Actin Assembly Factors Regulate the Gelation Kinetics and Architecture of F-actin Networks

The actin cytoskeleton generates forces on membranes for a wide range of cellular and subcellular morphogenic events, from cell migration to cytokinesis and membrane trafficking. For each of these processes, filamentous actin (F-actin) interacts with membranes and exerts force through its assembly, its associated myosin motors, or both. These two modes of force generation are well studied in isolation, but how they are coordinated in cells is mysterious. During clathrin-mediated endocytosis, F-actin assembly initiated by the Arp2/3 complex and several proteins that compose the WASP/myosin complex generates the force necessary to deform the plasma membrane into a pit. Here we present evidence that type I myosin is the key membrane anchor for endocytic actin assembly factors in budding yeast. By mooring actin assembly factors to the plasma membrane, this myosin organizes endocytic actin networks and couples actin-generated forces to the plasma membrane to drive invagination and scission. Through this unexpected mechanism, myosin facilitates force generation independent of its motor activity.

Download Full-text

Cell migration without a lamellipodium

The Journal of Cell Biology ◽

10.1083/jcb.200406063 ◽

2005 ◽

Vol 168 (4) ◽

pp. 619-631 ◽

Cited By ~ 206

Author(s):

Stephanie L. Gupton ◽

Karen L. Anderson ◽

Thomas P. Kole ◽

Robert S. Fischer ◽

Aaron Ponti ◽

...

Keyword(s):

Cell Migration ◽

Epithelial Cells ◽

Leading Edge ◽

Actin Binding ◽

Retrograde Flow ◽

Actin Assembly ◽

Migration Inhibition ◽

Actin Networks ◽

Actin Turnover ◽

High Concentrations

The actin cytoskeleton is locally regulated for functional specializations for cell motility. Using quantitative fluorescent speckle microscopy (qFSM) of migrating epithelial cells, we previously defined two distinct F-actin networks based on their F-actin–binding proteins and distinct patterns of F-actin turnover and movement. The lamellipodium consists of a treadmilling F-actin array with rapid polymerization-dependent retrograde flow and contains high concentrations of Arp2/3 and ADF/cofilin, whereas the lamella exhibits spatially random punctae of F-actin assembly and disassembly with slow myosin-mediated retrograde flow and contains myosin II and tropomyosin (TM). In this paper, we microinjected skeletal muscle αTM into epithelial cells, and using qFSM, electron microscopy, and immunolocalization show that this inhibits functional lamellipodium formation. Cells with inhibited lamellipodia exhibit persistent leading edge protrusion and rapid cell migration. Inhibition of endogenous long TM isoforms alters protrusion persistence. Thus, cells can migrate with inhibited lamellipodia, and we suggest that TM is a major regulator of F-actin functional specialization in migrating cells.

Download Full-text

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

10.1101/2020.09.21.306522 ◽

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.

Download Full-text

Lamellipodia-like actin networks in cells lacking WAVE Regulatory Complex

10.1101/2021.06.18.449030 ◽

2021 ◽

Author(s):

Frieda Kage ◽

Hermann Doering ◽

Magdalena Mietkowska ◽

Matthias Schaks ◽

Franziska Gruener ◽

...

Keyword(s):

Cell Migration ◽

Growth Factor ◽

Genome Editing ◽

Cell Periphery ◽

Actin Assembly ◽

Actin Remodeling ◽

Actin Networks ◽

Rac Gtpases ◽

Signaling Axis ◽

Regulatory Complex

Cell migration frequently involves the formation of lamellipodia induced by Rac GTPases mediating activation of WAVE Regulatory Complex (WRC) driving Arp2/3 complex-dependent actin assembly. Previous genome editing studies solidified the view of an essential, linear pathway employing aforementioned components. Using disruption of the WRC subunit Nap1 and its paralogue Hem1 followed by serum and growth factor stimulation or expression of active GTPases now revealed a pathway to formation of Arp2/3 complex-dependent, lamellipodia-like structures (LLS) that require both Rac and Cdc42, but not WRC. These observations were independent of WRC subunit eliminated and coincided with the lack of recruitment of Ena/VASP family actin polymerases. Moreover, aside from the latter, induced LLS contained all common lamellipodial regulators tested, including cortactin, the Ena/VASP ligand lamellipodin or FMNL subfamily formins. Our studies thus establish the existence of a signaling axis to Arp2/3 complex-dependent actin remodeling at the cell periphery operating without WRC and Ena/VASP.

Download Full-text

Regulation of Actin Assembly Associated With Protrusion and Adhesion in Cell Migration

Physiological Reviews ◽

10.1152/physrev.00021.2007 ◽

2008 ◽

Vol 88 (2) ◽

pp. 489-513 ◽

Cited By ~ 550

Author(s):

Christophe Le Clainche ◽

Marie-France Carlier

Keyword(s):

Cell Migration ◽

Actin Cytoskeleton ◽

Actin Filaments ◽

Actin Assembly ◽

Concerted Action ◽

Actin Networks ◽

Capping Proteins ◽

Cell Matrix ◽

A Cell ◽

Actomyosin Contraction

To migrate, a cell first extends protrusions such as lamellipodia and filopodia, forms adhesions, and finally retracts its tail. The actin cytoskeleton plays a major role in this process. The first part of this review (sect. ii) describes the formation of the lamellipodial and filopodial actin networks. In lamellipodia, the WASP-Arp2/3 pathways generate a branched filament array. This polarized dendritic actin array is maintained in rapid treadmilling by the concerted action of ADF, profilin, and capping proteins. In filopodia, formins catalyze the processive assembly of nonbranched actin filaments. Cell matrix adhesions mechanically couple actin filaments to the substrate to convert the treadmilling into protrusion and the actomyosin contraction into traction of the cell body and retraction of the tail. The second part of this review (sect. iii) focuses on the function and the regulation of major proteins (vinculin, talin, tensin, and α-actinin) that control the nucleation, the binding, and the barbed-end growth of actin filaments in adhesions.

Download Full-text

Activation of the Arp2/3 Complex by the Actin Filament Binding Protein Abp1p

The Journal of Cell Biology ◽

10.1083/jcb.153.3.627 ◽

2001 ◽

Vol 153 (3) ◽

pp. 627-634 ◽

Cited By ~ 139

Author(s):

Bruce L. Goode ◽

Avital A. Rodal ◽

Georjana Barnes ◽

David G. Drubin

Keyword(s):

Actin Filament ◽

Actin Filaments ◽

Mammalian Cells ◽

Actin Assembly ◽

Related Protein ◽

Genetic Studies ◽

Actin Networks ◽

Specific Step

The actin-related protein (Arp) 2/3 complex plays a central role in assembly of actin networks. Because distinct actin-based structures mediate diverse processes, many proteins are likely to make spatially and temporally regulated interactions with the Arp2/3 complex. We have isolated a new activator, Abp1p, which associates tightly with the yeast Arp2/3 complex. Abp1p contains two acidic sequences (DDW) similar to those found in SCAR/WASp proteins. We demonstrate that mutation of these sequences abolishes Arp2/3 complex activation in vitro. Genetic studies indicate that this activity is important for Abp1p functions in vivo. In contrast to SCAR/WASp proteins, Abp1p binds specifically to actin filaments, not monomers. Actin filament binding is mediated by the ADF/cofilin homology (ADF-H) domain of Abp1p and is required for Arp2/3 complex activation in vitro. We demonstrate that Abp1p recruits Arp2/3 complex to the sides of filaments, suggesting a novel mechanism of activation. Studies in yeast and mammalian cells indicate that Abp1p is involved functionally in endocytosis. Based on these results, we speculate that Abp1p may link Arp2/3-mediated actin assembly to a specific step in endocytosis.

Download Full-text

Inhibition of polar actin assembly by astral microtubules is required for cytokinesis

Nature Communications ◽

10.1038/s41467-021-22677-0 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Anan Chen ◽

Luisa Ulloa Severino ◽

Thomas C. Panagiotou ◽

Trevor F. Moraes ◽

Darren A. Yuen ◽

...

Keyword(s):

Cell Division ◽

Actin Cytoskeleton ◽

Actin Filament ◽

Cell Cortex ◽

Actin Assembly ◽

Actin Network ◽

Contractile Ring ◽

Actin Networks ◽

Membrane Blebbing ◽

Actin Isoform

AbstractDuring cytokinesis, the actin cytoskeleton is partitioned into two spatially distinct actin isoform specific networks: a β-actin network that generates the equatorial contractile ring, and a γ-actin network that localizes to the cell cortex. Here we demonstrate that the opposing regulation of the β- and γ-actin networks is required for successful cytokinesis. While activation of the formin DIAPH3 at the cytokinetic furrow underlies β-actin filament production, we show that the γ-actin network is specifically depleted at the cell poles through the localized deactivation of the formin DIAPH1. During anaphase, CLIP170 is delivered by astral microtubules and displaces IQGAP1 from DIAPH1, leading to formin autoinhibition, a decrease in cortical stiffness and localized membrane blebbing. The contemporaneous production of a β-actin contractile ring at the cell equator and loss of γ-actin from the poles is required to generate a stable cytokinetic furrow and for the completion of cell division.

Download Full-text

LC3 and STRAP regulate actin filament assembly by JMY during autophagosome formation

10.1101/451393 ◽

2018 ◽

Author(s):

Xiaohua Hu ◽

R. Dyche Mullins

Keyword(s):

Actin Filament ◽

Network Formation ◽

De Novo ◽

Actin Binding ◽

Actin Assembly ◽

Autophagosome Formation ◽

Actin Networks ◽

Filament Assembly ◽

Filament Networks ◽

Actin Comet Tails

AbstractDuring autophagy actin filament networks move and remodel cellular membranes to form autophagosomes that enclose and metabolize cytoplasmic contents. Two actin regulators, WHAMM and JMY, participate in autophagosome formation, but the signals linking autophagy to actin assembly are poorly understood. We show that, in non-starved cells, cytoplasmic JMY co-localizes with STRAP, a regulator of JMY’s nuclear functions, on non-motile vesicles with no associated actin networks. Upon starvation, JMY shifts to motile, LC3-containing membranes that move on actin comet tails. LC3 enhances JMY’s de novo actin nucleation activity via a cryptic actin-binding sequence near JMY’s N-terminus, and STRAP inhibits JMY’s ability to nucleate actin and activate the Arp2/3 complex. Cytoplasmic STRAP negatively regulates autophagy. Finally, we use purified proteins to reconstitute LC3‐ and JMY-dependent actin network formation on membranes, and inhibition of network formation by STRAP. We conclude that LC3 and STRAP regulate JMY’s actin assembly activities in trans during autophagy.eTOC BlurbThe actin regulator JMY creates filament networks that move membranes during autophagy. We find that, in unstarved cells, JMY is inhibited by interaction with the STRAP protein, but upon starvation JMY is recruited away from STRAP and activated by LC3.

Download Full-text

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

eLife ◽

10.7554/elife.60419 ◽

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.

Download Full-text