that Toxoplasma actin (TgACT1) rapidly polymerized into filaments at a critical concentration that was 3-4-fold lower than conventional actins, yet it failed to copolymerize with mammalian actin. Electron microscopic analysis revealed that TgACT1 filaments were 10 times shorter and less stable than rabbit actin. Phylogenetic comparison of actins revealed a limited number of apicomplexan-specific residues that likely govern the unusual behavior of parasite actin. Molecular modeling identified several key alterations that affect interactions between monomers and that are predicted to destabilize filaments. Our findings suggest that conserved molecular differences in parasite actin favor rapid cycles of assembly and disassembly that govern the unusual form of gliding motility utilized by apicomplexans.

scholarly journals Overexpression of cofilin stimulates bundling of actin filaments, membrane ruffling, and cell movement in Dictyostelium.

1996 ◽  
Vol 132 (3) ◽  
pp. 335-344 ◽  
Author(s):  
H Aizawa ◽  
K Sutoh ◽  
I Yahara
Keyword(s):  
Actin Filaments ◽  
Cell Movement ◽  
Structure And Function ◽  
Cross Linking ◽  
Low Molecular Weight ◽  
Filamentous Actin ◽  
Membrane Ruffling ◽  
And Function

Cofilin is a low molecular weight actin-modulating protein whose structure and function are conserved among eucaryotes. Cofilin exhibits in vitro both a monomeric actin-sequestering activity and a filamentous actin-severing activity. To investigate in vivo functions of cofilin, cofilin was overexpressed in Dictyostelium discoideum cells. An increase in the content of D. discoideum cofilin (d-cofilin) by sevenfold induced a co-overproduction of actin by threefold. In cells over-expressing d-cofilin, the amount of filamentous actin but not that of monomeric actin was increased. Overexpressed d-cofilin co-sedimented with actin filaments, suggesting that the sequestering activity of d-cofilin is weak in vivo. The overexpression of d-cofilin increased actin bundles just beneath ruffling membranes where d-cofilin was co-localized. The overexpression of d-cofilin also stimulated cell movement as well as membrane ruffling. We have demonstrated in vitro that d-cofilin transformed latticework of actin filaments cross-linked by alpha-actinin into bundles probably by severing the filaments. D. discoideum cofilin may sever actin filaments in vivo and induce bundling of the filaments in the presence of cross-linking proteins so as to generate contractile systems involved in membrane ruffling and cell movement.

scholarly journals Actin depolymerizing factor controls actin turnover and gliding motility in Toxoplasma gondii

2011 ◽  
Vol 22 (8) ◽  
pp. 1290-1299 ◽  
Author(s):  
Simren Mehta ◽  
L. David Sibley
Keyword(s):  
Toxoplasma Gondii ◽  
Actin Filaments ◽  
Gliding Motility ◽  
Conditional Knockout ◽  
Host Cells ◽  
Actin Binding ◽  
Actin Depolymerizing Factor

Apicomplexan parasites rely on actin-based gliding motility to move across the substratum, cross biological barriers, and invade their host cells. Gliding motility depends on polymerization of parasite actin filaments, yet ∼98% of actin is nonfilamentous in resting parasites. Previous studies suggest that the lack of actin filaments in the parasite is due to inherent instability, leaving uncertain the role of actin-binding proteins in controlling dynamics. We have previously shown that the single allele of Toxoplasma gondii actin depolymerizing factor (TgADF) has strong actin monomer–sequestering and weak filament-severing activities in vitro. Here we used a conditional knockout strategy to investigate the role of TgADF in vivo. Suppression of TgADF led to accumulation of actin-rich filaments that were detected by immunofluorescence and electron microscopy. Parasites deficient in TgADF showed reduced speed of motility, increased aberrant patterns of motion, and inhibition of sustained helical gliding. Lack of TgADF also led to severe defects in entry and egress from host cells, thus blocking infection in vitro. These studies establish that the absence of stable actin structures in the parasite are not simply the result of intrinsic instability, but that TgADF is required for the rapid turnover of parasite actin filaments, gliding motility, and cell invasion.

scholarly journals Characterization of an F-actin-binding domain in the cytoskeletal protein vinculin.

1994 ◽  
Vol 126 (5) ◽  
pp. 1231-1240 ◽  
Author(s):  
A R Menkel ◽  
M Kroemker ◽  
P Bubeck ◽  
M Ronsiek ◽  
G Nikolai ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Direct Interaction ◽  
Binding Domain ◽  
Actin Binding ◽  
Filamentous Actin ◽  
Permeabilized Cells ◽  
Cell Matrix ◽  
Actin Binding Domain ◽  
Major Structural Component

Vinculin, a major structural component of vertebrate cell-cell and cell-matrix adherens junctions, has been found to interact with several other junctional components. In this report, we have identified and characterized a binding site for filamentous actin. These results included studies with gizzard vinculin, its proteolytic head and tail fragments, and recombinant proteins containing various gizzard vinculin sequences fused to the maltose binding protein (MBP) of Escherichia coli. In cosedimentation assays, only the vinculin tail sequence mediated a direct interaction with actin filaments. The binding was saturable, with a dissociation constant value in the micromolar range. Experiments with deletion clones localized the actin-binding domain to a region confined by residues 893-1016 in the 170-residue-long carboxyterminal segment, while the proline-rich hinge connecting the globular head to the rodlike tail was not required for this interaction. In fixed and permeabilized cells (cell models), as well as after microinjection, proteins containing the actin-binding domain specifically decorated stress fibers and the cortical network of fibroblasts and epithelial cells, as well as of brush border type microvilli. These results corroborated the sedimentation experiments. Our data support and extend previous work showing that vinculin binds directly to actin filaments. They are consistent with a model suggesting that in adhesive cells, the NH2-terminal head piece of vinculin directs this molecule to the focal contact sites, while its tail segment causes bundling of the actin filament ends into the characteristic spear tip-shaped structures.

scholarly journals Microinjection of ADP-ribosylated actin inhibits actin synthesis in hepatocyte-hepatoma hybrid cells

1996 ◽  
Vol 319 (3) ◽  
pp. 843-849 ◽  
Author(s):  
Karl H. REUNER ◽  
Anke van der DOES ◽  
Petra DUNKER ◽  
Ingo JUST ◽  
Klaus AKTORIES ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Culture Medium ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Hybrid Cells ◽  
Filamentous Actin ◽  
Clostridium Botulinum C2 Toxin ◽  
Actin Mrna ◽  
Immortalized Hepatocytes ◽  
C2 Toxin

Treatment of hepatocyte-hepatoma hybrid cells with Clostridium botulinum C2 toxin led to a 167% increase in monomeric globular actin (G-actin) and to a 57% decrease in filamentous actin (F-actin) within 2 h. Simultaneously, the level of actin mRNA was specifically decreased to 49% and actin synthesis was significantly diminished. In contrast, treatment of hybrid cells with phalloidin led to a decrease in G-actin to 55% and to a reciprocal increase in actin mRNA to 244% and an increase in actin synthesis. These alterations of actin synthesis depending on the G-actin/F-actin ratio corresponded to the autoregulation of actin synthesis observed in primary cultures of rat hepatocytes. Microinjection of C2 toxin or of phalloidin into hepatocyte-hepatoma hybrid cells had the same effects on actin synthesis as incubation with either toxin in the culture medium. Microinjection of non-polymerizable ADP-ribosylated G-actin into hepatocyte-hepatoma hybrid cells specifically decreased the incorporation of [35S]methionine into newly synthesized actin within 1 h. This decrease continued for at least 19 h. Microinjection of ADP-ribosylated actin led to rounding of cells and obvious disaggregation of actin filaments, which might be due to capping of actin filaments by the ADP-ribosylated actin. Because stabilization of actin filaments by phalloidin before microinjection of ADP-ribosylated actin also resulted in decreased actin synthesis, the concentration of monomeric G-actin seems to be responsible for the regulation of actin synthesis in hepatocyte-hepatoma hybrid cells, which can be regarded as immortalized hepatocytes.

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