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

2017 ◽  
Author(s):  
Andrea Picco ◽  
Wanda Kukulski ◽  
Hetty E. Manenschijn ◽  
Tanja Specht ◽  
John A. G. Briggs ◽  
...  
Keyword(s):  
Hybrid Imaging ◽  
Correlative Microscopy ◽  
Actin Network ◽  
Filamentous Actin ◽  
Actin Networks ◽  
Cellular Processes ◽  
Membrane Invagination ◽  
Imaging Approach ◽  
Membrane Bending ◽  
Time Point

AbstractBranched and crosslinked actin networks mediate cellular processes that move and shape membranes. To understand how actin contributes during the different stages of endocytic membrane reshaping, we analysed deletion mutants of yeast actin network components using a hybrid imaging approach that combines live imaging with correlative microscopy. We could thereby 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 crosslinked. Once initiated, the membrane invagination process is driven by nucleation and polymerization of new actin filaments, independently of the degree of cross-linking and unaffected by a surplus of actin network components. A key transition occurs 2 seconds 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 assembled actin network. The expansion is sensitive to the amount of filamentous actin and its crosslinking. Our results suggest that the mechanism by which actin reshapes the membrane adapts to force requirements that vary during the progress of endocytosis.

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 MICAL2 acts through Arp3B isoform-specific Arp2/3 complexes to destabilize branched actin networks

2020 ◽  
Author(s):  
Chiara Galloni ◽  
Davide Carra ◽  
Jasmine V. G. Abella ◽  
Svend Kjær ◽  
Pavithra Singaravelu ◽  
...  
Keyword(s):  
Functional Properties ◽  
Actin Filament ◽  
Actin Assembly ◽  
Actin Network ◽  
Network Stability ◽  
Actin Networks ◽  
Cellular Processes ◽  
Actin Turnover ◽  
Filament Networks

AbstractThe Arp2/3 complex (Arp2, Arp3 and ARPC1-5) is essential to generate branched actin filament networks for many cellular processes. Human Arp3, ARPC1 and ARPC5 exist as two isoforms but the functional properties of Arp2/3 iso-complexes is largely unexplored. Here we show that Arp3B, but not Arp3 is subject to regulation by the methionine monooxygenase MICAL2, which is recruited to branched actin networks by coronin-1C. Although Arp3 and Arp3B iso-complexes promote actin assembly equally efficiently in vitro, they have different cellular properties. Arp3B turns over significantly faster than Arp3 within the network and upon its depletion actin turnover decreases. Substitution of Arp3B Met293 by Thr, the corresponding residue in Arp3 increases actin network stability, and conversely, replacing Arp3 Thr293 with Gln to mimic Met oxidation promotes network disassembly. Thus, MICAL2 regulates a subset of Arp2/3 complexes to control branched actin network disassembly.

scholarly journals Myosin 1b Flattens and Prunes Branched Actin Filaments

2020 ◽  
Author(s):  
Julien Pernier ◽  
Antoine Morchain ◽  
Valentina Caorsi ◽  
Aurélie Bertin ◽  
Hugo Bousquet ◽  
...  
Keyword(s):  
Total Internal Reflection ◽  
Molecular Motor ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Actin Network ◽  
Tirf Microscopy ◽  
Actin Networks ◽  
Cellular Processes

AbstractMotile and morphological cellular processes require a spatially and temporally coordinated branched actin network that is controlled by the activity of various regulatory proteins including the Arp2/3 complex, profilin, cofilin and tropomyosin. We have previously reported that myosin 1b regulates the density of the actin network in the growth cone. Using in vitro F-actin gliding assays and total internal reflection fluorescence (TIRF) microscopy we show in this report that this molecular motor flattens the Arp2/3-dependent actin branches up to breaking them and reduces the probability to form new branches. This experiment reveals that myosin 1b can produce force sufficient enough to break up the Arp2/3-mediated actin junction. Together with the former in vivo studies, this work emphasizes the essential role played by myosins in the architecture and in the dynamics of actin networks in different cellular regions.Short summaryUsing in vitro F-actin gliding assays and total internal reflection fluorescence (TIRF) microscopy we show that myosin flattens the Arp2/3-dependent actin branches up to breaking them and reduces the probability to form new branches

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 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 Multifunctional roles of Tropomodulin-3 in regulating actin dynamics

2018 ◽  
Author(s):  
Justin Parreno ◽  
Velia M Fowler
Keyword(s):  
Fetal Liver ◽  
Actin Dynamics ◽  
Cell Physiology ◽  
Actin Network ◽  
Actin Remodeling ◽  
Actin Networks ◽  
Cellular Processes ◽  
Definitive Erythropoiesis ◽  
Actin Capping ◽  
Slow Growing

Tropomodulins (Tmods) are proteins that cap the slow growing (pointed) ends of actin filaments (F-actin). The basis for our current understanding of Tmod function comes from studies in cells with relatively stable and highly organized F-actin networks, leading to the view that Tmod capping functions principally to preserve F-actin stability. However, not only is Tmod capping dynamic, but it also can play major roles in regulating diverse cellular processes involving F-actin remodeling. Here, we highlight the multifunctional roles of Tmod with a focus on Tmod3. Like other Tmods, Tmod3 binds tropomyosin (Tpm) and actin, capping pure F-actin at submicromolar and Tpm-coated F-actin at nanomolar concentrations. Unlike other Tmods, Tmod3 can also bind actin monomers and its ability to bind actin is inhibited by phosphorylation of Tmod3 by Akt2. Tmod3 is ubiquitously expressed and present in a diverse array of cytoskeletal structures, including contractile structures such as sarcomere-like units of actomyosin stress fibers and in the F-actin network encompassing adherens junctions. Tmod3 participates in F-actin network remodeling in lamellipodia during cell migration, and in the assembly of specialized F-actin networks during exocytosis. Furthermore, Tmod3 is required for development, regulating F-actin mesh formation during meiosis I of mouse oocytes, erythroblast enucleation in definitive erythropoiesis, and megakaryocyte morphogenesis in the mouse fetal liver. Thus, Tmod3 plays vital roles in dynamic and stable F-actin networks in cell physiology and development, with further research required to delineate the mechanistic details of Tmod3 regulation in the aforementioned processes, or in other yet to be discovered processes.

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 Myosin 1b flattens and prunes branched actin filaments

2020 ◽  
Vol 133 (18) ◽  
pp. jcs247403 ◽  
Author(s):  
Julien Pernier ◽  
Antoine Morchain ◽  
Valentina Caorsi ◽  
Aurélie Bertin ◽  
Hugo Bousquet ◽  
...  
Keyword(s):  
Total Internal Reflection ◽  
Essential Role ◽  
Molecular Motor ◽  
Regulatory Proteins ◽  
Actin Network ◽  
Actin Networks ◽  
Cellular Processes ◽  
Branch Angle

ABSTRACTMotile and morphological cellular processes require a spatially and temporally coordinated branched actin network that is controlled by the activity of various regulatory proteins, including the Arp2/3 complex, profilin, cofilin and tropomyosin. We have previously reported that myosin 1b regulates the density of the actin network in the growth cone. Here, by performing in vitro F-actin gliding assays and total internal reflection fluorescence (TIRF) microscopy, we show that this molecular motor flattens (reduces the branch angle) in the Arp2/3-dependent actin branches, resulting in them breaking, and reduces the probability of new branches forming. This experiment reveals that myosin 1b can produce force sufficient enough to break up the Arp2/3-mediated actin junction. Together with the former in vivo studies, this work emphasizes the essential role played by myosins in the architecture and dynamics of actin networks in different cellular regions.This article has an associated First Person interview with the first author of the paper.

scholarly journals Multifunctional roles of Tropomodulin-3 in regulating actin dynamics

2018 ◽  
Author(s):  
Justin Parreno ◽  
Velia M Fowler
Keyword(s):  
Fetal Liver ◽  
Actin Dynamics ◽  
Cell Physiology ◽  
Actin Network ◽  
Actin Remodeling ◽  
Actin Networks ◽  
Cellular Processes ◽  
Definitive Erythropoiesis ◽  
Actin Capping ◽  
Slow Growing

Tropomodulins (Tmods) are proteins that cap the slow growing (pointed) ends of actin filaments (F-actin). The basis for our current understanding of Tmod function comes from studies in cells with relatively stable and highly organized F-actin networks, leading to the view that Tmod capping functions principally to preserve F-actin stability. However, not only is Tmod capping dynamic, but it also can play major roles in regulating diverse cellular processes involving F-actin remodeling. Here, we highlight the multifunctional roles of Tmod with a focus on Tmod3. Like other Tmods, Tmod3 binds tropomyosin (Tpm) and actin, capping pure F-actin at submicromolar and Tpm-coated F-actin at nanomolar concentrations. Unlike other Tmods, Tmod3 can also bind actin monomers and its ability to bind actin is inhibited by phosphorylation of Tmod3 by Akt2. Tmod3 is ubiquitously expressed and present in a diverse array of cytoskeletal structures, including contractile structures such as sarcomere-like units of actomyosin stress fibers and in the F-actin network encompassing adherens junctions. Tmod3 participates in F-actin network remodeling in lamellipodia during cell migration, and in the assembly of specialized F-actin networks during exocytosis. Furthermore, Tmod3 is required for development, regulating F-actin mesh formation during meiosis I of mouse oocytes, erythroblast enucleation in definitive erythropoiesis, and megakaryocyte morphogenesis in the mouse fetal liver. Thus, Tmod3 plays vital roles in dynamic and stable F-actin networks in cell physiology and development, with further research required to delineate the mechanistic details of Tmod3 regulation in the aforementioned processes, or in other yet to be discovered processes.

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