Talin-activated vinculin interacts with branched actin networks to initiate bundles

Vinculin plays a fundamental role in integrin-mediated cell adhesion. Activated by talin, it interacts with diverse adhesome components, enabling mechanical coupling between the actin cytoskeleton and the extracellular matrix. Here we studied the interactions of activated full-length vinculin with actin and the way it regulates the organization and dynamics of the Arp2/3 complex-mediated branched actin network. Through a combination of surface patterning and light microscopy experiments we show that vinculin can bundle dendritic actin networks through rapid binding and filament crosslinking. We show that vinculin promotes stable but flexible actin bundles having a mixed-polarity organization, as confirmed by cryo-electron tomography. Adhesion-like synthetic design of vinculin activation by surface-bound talin revealed that clustered vinculin can initiate and immobilize bundles from mobile Arp2/3-branched networks. Our results provide a molecular basis for coordinate actin bundle formation at nascent adhesions.

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Cryo-electron Tomography Workflows for Quantitative Analysis of Actin Networks Involved in Cell Migration

Microscopy and Microanalysis ◽

10.1017/s1431927620021881 ◽

2020 ◽

Vol 26 (S2) ◽

pp. 2518-2519

Author(s):

Florian Fäßler ◽

Georgi Dimchev ◽

Victor-Valentin Hodirnau ◽

Bettina Zens ◽

Christoph Möhl ◽

...

Keyword(s):

Cell Migration ◽

Quantitative Analysis ◽

Electron Tomography ◽

Actin Networks ◽

Cryo Electron Tomography

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Mechanism of synergistic activation of Arp2/3 complex by cortactin and N-WASP

eLife ◽

10.7554/elife.00884 ◽

2013 ◽

Vol 2 ◽

Cited By ~ 59

Author(s):

Luke A Helgeson ◽

Brad J Nolen

Keyword(s):

Mathematical Modeling ◽

Actin Filament ◽

Single Molecule ◽

Molecular Basis ◽

Single Molecule Imaging ◽

Actin Network ◽

Actin Networks ◽

Synergistic Activation ◽

Complex Regulation

Nucleation promoting factors (NPFs) initiate branched actin network assembly by activating Arp2/3 complex, a branched actin filament nucleator. Cellular actin networks contain multiple NPFs, but how they coordinately regulate Arp2/3 complex is unclear. Cortactin is an NPF that activates Arp2/3 complex weakly on its own, but with WASP/N-WASP, another class of NPFs, potently activates. We dissect the mechanism of synergy and propose a model in which cortactin displaces N-WASP from nascent branches as a prerequisite for nucleation. Single-molecule imaging revealed that unlike WASP/N-WASP, cortactin remains bound to junctions during nucleation, and specifically targets junctions with a ∼160-fold increased on rate over filament sides. N-WASP must be dimerized for potent synergy, and targeted mutations indicate release of dimeric N-WASP from nascent branches limits nucleation. Mathematical modeling shows cortactin-mediated displacement but not N-WASP recycling or filament recruitment models can explain synergy. Our results provide a molecular basis for coordinate Arp2/3 complex regulation.

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A helical inner scaffold provides a structural basis for centriole cohesion

Science Advances ◽

10.1126/sciadv.aaz4137 ◽

2020 ◽

Vol 6 (7) ◽

pp. eaaz4137 ◽

Cited By ~ 21

Author(s):

Maeva Le Guennec ◽

Nikolai Klena ◽

Davide Gambarotto ◽

Marine H. Laporte ◽

Anne-Marie Tassin ◽

...

Keyword(s):

Mitotic Spindle ◽

Molecular Basis ◽

Structural Integrity ◽

Electron Tomography ◽

Structural Basis ◽

Ciliary Beating ◽

Cryo Electron Tomography ◽

Scaffold Structure ◽

Subtomogram Averaging ◽

Radial Arrangement

The ninefold radial arrangement of microtubule triplets (MTTs) is the hallmark of the centriole, a conserved organelle crucial for the formation of centrosomes and cilia. Although strong cohesion between MTTs is critical to resist forces applied by ciliary beating and the mitotic spindle, how the centriole maintains its structural integrity is not known. Using cryo–electron tomography and subtomogram averaging of centrioles from four evolutionarily distant species, we found that MTTs are bound together by a helical inner scaffold covering ~70% of the centriole length that maintains MTTs cohesion under compressive forces. Ultrastructure Expansion Microscopy (U-ExM) indicated that POC5, POC1B, FAM161A, and Centrin-2 localize to the scaffold structure along the inner wall of the centriole MTTs. Moreover, we established that these four proteins interact with each other to form a complex that binds microtubules. Together, our results provide a structural and molecular basis for centriole cohesion and geometry.

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Principles of self-organization and load adaptation by the actin cytoskeleton during clathrin-mediated endocytosis

eLife ◽

10.7554/elife.49840 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 15

Author(s):

Matthew Akamatsu ◽

Ritvik Vasan ◽

Daniel Serwas ◽

Michael A Ferrin ◽

Padmini Rangamani ◽

...

Keyword(s):

Electron Tomography ◽

Force Production ◽

Multiscale Model ◽

Membrane Tension ◽

Actin Assembly ◽

Actin Network ◽

Physical Constraints ◽

Filament Assembly ◽

Attachment Sites ◽

Cryo Electron Tomography

Force generation by actin assembly shapes cellular membranes. An experimentally constrained multiscale model shows that a minimal branched actin network is sufficient to internalize endocytic pits against membrane tension. Around 200 activated Arp2/3 complexes are required for robust internalization. A newly developed molecule-counting method determined that ~200 Arp2/3 complexes assemble at sites of clathrin-mediated endocytosis in human cells. Simulations predict that actin self-organizes into a radial branched array with growing ends oriented toward the base of the pit. Long actin filaments bend between attachment sites in the coat and the base of the pit. Elastic energy stored in bent filaments, whose presence was confirmed by cryo-electron tomography, contributes to endocytic internalization. Elevated membrane tension directs more growing filaments toward the base of the pit, increasing actin nucleation and bending for increased force production. Thus, spatially constrained actin filament assembly utilizes an adaptive mechanism enabling endocytosis under varying physical constraints.

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Cofilin Regulates Filopodial Structure and Flexibility in Neuronal Growth Cones

10.1101/2021.09.17.460569 ◽

2021 ◽

Author(s):

Ryan K. Hylton ◽

Jessica Heebner ◽

Michael Grillo ◽

Matthew T Swulius

Keyword(s):

Electron Tomography ◽

Leading Edge ◽

Growth Cones ◽

Axonal Guidance ◽

Neuronal Growth ◽

Cellular Targets ◽

Actin Networks ◽

Motile Cells ◽

Cryo Electron Tomography ◽

Neuronal Growth Cones

Filopodia are actin-rich cytoskeletal protrusions at the leading edge of motile cells. In neuronal growth cones they function as antennae, guiding axonal growth toward the appropriate cellular targets. Proper brain development relies on robust axonal guidance mechanisms, so it is imperative to understand how the actin cytoskeleton functions in remodeling to meet the demands of growth cone exploration. Here we show by cryo-electron tomography and fluorescence imaging that filopodia in neuronal growth cones switch between fascin-linked and cofilin-decorated states, and that this transition regulates the exclusion of fascin from the cofilactin bundle at the filopodial base by hyper-twisting individual filaments and rearranging their packing. Additionally, we show that cofilactin bundles contribute to the flexibility of filopodial actin networks, thus, likely regulating the efficiency of targeted neurite outgrowth.

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Principles of self-organization and load adaptation by the actin cytoskeleton during clathrin-mediated endocytosis

10.1101/678797 ◽

2019 ◽

Author(s):

Matthew Akamatsu ◽

Ritvik Vasan ◽

Daniel Serwas ◽

Michael Ferrin ◽

Padmini Rangamani ◽

...

Keyword(s):

Actin Filaments ◽

Mammalian Cells ◽

Electron Tomography ◽

Force Production ◽

Fundamental Aspect ◽

Membrane Tension ◽

Actin Network ◽

Physical Constraints ◽

Actin Networks ◽

Attachment Sites

SUMMARYForce generation due to actin assembly is a fundamental aspect of membrane sculpting for many essential processes. In this work, we use a multiscale computational model constrained by experimental measurements to show that a minimal branched actin network is sufficient to internalize endocytic pits against physiological membrane tension. A parameter sweep identified the number of Arp2/3 complexes as particularly important for robust internalization, which prompted the development of a molecule-counting method in live mammalian cells. Using this method, we found that ~200 Arp2/3 complexes assemble at sites of clathrin-mediated endocytosis in human cells. Our simulations also revealed that actin networks self-organize in a radial branched array with barbed filament ends oriented to grow toward the base of the pit, and that the distribution of linker proteins around the endocytic pit is critical for this organization. Surprisingly, our model predicted that long actin filaments bend from their attachment sites in the coat to the base of the pit and store elastic energy that can be harnessed to drive endocytosis. This prediction was validated using cryo-electron tomography on cells, which revealed the presence of bent actin filaments along the endocytic site. Furthermore, we predict that under elevated membrane tension, the self-organized actin network directs more growing filaments toward the base of the pit, increasing actin nucleation and bending for increased force production. Thus, our study reveals that spatially constrained actin filament assembly utilizes an adaptive mechanism that enables endocytosis under varying physical constraints.

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