Reptation of Microtubules in F-Actin Networks : Effects of Filament Stiffness and Network Topology on Reptation Dynamics

1997 ◽  
Vol 489 ◽  
Author(s):  
Jagesh V. Shah ◽  
Lisa A. Flanagan ◽  
David Bahk ◽  
Paul A. Janmey
Keyword(s):  
Network Topology ◽  
Actin Filaments ◽  
Diffusion Constant ◽  
Actin Network ◽  
Filamentous Actin ◽  
Actin Networks ◽  
Apparent Diffusion ◽  
Thermally Driven

AbstractThe thermally driven motions of fluorescently labeled microtubules embedded in a network of filamentous actin polymers are analysed as the diffusion of a rod-like polymer within a virtual tube formed by the surrounding semiflexible actin filaments. The apparent diffusion constant parallel to the tube scales with the inverse of the microtubule length and the magnitude is consistant with diffusion through a medium with a viscosity of approximately 10 centipoise. Introduction of crosslinks between the actin filaments does not alter the diffusion of the microtubules in the actin network.

scholarly journals Synergy between Cyclase-associated protein and Cofilin accelerates actin filament depolymerization by two orders of magnitude

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Shashank Shekhar ◽  
Johnson Chung ◽  
Jane Kondev ◽  
Jeff Gelles ◽  
Bruce L. Goode
Keyword(s):  
Actin Filament ◽  
Single Molecule ◽  
Actin Filaments ◽  
Binding Protein ◽  
Actin Dynamics ◽  
Actin Network ◽  
Cellular Mechanisms ◽  
Actin Networks ◽  
Binding Event

AbstractCellular actin networks can be rapidly disassembled and remodeled in a few seconds, yet in vitro actin filaments depolymerize slowly over minutes. The cellular mechanisms enabling actin to depolymerize this fast have so far remained obscure. Using microfluidics-assisted TIRF, we show that Cyclase-associated protein (CAP) and Cofilin synergize to processively depolymerize actin filament pointed ends at a rate 330-fold faster than spontaneous depolymerization. Single molecule imaging further reveals that hexameric CAP molecules interact with the pointed ends of Cofilin-decorated filaments for several seconds at a time, removing approximately 100 actin subunits per binding event. These findings establish a paradigm, in which a filament end-binding protein and a side-binding protein work in concert to control actin dynamics, and help explain how rapid actin network depolymerization is achieved in cells.

scholarly journals A barbed end interference mechanism reveals how capping protein promotes nucleation in branched actin networks

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Johanna Funk ◽  
Felipe Merino ◽  
Matthias Schaks ◽  
Klemens Rottner ◽  
Stefan Raunser ◽  
...  
Keyword(s):  
Structural Biology ◽  
Actin Filaments ◽  
Actin Network ◽  
Essential Factor ◽  
Actin Networks ◽  
Capping Protein ◽  
Cellular Processes ◽  
In Vitro Reconstitution ◽  
Interference Mechanism

AbstractHeterodimeric capping protein (CP/CapZ) is an essential factor for the assembly of branched actin networks, which push against cellular membranes to drive a large variety of cellular processes. Aside from terminating filament growth, CP potentiates the nucleation of actin filaments by the Arp2/3 complex in branched actin networks through an unclear mechanism. Here, we combine structural biology with in vitro reconstitution to demonstrate that CP not only terminates filament elongation, but indirectly stimulates the activity of Arp2/3 activating nucleation promoting factors (NPFs) by preventing their association to filament barbed ends. Key to this function is one of CP’s C-terminal “tentacle” extensions, which sterically masks the main interaction site of the terminal actin protomer. Deletion of the β tentacle only modestly impairs capping. However, in the context of a growing branched actin network, its removal potently inhibits nucleation promoting factors by tethering them to capped filament ends. End tethering of NPFs prevents their loading with actin monomers required for activation of the Arp2/3 complex and thus strongly inhibits branched network assembly both in cells and reconstituted motility assays. Our results mechanistically explain how CP couples two opposed processes—capping and nucleation—in branched actin network assembly.

Scinderin and cortical F-actin are components of the secretory machinery

1999 ◽  
Vol 77 (9) ◽  
pp. 660-671 ◽  
Author(s):  
J -M Trifaró
Keyword(s):  
Chromaffin Cell ◽  
Secretory Vesicle ◽  
Secretory Vesicles ◽  
Actin Network ◽  
Filamentous Actin ◽  
Actin Networks ◽  
Reserve Pool ◽  
Vesicle Exocytosis

Secretory vesicle exocytosis is the mechanism of release of neurotransmitters and neuropeptides. Secretory vesicles are localized in at least two morphologically and functionally distinct compartments: the reserve pool and the release-ready pool. Filamentous actin networks play an important role in this compartmentalization and in the trafficking of vesicles between these compartments. The cortical F-actin network constitutes a barrier (negative clamp) to the movement of secretory vesicles to release sites, and it must be locally disassembled to allow translocation of secretory vesicles in preparation for exocytosis. The disassembly of the cortical F-actin network is controlled by scinderin (a Ca2+-dependent F-actin severing protein) upon activation by Ca2+ entering the cells during stimulation. There are several factors that regulate scinderin activation (i.e., Ca2+ levels, phosphatidylinositol 4,5-bisphosphate (PIP2), etc.). The results suggest that scinderin and the cortical F-actin network are components of the secretory machinery.Key words: F-actin, scinderin, exocytosis, cytoskeleton, chromaffin cell.

scholarly journals The Arp2/3 Regulatory System and Its Deregulation in Cancer

2018 ◽  
Vol 98 (1) ◽  
pp. 215-238 ◽  
Author(s):  
Nicolas Molinie ◽  
Alexis Gautreau
Keyword(s):  
Cell Migration ◽  
Cancer Progression ◽  
Actin Filaments ◽  
Patient Survival ◽  
Regulatory System ◽  
Direct Impact ◽  
Actin Network ◽  
Actin Networks ◽  
Cross Links ◽  
The Stability

The Arp2/3 complex is an evolutionary conserved molecular machine that generates branched actin networks. When activated, the Arp2/3 complex contributes the actin branched junction and thus cross-links the polymerizing actin filaments in a network that exerts a pushing force. The different activators initiate branched actin networks at the cytosolic surface of different cellular membranes to promote their protrusion, movement, or scission in cell migration and membrane traffic. Here we review the structure, function, and regulation of all the direct regulators of the Arp2/3 complex that induce or inhibit the initiation of a branched actin network and that controls the stability of its branched junctions. Our goal is to present recent findings concerning novel inhibitory proteins or the regulation of the actin branched junction and place these in the context of what was previously known to provide a global overview of how the Arp2/3 complex is regulated in human cells. We focus on the human set of Arp2/3 regulators to compare normal Arp2/3 regulation in untransformed cells to the deregulation of the Arp2/3 system observed in patients affected by various cancers. In many cases, these deregulations promote cancer progression and have a direct impact on patient survival.

scholarly journals Synergy between Wsp1 and Dip1 may initiate assembly of endocytic actin networks

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Connor J Balzer ◽  
Michael L James ◽  
Heidy Y Narvaez-Ortiz ◽  
Luke A Helgeson ◽  
Vladimir Sirotkin ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Schizosaccharomyces Pombe ◽  
Actin Filament ◽  
Single Molecule ◽  
Actin Filaments ◽  
Total Internal Reflection ◽  
Actin Assembly ◽  
Actin Network ◽  
Actin Networks ◽  
Co Activation

The actin filament nucleator Arp2/3 complex is activated at cortical sites in Schizosaccharomyces pombe to assemble branched actin networks that drive endocytosis. Arp2/3 complex activators Wsp1 and Dip1 are required for proper actin assembly at endocytic sites, but how they coordinately control Arp2/3-mediated actin assembly is unknown. Alone, Dip1 activates Arp2/3 complex without preexisting actin filaments to nucleate ‘seed’ filaments that activate Wsp1-bound Arp2/3 complex, thereby initiating branched actin network assembly. In contrast, because Wsp1 requires preexisting filaments to activate, it has been assumed to function exclusively in propagating actin networks by stimulating branching from preexisting filaments. Here we show that Wsp1 is important not only for propagation but also for initiation of endocytic actin networks. Using single molecule total internal reflection fluorescence microscopy we show that Wsp1 synergizes with Dip1 to co-activate Arp2/3 complex. Synergistic co-activation does not require preexisting actin filaments, explaining how Wsp1 contributes to actin network initiation in cells.

scholarly journals The contributions of the actin machinery to endocytic membrane bending and vesicle formation

2018 ◽  
Vol 29 (11) ◽  
pp. 1346-1358 ◽  
Author(s):  
Andrea Picco ◽  
Wanda Kukulski ◽  
Hetty E. Manenschijn ◽  
Tanja Specht ◽  
John A. G. Briggs ◽  
...  
Keyword(s):  
Cross Linking ◽  
Actin Network ◽  
Filamentous Actin ◽  
Actin Networks ◽  
Cellular Processes ◽  
Membrane Invagination ◽  
Imaging Approach ◽  
Proportional Increase ◽  
Membrane Bending ◽  
Membrane Changes

Branched and cross-linked actin networks mediate cellular processes that move and shape membranes. To understand how actin contributes during the different stages of endocytic membrane reshaping, we analyzed deletion mutants of yeast actin network components using a hybrid imaging approach that combines live imaging with correlative microscopy. We could thus temporally dissect the effects of different actin network perturbations, revealing distinct stages of actin-based membrane reshaping. Our data show that initiation of membrane bending requires the actin network to be physically linked to the plasma membrane and to be optimally cross-linked. Once initiated, the membrane invagination process is driven by nucleation and polymerization of new actin filaments, independent of the degree of cross-linking and unaffected by a surplus of actin network components. A key transition occurs 2 s before scission, when the filament nucleation rate drops. From that time point on, invagination growth and vesicle scission are driven by an expansion of the actin network without a proportional increase of net actin amounts. The expansion is sensitive to the amount of filamentous actin and its cross-linking. Our results suggest that the mechanism by which actin reshapes the membrane changes during the progress of endocytosis, possibly adapting to varying force requirements.

scholarly journals Synergy between Wsp1 and Dip1 may initiate assembly of endocytic actin networks

2020 ◽  
Author(s):  
Connor J. Balzer ◽  
Michael L. James ◽  
Luke A. Helgeson ◽  
Vladimir Sirotkin ◽  
Brad J. Nolen
Keyword(s):  
Actin Filament ◽  
Single Molecule ◽  
Actin Filaments ◽  
Actin Assembly ◽  
Actin Network ◽  
Tirf Microscopy ◽  
Actin Networks ◽  
In Cells

AbstractThe actin filament nucleator Arp2/3 complex is activated at cortical sites in S. pombe to assemble branched actin networks that drive endocytosis. Arp2/3 complex activators Wsp1 and Dip1 are required for proper actin assembly at endocytic sites, but how they coordinately control Arp2/3-mediated actin assembly is unknown. Alone, Dip1 activates Arp2/3 complex without preexisting actin filaments to nucleate “seed” filaments that activate Wsp1-bound Arp2/3 complex, thereby initiating branched actin network assembly. In contrast, because Wsp1 requires pre-existing filaments to activate, it has been assumed to function exclusively in propagating actin networks by stimulating branching from pre-existing filaments. Here we show that Wsp1 is important not only for propagation, but also for initiation of endocytic actin networks. Using single molecule TIRF microscopy we show that Wsp1 synergizes with Dip1 to co-activate Arp2/3 complex. Synergistic coactivation does not require pre-existing actin filaments, explaining how Wsp1 contributes to actin network initiation in cells.

scholarly journals Toxoplasma gondii F-actin forms an extensive filamentous network required for material exchange and parasite maturation

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Javier Periz ◽  
Jamie Whitelaw ◽  
Clare Harding ◽  
Simon Gras ◽  
Mario Igor Del Rosario Minina ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Actin Filaments ◽  
Parasitophorous Vacuole ◽  
Lytic Cycle ◽  
Actin Dynamics ◽  
Residual Body ◽  
Actin Network ◽  
Filamentous Actin ◽  
Material Exchange

Apicomplexan actin is important during the parasite's life cycle. Its polymerization kinetics are unusual, permitting only short, unstable F-actin filaments. It has not been possible to study actin in vivo and so its physiological roles have remained obscure, leading to models distinct from conventional actin behaviour. Here a modified version of the commercially available actin-chromobody was tested as a novel tool for visualising F-actin dynamics in Toxoplasma gondii. Cb labels filamentous actin structures within the parasite cytosol and labels an extensive F-actin network that connects parasites within the parasitophorous vacuole and allows vesicles to be exchanged between parasites. In the absence of actin, parasites lack a residual body and inter-parasite connections and grow in an asynchronous and disorganized manner. Collectively, these data identify new roles for actin in the intracellular phase of the parasites lytic cycle and provide a robust new tool for imaging parasitic F-actin dynamics.

scholarly journals A structure-derived mechanism reveals how capping protein promotes nucleation in branched actin networks

2021 ◽  
Author(s):  
Johanna Funk ◽  
Felipe Merino ◽  
Matthias Schaks ◽  
Klemens Rottner ◽  
Stefan Raunser ◽  
...  
Keyword(s):  
Binding Site ◽  
Actin Filament ◽  
Actin Filaments ◽  
Actin Network ◽  
Essential Factor ◽  
Interaction Site ◽  
Actin Networks ◽  
Capping Protein ◽  
Cellular Processes ◽  
Terminal Filament

Heterodimeric capping protein (CP/CapZ) is an essential factor for the assembly of branched actin networks, which push against cellular membranes to drive a large variety of cellular processes. Aside from terminating filament growth, CP stimulates the nucleation of actin filaments by the Arp2/3 complex in branched actin networks through an unclear mechanism. Here, we report the structure of capped actin filament barbed ends, which reveals how CP not only prevents filament elongation, but also controls access to both terminal filament subunits. In addition to its primary binding site that blocks the penultimate subunit, we find that the CP sterically occludes the central interaction site of the terminal actin protomer through one of its C-terminal tentacle extensions. Deletion of this β tentacle only modestly impairs capping. However in the context of a growing branched actin network, its removal potently inhibits nucleation promoting factors (NPFs) by tethering them to capped filament ends. End tethering of NPFs prevents their loading with actin monomers required for activation of the Arp2/3 complex and thus strongly inhibits branched network assembly both in cells and reconstituted motility assays. Our results mechanistically explain how CP couples two opposed processes -capping and nucleation- in branched actin network assembly.

scholarly journals Competition between Tropomyosin, Fimbrin, and ADF/Cofilin drives their sorting to distinct actin filament networks

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Jenna R Christensen ◽  
Glen M Hocky ◽  
Kaitlin E Homa ◽  
Alisha N Morganthaler ◽  
Sarah E Hitchcock-DeGregori ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Actin Binding ◽  
Actin Network ◽  
Actin Networks ◽  
Cooperative Interactions ◽  
Filament Networks ◽  
Collective Interactions ◽  
Functionally Diverse ◽  
Cooperative Association

The fission yeast actin cytoskeleton is an ideal, simplified system to investigate fundamental mechanisms behind cellular self-organization. By focusing on the stabilizing protein tropomyosin Cdc8, bundling protein fimbrin Fim1, and severing protein coffin Adf1, we examined how their pairwise and collective interactions with actin filaments regulate their activity and segregation to functionally diverse F-actin networks. Utilizing multi-color TIRF microscopy of in vitro reconstituted F-actin networks, we observed and characterized two distinct Cdc8 cables loading and spreading cooperatively on individual actin filaments. Furthermore, Cdc8, Fim1, and Adf1 all compete for association with F-actin by different mechanisms, and their cooperative association with actin filaments affects their ability to compete. Finally, competition between Fim1 and Adf1 for F-actin synergizes their activities, promoting rapid displacement of Cdc8 from a dense F-actin network. Our findings reveal that competitive and cooperative interactions between actin binding proteins help define their associations with different F-actin networks.

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