scholarly journals Dimer arrangement and monomer flattening determine actin filament formation

2018 ◽  
Author(s):  
Maria Hoyer ◽  
Jose Rafael Cabral Correia ◽  
Don C. Lamb ◽  
Alvaro H. Crevenna
Keyword(s):  
Actin Filament ◽  
Zero Mode ◽  
Filament Formation ◽  
Cellular Processes ◽  
The Past ◽  
Filament Dynamics ◽  
Filament Assembly ◽  
Initial Monomer ◽  
Pointed End ◽  
Two Populations

ABSTRACTActin filament dynamics underlie key cellular processes, such as cell motility. Although actin filament elongation has been extensively studied under the past decades, the mechanism of filament nucleation remains unclear. Here, we immobilized gelsolin, a pointed-end nucleator, at the bottom of zero-mode waveguides to directly monitor the early steps of filament assembly. Our data revealed extensive dynamics and that only one, of two populations, elongates. Annalysis of the kinetics revealed a more stable trimer but a less stable tetramer in the elongating population compared to the non-elongating one. Furthermore, blocking flattening, the conformational change associated with filament formation, prevented the formation of both types of assemblies. Thus, flattening and the initial monomer arrangement determine gelsolin-mediated filament initiation.

scholarly journals Side-binding proteins modulate actin filament dynamics

2014 ◽  
Author(s):  
Alvaro H. Crevenna ◽  
Marcelino Arciniega ◽  
Aurelie Dupont ◽  
Kaja Kowalska ◽  
Oliver Lange ◽  
...  
Keyword(s):  
Actin Filament ◽  
Brownian Dynamics ◽  
Actin Polymerization ◽  
Actin Dynamics ◽  
Central Feature ◽  
Filament Dynamics ◽  
Filament Structure ◽  
The Rich ◽  
Dynamics Simulations ◽  
Pointed End

Actin filament dynamics govern many key physiological processes from cell motility to tissue morphogenesis. A central feature of actin dynamics is the capacity of the filament to polymerize and depolymerize at its ends in response to cellular conditions. It is currently thought that filament kinetics can be described by a single rate constant for each end. Here, using direct visualization of single actin filament elongation, we show that actin polymerization kinetics at both filament ends are strongly influenced by proteins that bind to the lateral filament surface. We also show that the less dynamic end, called the pointed-end, has a non-elongating state that dominates the observed filament kinetic asymmetry. Estimates of filament flexibility and Brownian dynamics simulations suggest that the observed kinetic diversity arises from structural alteration. Tuning filament kinetics by exploiting the natural malleability of the actin filament structure may be a ubiquitous mechanism to generate the rich variety of observed cellular actin dynamics.

scholarly journals Mechanism of synergistic actin filament pointed end depolymerization by cyclase-associated protein and cofilin

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Tommi Kotila ◽  
Hugo Wioland ◽  
Giray Enkavi ◽  
Konstantin Kogan ◽  
Ilpo Vattulainen ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Actin Filament ◽  
Actin Filaments ◽  
Monomeric Form ◽  
Actin Monomer ◽  
Filament Assembly ◽  
Structural Work ◽  
Cytoskeletal Dynamics ◽  
Pointed End ◽  
Dependent Processes

AbstractThe ability of cells to generate forces through actin filament turnover was an early adaptation in evolution. While much is known about how actin filaments grow, mechanisms of their disassembly are incompletely understood. The best-characterized actin disassembly factors are the cofilin family proteins, which increase cytoskeletal dynamics by severing actin filaments. However, the mechanism by which severed actin filaments are recycled back to monomeric form has remained enigmatic. We report that cyclase-associated-protein (CAP) works in synergy with cofilin to accelerate actin filament depolymerization by nearly 100-fold. Structural work uncovers the molecular mechanism by which CAP interacts with actin filament pointed end to destabilize the interface between terminal actin subunits, and subsequently recycles the newly-depolymerized actin monomer for the next round of filament assembly. These findings establish CAP as a molecular machine promoting rapid actin filament depolymerization and monomer recycling, and explain why CAP is critical for actin-dependent processes in all eukaryotes.

scholarly journals Filament dynamics driven by ATP hydrolysis modulates membrane binding of the bacterial actin MreB

2021 ◽  
Author(s):  
Vani Pande ◽  
Nivedita Mitra ◽  
Saket Rahul Bagde ◽  
Ramanujam Srinivasan ◽  
Pananghat Gayathri
Keyword(s):  
Atp Hydrolysis ◽  
Membrane Binding ◽  
Bound State ◽  
Filament Length ◽  
Lateral Interactions ◽  
Filament Formation ◽  
Wild Type ◽  
Spiroplasma Citri ◽  
Filament Dynamics ◽  
Filament Assembly

MreB, the bacterial ancestor of eukaryotic actin, is responsible for shape in most rod- shaped bacteria. While the eukaryotic actin utilizes ATP hydrolysis to drive filament treadmilling, the relevance of nucleotide-driven polymerization dynamics for MreB function is unclear. Here, we report mechanistic insights into the interplay between nucleotide-binding, ATP hydrolysis and membrane-binding of Spiroplasma citri MreB5 (ScMreB5). Antiparallel double protofilament assembly of ScMreB5WT with ATP, ADP or AMPPNP and an ATPase deficient mutant ScMreB5E134A demonstrate that the filaments assemble independent of ATP hydrolysis. However, capture of the filament dynamics revealed that efficient filament formation, bundling through lateral interactions and filament disassembly are affected in ScMreB5E134A. Hence, the catalytic glutamate (Glu134 in ScMreB5) plays a dual role - it functions as a switch by sensing the ATP-bound state for filament assembly, and by assisting hydrolysis for triggering disassembly. Glu134 mutation also exhibits an allosteric effect on membrane binding, as observed from the reduced liposome binding compared to that of the wild type. Thus, ATP hydrolysis can modulate filament length and bundling, and consequently the orientation of MreB filaments on the cell membrane depending on the curvature. Binding of ScMreB5 with liposomes is mediated by surface charge-based interactions, demonstrating paralog and organism specific features for MreB function. We conclude that the conserved ATP-dependent polymerization and disassembly upon ATP hydrolysis has been repurposed for modulating curvature-dependent organization of filaments on the membrane.

scholarly journals Erythroid differentiation in mouse erythroleukemia cells is driven via actin filament-tropomodulin3-tropomyosin networks

2021 ◽  
Author(s):  
Arit Ghosh ◽  
Megan Coffin ◽  
Richard West ◽  
Velia M Fowler
Keyword(s):  
Actin Filament ◽  
Terminal Differentiation ◽  
Erythroid Differentiation ◽  
Erythroid Terminal Differentiation ◽  
Capping Proteins ◽  
Filament Assembly ◽  
Maturation Stages ◽  
Pointed End ◽  
Triton X ◽  
Mouse Erythroleukemia

Erythroid differentiation (ED) is a complex cellular process entailing morphologically distinct maturation stages of erythroblasts during terminal differentiation. Studies of actin filament assembly and organization during terminal ED have revealed essential roles for the pointed-end actin filament capping proteins, tropomodulins (Tmod1 and Tmod3). Additionally, tropomyosin (Tpm) binding to Tmods is a key feature promoting Tmod-mediated actin filament capping. Global deletion of Tmod3 leads to embryonic lethality in mice with impaired ED. To test a cell autonomous function for Tmod3 and further decipher its biochemical function during ED, we generated a Tmod3 knockout in a mouse erythroleukemia cell line (Mel ds19). Tmod3 knockout cells appeared normal prior to ED, but showed defects during progression of ED, characterized by a marked failure to reduce cell and nuclear size, reduced viability and increased apoptosis. In Mel ds19 cells, both Tpms and actin were preferentially associated with the Triton-X 100 insoluble cytoskeleton during ED, indicating Tpm-coated actin filament assembly during ED. While loss of Tmod3 did not lead to a change in total actin levels, it led to a severe reduction in the proportion of Tpms and actin associated with the Triton-X 100 insoluble cytoskeleton during ED. We conclude that Tmod3-regulation of actin cytoskeleton assembly via Tpms is integral to morphological maturation and cell survival during normal erythroid terminal differentiation.

scholarly journals Side-binding proteins modulate actin filament dynamics

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Alvaro H Crevenna ◽  
Marcelino Arciniega ◽  
Aurélie Dupont ◽  
Naoko Mizuno ◽  
Kaja Kowalska ◽  
...  
Keyword(s):  
Actin Filament ◽  
Actin Polymerization ◽  
Actin Dynamics ◽  
Central Feature ◽  
Filament Dynamics ◽  
Physiological Processes ◽  
Filament Structure ◽  
Pointed End ◽  
Filament Surface ◽  
As Effects

Actin filament dynamics govern many key physiological processes from cell motility to tissue morphogenesis. A central feature of actin dynamics is the capacity of filaments to polymerize and depolymerize at their ends in response to cellular conditions. It is currently thought that filament kinetics can be described by a single rate constant for each end. In this study, using direct visualization of single actin filament elongation, we show that actin polymerization kinetics at both filament ends are strongly influenced by the binding of proteins to the lateral filament surface. We also show that the pointed-end has a non-elongating state that dominates the observed filament kinetic asymmetry. Estimates of flexibility as well as effects on fragmentation and growth suggest that the observed kinetic diversity arises from structural alteration. Tuning elongation kinetics by exploiting the malleability of the filament structure may be a ubiquitous mechanism to generate a rich variety of cellular actin dynamics.

Zero-mode waveguides visualize the first steps during gelsolin-mediated actin filament formation

2021 ◽  
Author(s):  
Maria Hoyer ◽  
Alvaro H. Crevenna ◽  
Jose Rafael Cabral Correia ◽  
Andrea G. Quezada ◽  
Don C. Lamb
Keyword(s):  
Actin Filament ◽  
Zero Mode ◽  
Filament Formation

scholarly journals Dynamics of Tpm1.8 domains on actin filaments with single-molecule resolution

2020 ◽  
Vol 31 (22) ◽  
pp. 2452-2462
Author(s):  
Ilina Bareja ◽  
Hugo Wioland ◽  
Miro Janco ◽  
Philip R. Nicovich ◽  
Antoine Jégou ◽  
...  
Keyword(s):  
Actin Filament ◽  
Single Molecule ◽  
Actin Filaments ◽  
Filament Formation ◽  
Filament Dynamics ◽  
Kinetics Of ◽  
Insight Into

Characterization of the kinetics of Tpm1.8 binding to actin filaments with single-molecule resolution. This work provides molecular insight into actin–tropomyosin filament formation and the role of tropomyosins in regulating actin filament dynamics.

scholarly journals Interactions with PIP2, ADP-actin monomers, and capping protein regulate the activity and localization of yeast twinfilin

2001 ◽  
Vol 155 (2) ◽  
pp. 251-260 ◽  
Author(s):  
Sandra Palmgren ◽  
Pauli J. Ojala ◽  
Martin A. Wear ◽  
John A. Cooper ◽  
Pekka Lappalainen
Keyword(s):  
Actin Filament ◽  
Mammalian Cells ◽  
Yeast Cells ◽  
Actin Monomer ◽  
Cortical Actin ◽  
Gene Encoding ◽  
Capping Protein ◽  
Filament Dynamics ◽  
Filament Assembly

Twinfilin is a ubiquitous actin monomer–binding protein that regulates actin filament turnover in yeast and mammalian cells. To elucidate the mechanism by which twinfilin contributes to actin filament dynamics, we carried out an analysis of yeast twinfilin, and we show here that twinfilin is an abundant protein that localizes to cortical actin patches in wild-type yeast cells. Native gel assays demonstrate that twinfilin binds ADP-actin monomers with higher affinity than ATP-actin monomers. A mutant twinfilin that does not interact with actin monomers in vitro no longer localizes to cortical actin patches when expressed in yeast, suggesting that the ability to interact with actin monomers may be essential for the localization of twinfilin. The localization of twinfilin to the cortical actin cytoskeleton is also disrupted in yeast strains where either the CAP1 or CAP2 gene, encoding for the α and β subunits of capping protein, is deleted. Purified twinfilin and capping protein form a complex on native gels. Twinfilin also interacts with phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2), and its actin monomer–sequestering activity is inhibited by PI(4,5)P2. Based on these results, we propose a model for the biological role of twinfilin as a protein that localizes actin monomers to the sites of rapid filament assembly in cells.

scholarly journals The Nck-interacting kinase NIK increases Arp2/3 complex activity by phosphorylating the Arp2 subunit

2015 ◽  
Vol 208 (2) ◽  
pp. 161-170 ◽  
Author(s):  
Lawrence L. LeClaire ◽  
Manish Rana ◽  
Martin Baumgartner ◽  
Diane L. Barber
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Actin Filament ◽  
Carcinoma Cells ◽  
Membrane Protrusion ◽  
Complex Activity ◽  
Filament Dynamics ◽  
Filament Assembly ◽  
Mammary Carcinoma Cells ◽  
Epidermal Growth

The nucleating activity of the Arp2/3 complex promotes the assembly of branched actin filaments that drive plasma membrane protrusion in migrating cells. Arp2/3 complex binding to nucleation-promoting factors of the WASP and WAVE families was previously thought to be sufficient to increase nucleating activity. However, phosphorylation of the Arp2 subunit was recently shown to be necessary for Arp2/3 complex activity. We show in mammary carcinoma cells that mutant Arp2 lacking phosphorylation assembled with endogenous subunits and dominantly suppressed actin filament assembly and membrane protrusion. We also report that Nck-interacting kinase (NIK), a MAP4K4, binds and directly phosphorylates the Arp2 subunit, which increases the nucleating activity of the Arp2/3 complex. In cells, NIK kinase activity was necessary for increased Arp2 phosphorylation and plasma membrane protrusion in response to epidermal growth factor. NIK is the first kinase shown to phosphorylate and increase the activity of the Arp2/3 complex, and our findings suggest that it integrates growth factor regulation of actin filament dynamics.

scholarly journals Actin filament assembly by bacterial factors VopL/F: Which end is up?

2017 ◽  
Vol 216 (5) ◽  
pp. 1211-1213
Author(s):  
Christina L. Vizcarra ◽  
Margot E. Quinlan
Keyword(s):  
Actin Filament ◽  
Direct Observation ◽  
Actin Filaments ◽  
Bacterial Proteins ◽  
Filament Assembly ◽  
Cell Biol ◽  
Competing Models ◽  
Pointed End

Competing models have been proposed for actin filament nucleation by the bacterial proteins VopL/F. In this issue, Burke et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201608104) use direct observation to demonstrate that VopL/F bind the barbed and pointed ends of actin filaments but only nucleate new filaments from the pointed end.

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