scholarly journals A mechanism with severing near barbed ends and annealing explains structure and dynamics of dendritic actin networks

2021 ◽  
Author(s):  
Danielle Holz ◽  
Aaron R Hall ◽  
Eiji Usukura ◽  
Sawako Yamashiro ◽  
Naoki Watanabe ◽  
...  
Keyword(s):  
Single Molecule ◽  
Leading Edge ◽  
Lifetime Distribution ◽  
Diffusion Annealing ◽  
Actin Networks ◽  
Structure And Dynamics ◽  
In Vitro Measurements ◽  
Filament Network ◽  
Orientation Pattern

Single molecule imaging has shown that part of actin disassembles within a few seconds after incorporation into the dendritic filament network in lamellipodia, suggestive of frequent destabilization near barbed ends. To investigate the mechanisms behind network remodeling, we created a stochastic model with polymerization, depolymerization, branching, capping, uncapping, severing, oligomer diffusion, annealing, and debranching. We find that filament severing, enhanced near barbed ends, can explain the single molecule actin lifetime distribution, if oligomer fragments reanneal to free ends with rate constants comparable to in vitro measurements. The same mechanism leads to actin networks consistent with measured filament, end, and branch concentrations. These networks undergo structural remodeling, leading to longer filaments away from the leading edge, at the +/- 35$^o$ orientation pattern. Imaging of actin speckle lifetimes at sub-second resolution verifies frequent disassembly of newly-assembled actin. We thus propose a unified mechanism that fits a diverse set of basic lamellipodia phenomenology.

scholarly journals Dissection of molecular assembly dynamics by tracking orientation and position of single molecules in live cells

2016 ◽  
Vol 113 (42) ◽  
pp. E6352-E6361 ◽  
Author(s):  
Shalin B. Mehta ◽  
Molly McQuilken ◽  
Patrick J. La Riviere ◽  
Patricia Occhipinti ◽  
Amitabh Verma ◽  
...  
Keyword(s):  
Single Molecule ◽  
Leading Edge ◽  
Living Cells ◽  
Molecular Assembly ◽  
Higher Order ◽  
Live Cells ◽  
Actin Network ◽  
Position And Orientation ◽  
Orientation Of Molecules

Regulation of order, such as orientation and conformation, drives the function of most molecular assemblies in living cells but remains difficult to measure accurately through space and time. We built an instantaneous fluorescence polarization microscope, which simultaneously images position and orientation of fluorophores in living cells with single-molecule sensitivity and a time resolution of 100 ms. We developed image acquisition and analysis methods to track single particles that interact with higher-order assemblies of molecules. We tracked the fluctuations in position and orientation of molecules from the level of an ensemble of fluorophores down to single fluorophores. We tested our system in vitro using fluorescently labeled DNA and F-actin, in which the ensemble orientation of polarized fluorescence is known. We then tracked the orientation of sparsely labeled F-actin network at the leading edge of migrating human keratinocytes, revealing the anisotropic distribution of actin filaments relative to the local retrograde flow of the F-actin network. Additionally, we analyzed the position and orientation of septin-GFP molecules incorporated in septin bundles in growing hyphae of a filamentous fungus. Our data indicate that septin-GFP molecules undergo positional fluctuations within ∼350 nm of the binding site and angular fluctuations within ∼30° of the central orientation of the bundle. By reporting position and orientation of molecules while they form dynamic higher-order structures, our approach can provide insights into how micrometer-scale ordered assemblies emerge from nanoscale molecules in living cells.

scholarly journals Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks

2019 ◽  
Author(s):  
Markku Hakala ◽  
Hugo Wioland ◽  
Mari Tolonen ◽  
Antoine Jegou ◽  
Guillaume Romet-Lemonne ◽  
...  
Keyword(s):  
Leading Edge ◽  
Underlying Mechanism ◽  
Actin Dynamics ◽  
Dissociation Kinetics ◽  
Single Filament ◽  
Actin Networks ◽  
Capping Protein ◽  
Specific Localization ◽  
In Cells

AbstractCoordinated polymerization of actin filaments provides force for cell migration, morphogenesis, and endocytosis. Capping Protein (CP) is central regulator of actin dynamics in all eukaryotes. It binds actin filament (F-actin) barbed ends with high affinity and slow dissociation kinetics to prevent filament polymerization and depolymerization. In cells, however, CP displays remarkably rapid dynamics within F-actin networks, but the underlying mechanism has remained enigmatic. We report that a conserved cytoskeletal regulator, twinfilin, is responsible for CP’s rapid dynamics and specific localization in cells. Depletion of twinfilin led to stable association of CP with cellular F-actin arrays and its treadmilling throughout leading-edge lamellipodium. These were accompanied by diminished F-actin disassembly rates. In vitro single filament imaging approaches revealed that twinfilin directly promotes dissociation of CP from filament barbed ends, while allowing subsequent filament depolymerization. These results uncover an evolutionary conserved bipartite mechanism that controls how actin cytoskeleton-mediated forces are generated in cells.

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 Arp2/3 complex ATP hydrolysis promotes lamellipodial actin network disassembly but is dispensable for assembly

2013 ◽  
Vol 200 (5) ◽  
pp. 619-633 ◽  
Author(s):  
Elena Ingerman ◽  
Jennifer Ying Hsiao ◽  
R. Dyche Mullins
Keyword(s):  
Atp Hydrolysis ◽  
Leading Edge ◽  
S2 Cells ◽  
Actin Network ◽  
Wild Type ◽  
Actin Networks ◽  
Dendritic Networks ◽  
A Cell

We examined the role of ATP hydrolysis by the Arp2/3 complex in building the leading edge of a cell by studying the effects of hydrolysis defects on the behavior of the complex in the lamellipodial actin network of Drosophila S2 cells and in a reconstituted, in vitro, actin-based motility system. In S2 cells, nonhydrolyzing Arp2 and Arp3 subunits expanded and delayed disassembly of lamellipodial actin networks and the effect of mutant subunits was additive. Arp2 and Arp3 ATP hydrolysis mutants remained in lamellipodial networks longer and traveled greater distances from the plasma membrane, even in networks still containing wild-type Arp2/3 complex. In vitro, wild-type and ATP hydrolysis mutant Arp2/3 complexes each nucleated actin and built similar dendritic networks. However, networks constructed with Arp2/3 hydrolysis-defective mutants were more resistant to disassembly by cofilin. Our results indicate that ATP hydrolysis on both Arp2 and Arp3 contributes to dissociation of the complex from the actin network but is not strictly necessary for lamellipodial network disassembly.

scholarly journals Insights into chromatin fibre structure by in vitro and in silico single-molecule stretching experiments

2013 ◽  
Vol 41 (2) ◽  
pp. 494-500 ◽  
Author(s):  
Rosana Collepardo-Guevara ◽  
Tamar Schlick
Keyword(s):  
Gene Regulation ◽  
Single Molecule ◽  
Fibre Structure ◽  
Detailed Structure ◽  
Single Molecule Force Spectroscopy ◽  
Linker Histones ◽  
Chromatin Fibre ◽  
Structure And Dynamics ◽  
Experimental Findings

The detailed structure and dynamics of the chromatin fibre and their relation to gene regulation represent important open biological questions. Recent advances in single-molecule force spectroscopy experiments have addressed these questions by directly measuring the forces that stabilize and alter the folded states of chromatin, and by investigating the mechanisms of fibre unfolding. We present examples that demonstrate how complementary modelling approaches have helped not only to interpret the experimental findings, but also to advance our knowledge of force-induced events such as unfolding of chromatin with dynamically bound linker histones and nucleosome unwrapping.

scholarly journals Myosin Va transport of liposomes in three-dimensional actin networks is modulated by actin filament density, position, and polarity

2019 ◽  
Vol 116 (17) ◽  
pp. 8326-8335 ◽  
Author(s):  
Andrew T. Lombardo ◽  
Shane R. Nelson ◽  
Guy G. Kennedy ◽  
Kathleen M. Trybus ◽  
Sam Walcott ◽  
...  
Keyword(s):  
Actin Filament ◽  
Molecular Motors ◽  
Actin Filaments ◽  
Three Dimensional ◽  
Fluorescent Reporter ◽  
Actin Networks ◽  
Cargo Transport ◽  
Myosin Va ◽  
Filament Network

The cell’s dense 3D actin filament network presents numerous challenges to vesicular transport by teams of myosin Va (MyoVa) molecular motors. These teams must navigate their cargo through diverse actin structures ranging from Arp2/3-branched lamellipodial networks to the dense, unbranched cortical networks. To define how actin filament network organization affects MyoVa cargo transport, we created two different 3D actin networks in vitro. One network was comprised of randomly oriented, unbranched actin filaments; the other was comprised of Arp2/3-branched actin filaments, which effectively polarized the network by aligning the actin filament plus-ends. Within both networks, we defined each actin filament’s 3D spatial position using superresolution stochastic optical reconstruction microscopy (STORM) and its polarity by observing the movement of single fluorescent reporter MyoVa. We then characterized the 3D trajectories of fluorescent, 350-nm fluid-like liposomes transported by MyoVa teams (∼10 motors) moving within each of the two networks. Compared with the unbranched network, we observed more liposomes with directed and fewer with stationary motion on the Arp2/3-branched network. This suggests that the modes of liposome transport by MyoVa motors are influenced by changes in the local actin filament polarity alignment within the network. This mechanism was supported by an in silico 3D model that provides a broader platform to understand how cellular regulation of the actin cytoskeletal architecture may fine tune MyoVa-based intracellular cargo transport.

scholarly journals In Vitro Measurements of Single-Molecule Transport Across an Individual Biomimetic Nuclear Pore Complex

2011 ◽  
Vol 100 (3) ◽  
pp. 521a ◽  
Author(s):  
Cees Dekker ◽  
Stefan Kowalczyk ◽  
Larissa Kapinos ◽  
Roderick Y.M. Lim
Keyword(s):  
Single Molecule ◽  
Nuclear Pore Complex ◽  
Nuclear Pore ◽  
In Vitro Measurements ◽  
Molecule Transport

scholarly journals MYOSIN VA TRANSPORT OF LIPOSOMES IN THREE-DIMENSIONAL ACTIN NETWORKS IS MODULATED BY ACTIN FILAMENT DENSITY, POSITION, AND POLARITY

2019 ◽  
Author(s):  
Andrew T. Lombardo ◽  
Shane R. Nelson ◽  
Guy G. Kennedy ◽  
Kathleen M. Trybus ◽  
Sam Walcott ◽  
...  
Keyword(s):  
Actin Filament ◽  
Molecular Motors ◽  
Actin Filaments ◽  
Three Dimensional ◽  
Fluorescent Reporter ◽  
Actin Networks ◽  
Cargo Transport ◽  
Myosin Va ◽  
Filament Network

The cell's dense three-dimensional (3D) actin filament network presents numerous challenges to vesicular transport by teams of myosin Va (MyoVa) molecular motors. These teams must navigate their cargo through diverse actin structures ranging from Arp2/3-branched lamellipodial networks to the dense, unbranched cortical networks. To define how actin filament network organization affects MyoVa cargo transport, we created two different 3D actin networks in vitro. One network was comprised of randomly oriented, unbranched actin filaments; the other was comprised of Arp2/3-branched actin filaments, which effectively polarized the network by aligning the actin filament plus-ends. Within both networks, we defined each actin filament's 3D spatial position, using STORM microscopy, and its polarity by observing the movement of single fluorescent, reporter MyoVa. We then characterized the 3D trajectories of fluorescent, 350 nm fluid-like, liposomes transported by MyoVa teams (~10 motors) moving within each of the two networks. Compared to the unbranched network, we observed more liposomes with directed and fewer with stationary motion on the Arp2/3-branched network. This suggests that the modes of liposome transport by MyoVa motors are influenced by changes in the local actin filament polarity alignment within the network. This mechanism was supported by an in silico 3D model that provides a broader platform to understand how cellular regulation of the actin cytoskeletal architecture may fine-tune MyoVa-based intracellular cargo transport.

Actin filament debranching regulates cell polarity during cell migration and asymmetric cell division

2021 ◽  
Vol 118 (37) ◽  
pp. e2100805118
Author(s):  
Chao Xie ◽  
Yuxiang Jiang ◽  
Zhiwen Zhu ◽  
Shanjin Huang ◽  
Wei Li ◽  
...  
Keyword(s):  
Cell Migration ◽  
Cell Fate ◽  
Cell Polarity ◽  
Actin Filaments ◽  
Asymmetric Cell Division ◽  
Leading Edge ◽  
Cell Stage ◽  
Actin Networks ◽  
C Elegans

The formation of the branched actin networks is essential for cell polarity, but it remains unclear how the debranching activity of actin filaments contributes to this process. Here, we showed that an evolutionarily conserved coronin family protein, the Caenorhabditis elegans POD-1, debranched the Arp2/3-nucleated actin filaments in vitro. By fluorescence live imaging analysis of the endogenous POD-1 protein, we found that POD-1 colocalized with Arp2/3 at the leading edge of the migrating C. elegans neuroblasts. Conditional mutations of POD-1 in neuroblasts caused aberrant actin assembly, disrupted cell polarity, and impaired cell migration. In C. elegans one-cell−stage embryos, POD-1 and Arp2/3, moved together during cell polarity establishment, and inhibition of POD-1 blocked Arp2/3 motility and affected the polarized cortical flow, leading to symmetric segregation of cell fate determinants. Together, these results indicate that F-actin debranching organizes actin network and cell polarity in migrating neuroblasts and asymmetrically dividing embryos.

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