Cytoskeletal Bundle Mechanics

2007 ◽  
Author(s):  
Mark Bathe ◽  
Claus Heussinger ◽  
Mireille Claessens ◽  
Andreas Bausch ◽  
Erwin Frey
Keyword(s):  
Acoustic Waves ◽  
Actin Binding ◽  
Mechanical Resistance ◽  
Mechanical Forces ◽  
Mechanical Stimuli ◽  
Filamentous Actin ◽  
Cellular Functions ◽  
Mechanochemical Transduction ◽  
Cellular Locomotion

Filamentous actin (F-actin) is a stiff biopolymer that is tightly crosslinked in vivo by actin-binding proteins (ABPs) to form stiff bundles that form major constituents of a multitude of slender cytoskeletal processes including stereocilia, filopodia, microvilli, neurosensory bristles, cytoskeletal stress fibers, and the acrosomal process of sperm cells (Fig. 1). The mechanical properties of these cytoskeletal processes play key roles in a broad range of cellular functions — the bending stiffness of stereocilia mediates the mechanochemical transduction of mechanical stimuli such as acoustic waves to detect sound, the critical buckling load of filopodia and acrosomal processes determines their ability to withstand compressive mechanical forces generated during cellular locomotion and fertilization, and the entropic stretching stiffness of cytoskeletal bundles mediates cytoskeletal mechanical resistance to cellular deformation. Thus, a detailed understanding of F-actin bundle mechanics is fundamental to gaining a mechanistic understanding of cytoskeletal function.

scholarly journals Structure of the Lifeact–F-actin complex

PLoS Biology ◽  
2020 ◽  
Vol 18 (11) ◽  
pp. e3000925 ◽  
Author(s):  
Alexander Belyy ◽  
Felipe Merino ◽  
Oleg Sitsel ◽  
Stefan Raunser
Keyword(s):  
Hypervariable Region ◽  
Binding Pocket ◽  
Actin Binding ◽  
Filamentous Actin ◽  
Activity Measurements ◽  
High Resolution Structure ◽  
D Loop ◽  
Hydrophobic Binding

Lifeact is a short actin-binding peptide that is used to visualize filamentous actin (F-actin) structures in live eukaryotic cells using fluorescence microscopy. However, this popular probe has been shown to alter cellular morphology by affecting the structure of the cytoskeleton. The molecular basis for such artefacts is poorly understood. Here, we determined the high-resolution structure of the Lifeact–F-actin complex using electron cryo-microscopy (cryo-EM). The structure reveals that Lifeact interacts with a hydrophobic binding pocket on F-actin and stretches over 2 adjacent actin subunits, stabilizing the DNase I-binding loop (D-loop) of actin in the closed conformation. Interestingly, the hydrophobic binding site is also used by actin-binding proteins, such as cofilin and myosin and actin-binding toxins, such as the hypervariable region of TccC3 (TccC3HVR) from Photorhabdus luminescens and ExoY from Pseudomonas aeruginosa. In vitro binding assays and activity measurements demonstrate that Lifeact indeed competes with these proteins, providing an explanation for the altering effects of Lifeact on cell morphology in vivo. Finally, we demonstrate that the affinity of Lifeact to F-actin can be increased by introducing mutations into the peptide, laying the foundation for designing improved actin probes for live cell imaging.

scholarly journals Coronin 1C harbours a second actin-binding site that confers co-operative binding to F-actin

2012 ◽  
Vol 444 (1) ◽  
pp. 89-96 ◽  
Author(s):  
Keefe T. Chan ◽  
David W. Roadcap ◽  
Nicholas Holoweckyj ◽  
James E. Bear
Keyword(s):  
Binding Site ◽  
Leading Edge ◽  
Actin Binding ◽  
Filamentous Actin ◽  
Unique Region ◽  
Equivalent Residue ◽  
Filament Networks ◽  
Actin Binding Site

Dynamic rearrangement of actin filament networks is critical for cell motility, phagocytosis and endocytosis. Coronins facilitate these processes, in part, by their ability to bind F-actin (filamentous actin). We previously identified a conserved surface-exposed arginine (Arg30) in the β-propeller of Coronin 1B required for F-actin binding in vitro and in vivo. However, whether this finding translates to other coronins has not been well defined. Using quantitative actin-binding assays, we show that mutating the equivalent residue abolishes F-actin binding in Coronin 1A, but not Coronin 1C. By mutagenesis and biochemical competition, we have identified a second actin-binding site in the unique region of Coronin 1C. Interestingly, leading-edge localization of Coronin 1C in fibroblasts requires the conserved site in the β-propeller, but not the site in the unique region. Furthermore, in contrast with Coronin 1A and Coronin 1B, Coronin 1C displays highly co-operative binding to actin filaments. In the present study, we highlight a novel mode of coronin regulation, which has implications for how coronins orchestrate cytoskeletal dynamics.

High turnover of ezrin T567 phosphorylation: conformation, activity, and cellular function

2007 ◽  
Vol 293 (3) ◽  
pp. C874-C884 ◽  
Author(s):  
Lixin Zhu ◽  
Rihong Zhou ◽  
Shelley Mettler ◽  
Tim Wu ◽  
Aennes Abbas ◽  
...  
Keyword(s):  
Membrane Surface ◽  
Resonance Energy ◽  
Actin Binding ◽  
Live Cells ◽  
Filamentous Actin ◽  
High Turnover ◽  
Binding Conformation ◽  
Phosphorylated Form

In its dormant state, the membrane cytoskeletal linker protein ezrin takes on a NH2 terminal-to-COOH terminal (N-C) binding conformation. In vitro evidence suggests that eliminating the N-C binding conformation by Thr567 phosphorylation leads to ezrin activation. Here, we found for resting gastric parietal cells that the levels of ezrin phosphorylation on Thr567 are low and can be increased to a small extent (∼40%) by stimulating secretion via the cAMP pathway. Treatment of cells with protein phosphatase inhibitors led to a rapid, dramatic increase in Thr567 phosphorylation by 400% over resting levels, prompting the hypothesis that ezrin activity is regulated by turnover of phosphorylation on Thr567. In vitro and in vivo fluorescence resonance energy transfer analysis demonstrated that Thr567 phosphorylation opens the N-C interaction. However, even in the closed conformation, ezrin localizes to membranes by an exposed NH2 terminal binding site. Importantly, the opened phosphorylated form of ezrin more readily cosediments with F-actin and binds more tightly to membrane than the closed forms. Furthermore, fluorescence recovery after photobleaching analysis in live cells showed that the Thr567Asp mutant had longer recovery times than the wild type or the Thr567Ala mutant, indicating the Thr567-phosphorylated form of ezrin is tightly associated with F-actin and the membrane, restricting normal activity. These data demonstrate and emphasize the functional importance of reversible phosphorylation of ezrin on F-actin binding. A novel model is proposed whereby ezrin and closely associated kinase and phosphatase proteins represent a motor complex to maintain a dynamic relationship between the varying membrane surface area and filamentous actin length.

Enolase exists in the fluid phase of cytoplasm in 3T3 cells

1989 ◽  
Vol 94 (2) ◽  
pp. 333-342
Author(s):  
L. Pagliaro ◽  
K. Kerr ◽  
D.L. Taylor
Keyword(s):  
Diffusion Coefficient ◽  
Fluid Phase ◽  
Glycolytic Enzyme ◽  
Binding Activity ◽  
Actin Binding ◽  
Filamentous Actin ◽  
3T3 Cells ◽  
Ratio Imaging

We have investigated the intracellular distribution and mobility of the glycolytic enzyme enolase, using functional fluorescent analogs labeled with the succinimidyl esters of carboxyfluorescein (F1-enolase) and carboxytetramethylrhodamine (Rh-enolase) In contrast to aldolase, neither native enolase nor labeled enolase gelled filamentous actin (F-actin), as measured by falling-ball viscometry, indicating a lack of interaction between enolase and F-actin. Fluorescence redistribution after photo-bleaching (FRAP) measurements of the diffusion coefficient (D) of F1-enolase in aqueous solutions gave a value of D37,aq = 6.08 × 10(−7) cm2s-1, and no immobile fraction, consistent with a native molecular weight of 90,000. These values were not significantly different with Rh-enolase, or in the presence of F-actin, 2-phosphoglycerate or F-actin-aldolase gels, demonstrating that neither F1-enolase nor Rh-enolase binds to F-actin or aldolase in vitro. FRAP measurements of F1- and Rh-enolase microinjected into living Swiss 3T3 cells revealed spatial differences in the diffusion coefficient, but not the mobile fraction. In the perinuclear cytoplasm, we measured an apparent diffusion coefficient of 1.1 × 10(−7) cm2s-1, compared to 7.1 × 10(−8) cm2s-1 in the peripheral cytoplasm, with approximately 100% mobility of F1- or Rh-enolase in both regions. Imaging of cells co-injected with Rh-enolase and size-fractionated FITC-dextran (FD-90) revealed that Rh-enolase entered the nucleus, while FD-90 was excluded. Ratio imaging showed a relatively high nuclear ratio of Rh-enolase/FD-90, and a uniform cytoplasmic ratio, with no indication of increased concentration of enolase around stress fibers. These data demonstrate that Rh- and F1-enolase do not bind to F-actin in vitro, and are 100% mobile in vivo. Together with our recent finding that a significant fraction of aldolase binds to F-actin in vitro and is immobile in vivo, these data suggest a correlation between actin-binding activity and cytoplasmic mobility of glycolytic enzymes.

scholarly journals F-Actin Cytoskeleton Network Self-Organization Through Competition and Cooperation

2020 ◽  
Vol 36 (1) ◽  
pp. 35-60
Author(s):  
Rachel S. Kadzik ◽  
Kaitlin E. Homa ◽  
David R. Kovar
Keyword(s):  
Actin Binding ◽  
Filamentous Actin ◽  
Cellular Functions ◽  
Actin Organization ◽  
Actin Networks ◽  
Cellular Processes ◽  
Competition And Cooperation ◽  
A Cell ◽  
Overlapping Sets

Many fundamental cellular processes such as division, polarization, endocytosis, and motility require the assembly, maintenance, and disassembly of filamentous actin (F-actin) networks at specific locations and times within the cell. The particular function of each network is governed by F-actin organization, size, and density as well as by its dynamics. The distinct characteristics of different F-actin networks are determined through the coordinated actions of specific sets of actin-binding proteins (ABPs). Furthermore, a cell typically assembles and uses multiple F-actin networks simultaneously within a common cytoplasm, so these networks must self-organize from a common pool of shared globular actin (G-actin) monomers and overlapping sets of ABPs. Recent advances in multicolor imaging and analysis of ABPs and their associated F-actin networks in cells, as well as the development of sophisticated in vitro reconstitutions of networks with ensembles of ABPs, have allowed the field to start uncovering the underlying principles by which cells self-organize diverse F-actin networks to execute basic cellular functions.

scholarly journals Actin activatesPseudomonas aeruginosaExoY nucleotidyl cyclase toxin and ExoY-like effector domains from MARTX toxins

2015 ◽  
Author(s):  
Dorothee Raoux-Barbot ◽  
Cosmin Saveanu ◽  
Abdelkader Namane ◽  
Vasily Ogryzko ◽  
Lina Worpenberg ◽  
...  
Keyword(s):  
Actin Binding ◽  
Target Cells ◽  
Actin Assembly ◽  
Filamentous Actin ◽  
Severe Damage ◽  
Chronic Infections ◽  
Effector Domains ◽  
Host Target

Pseudomonas aeruginosa is a major cause of chronic infections in cystic fibrosis patients. The nucleotidyl cyclase toxin ExoY is a virulence factors injected by the pathogen and associated with severe damage to lung tissue. ExoY-like cyclases are also found in other Gram-negative pathogens and shown to contribute to virulence, although they remained poorly characterized. Here we demonstrate that filamentous actin (F-actin) is the hitherto unknown co-factor that activates P. aeruginosa ExoY within host target cells. Highly purified actin, when polymerized into filaments, potently stimulates (>10,000 fold) ExoY activity. ExoY co-localizes in vivo with actin filaments in transfected cells and, in vitro, it interferes with the regulation of actin assembly/disassembly-dynamics mediated by important F-actin-binding proteins. We further show that actin also activates an ExoY-like adenylate cyclase from a Vibrio species. Our results thus highlight a new sub-class within the class II adenylyl cyclase family, defined as actin-activated nucleotidyl cyclase (AA-NC) toxins.

scholarly journals Visualization and Manipulation of Actin Cytoskeleton with Small-Molecular Probes

2020 ◽  
Author(s):  
Takeru Takagi ◽  
Tasuku Ueno ◽  
Keisuke Ikawa ◽  
Daisuke Asanuma ◽  
Yusuke Nomura ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Green Light ◽  
Dynamic Network ◽  
Super Resolution ◽  
Molecular Probes ◽  
Actin Binding ◽  
Mechanical Forces ◽  
Cellular Functions ◽  
New Class ◽  
Resolution Imaging

Actin is a ubiquitous cytoskeletal protein, forming a dynamic network that generates mechanical forces in the cell. Here, in order to dissect the complex mechanisms of actin-related cellular functions, we introduce two powerful tools based on a new class of actin-binding small molecule: one enables visualization of the actin cytoskeleton, including super-resolution imaging, and the other enables highly specific green-light-controlled fragmentation of actin filaments, affording unprecedented control of the actin cytoskeleton and its force network in living cells.

scholarly journals Visualization and Manipulation of Actin Cytoskeleton with Small-Molecular Probes

2020 ◽  
Author(s):  
Takeru Takagi ◽  
Tasuku Ueno ◽  
Keisuke Ikawa ◽  
Daisuke Asanuma ◽  
Yusuke Nomura ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Green Light ◽  
Dynamic Network ◽  
Super Resolution ◽  
Molecular Probes ◽  
Actin Binding ◽  
Mechanical Forces ◽  
Cellular Functions ◽  
New Class ◽  
Resolution Imaging

Actin is a ubiquitous cytoskeletal protein, forming a dynamic network that generates mechanical forces in the cell. Here, in order to dissect the complex mechanisms of actin-related cellular functions, we introduce two powerful tools based on a new class of actin-binding small molecule: one enables visualization of the actin cytoskeleton, including super-resolution imaging, and the other enables highly specific green-light-controlled fragmentation of actin filaments, affording unprecedented control of the actin cytoskeleton and its force network in living cells.

scholarly journals Formin-2 drives polymerisation of actin filaments enabling segregation of apicoplasts and cytokinesis in Plasmodium falciparum

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Johannes Felix Stortz ◽  
Mario Del Rosario ◽  
Mirko Singer ◽  
Jonathan M Wilkes ◽  
Markus Meissner ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Comparative Approach ◽  
Filamentous Actin ◽  
Erythrocyte Invasion ◽  
Actin Regulatory Proteins ◽  
Local Intensity ◽  
Actin Nucleator

In addition to its role in erythrocyte invasion, Plasmodium falciparum actin is implicated in endocytosis, cytokinesis and inheritance of the chloroplast-like organelle called the apicoplast. Previously, the inability to visualise filamentous actin (F-actin) dynamics had restricted the characterisation of both F-actin and actin regulatory proteins, a limitation we recently overcame for Toxoplasma (Periz et al, 2017). Here, we have expressed and validated actin-binding chromobodies as F-actin-sensors in Plasmodium falciparum and characterised in-vivo actin dynamics. F-actin could be chemically modulated, and genetically disrupted upon conditionally deleting actin-1. In a comparative approach, we demonstrate that Formin-2, a predicted nucleator of F-actin, is responsible for apicoplast inheritance in both Plasmodium and Toxoplasma, and additionally mediates efficient cytokinesis in Plasmodium. Finally, time-averaged local intensity measurements of F-actin in Toxoplasma conditional mutants revealed molecular determinants of spatiotemporally regulated F-actin flow. Together, our data indicate that Formin-2 is the primary F-actin nucleator during apicomplexan intracellular growth, mediating multiple essential functions.

scholarly journals Phosphoinositide binding and phosphorylation act sequentially in the activation mechanism of ezrin

2004 ◽  
Vol 164 (5) ◽  
pp. 653-659 ◽  
Author(s):  
Bruno T. Fievet ◽  
Alexis Gautreau ◽  
Christian Roy ◽  
Laurence Del Maestro ◽  
Paul Mangeat ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Binding Sites ◽  
Intramolecular Interaction ◽  
Activation Mechanism ◽  
Actin Binding ◽  
Activation Process ◽  
Cell Morphogenesis ◽  
Cellular Functions ◽  
Apical Localization

Ezrin, a membrane–actin cytoskeleton linker, which participates in epithelial cell morphogenesis, is held inactive in the cytoplasm through an intramolecular interaction. Phosphatidylinositol 4,5-bisphosphate (PIP2) binding and the phosphorylation of threonine 567 (T567) are involved in the activation process that unmasks both membrane and actin binding sites. Here, we demonstrate that ezrin binding to PIP2, through its NH2-terminal domain, is required for T567 phosphorylation and thus for the conformational activation of ezrin in vivo. Furthermore, we found that the T567D mutation mimicking T567 phosphorylation bypasses the need for PIP2 binding for unmasking both membrane and actin binding sites. However, PIP2 binding and T567 phosphorylation are both necessary for the correct apical localization of ezrin and for its role in epithelial cell morphogenesis. These results establish that PIP2 binding and T567 phosphorylation act sequentially to allow ezrin to exert its cellular functions.

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