scholarly journals PI(3,5)P2 controls endosomal branched actin dynamics by regulating cortactin–actin interactions

2015 ◽  
Vol 210 (5) ◽  
pp. 753-769 ◽  
Author(s):  
Nan Hyung Hong ◽  
Aidong Qi ◽  
Alissa M. Weaver
Keyword(s):  
Membrane Trafficking ◽  
Membrane Curvature ◽  
Actin Dynamics ◽  
Complex Activity ◽  
Actin Networks ◽  
Binding Partner ◽  
Late Endosomes ◽  
Actin Turnover ◽  
Direct Binding

Branched actin critically contributes to membrane trafficking by regulating membrane curvature, dynamics, fission, and transport. However, how actin dynamics are controlled at membranes is poorly understood. Here, we identify the branched actin regulator cortactin as a direct binding partner of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) and demonstrate that their interaction promotes turnover of late endosomal actin. In vitro biochemical studies indicated that cortactin binds PI(3,5)P2 via its actin filament-binding region. Furthermore, PI(3,5)P2 competed with actin filaments for binding to cortactin, thereby antagonizing cortactin activity. These findings suggest that PI(3,5)P2 formation on endosomes may remove cortactin from endosome-associated branched actin. Indeed, inhibition of PI(3,5)P2 production led to cortactin accumulation and actin stabilization on Rab7+ endosomes. Conversely, inhibition of Arp2/3 complex activity greatly reduced cortactin localization to late endosomes. Knockdown of cortactin reversed PI(3,5)P2-inhibitor–induced actin accumulation and stabilization on endosomes. These data suggest a model in which PI(3,5)P2 binding removes cortactin from late endosomal branched actin networks and thereby promotes net actin turnover.

N-WASP inhibitor wiskostatin nonselectively perturbs membrane transport by decreasing cellular ATP levels

2007 ◽  
Vol 292 (4) ◽  
pp. C1562-C1566 ◽  
Author(s):  
Christopher J. Guerriero ◽  
Ora A. Weisz
Keyword(s):  
Membrane Trafficking ◽  
Actin Polymerization ◽  
Cell Function ◽  
Actin Dynamics ◽  
Cellular Functions ◽  
Intact Cells ◽  
Atp Levels ◽  
Dependent Inhibition

Wiskott-Aldrich syndrome protein (WASP) and WAVE stimulate actin-related protein (Arp)2/3-mediated actin polymerization, leading to diverse downstream effects, including the formation and remodeling of cell surface protrusions, modulation of cell migration, and intracytoplasmic propulsion of organelles and pathogens. Selective inhibitors of individual Arp2/3 activators would enable more exact dissection of WASP- and WAVE-dependent cellular pathways and are potential therapeutic targets for viral pathogenesis. Wiskostatin is a recently described chemical inhibitor that selectively inhibits neuronal WASP (N-WASP)-mediated actin polymerization in vitro. A growing number of recent studies have utilized this drug in vivo to uncover novel cellular functions for N-WASP; however, the selectivity of wiskostatin in intact cells has not been carefully explored. In our studies with this drug, we observed rapid and dose-dependent inhibition of N-WASP-dependent membrane trafficking steps. Additionally, however, we found that addition of wiskostatin inhibited numerous other cellular functions that are not believed to be N-WASP dependent. Further studies revealed that wiskostatin treatment caused a rapid, profound, and irreversible decrease in cellular ATP levels, consistent with its global effects on cell function. Our data caution against the use of this drug as a selective perturbant of N-WASP-dependent actin dynamics in vivo.

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 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 Comparative study of curvature sensing mediated by F-BAR domain and an intrinsically disordered region of FBP17

2020 ◽  
Author(s):  
Maohan Su ◽  
Yinyin Zhuang ◽  
Xinwen Miao ◽  
Yongpeng Zeng ◽  
Weibo Gao ◽  
...  
Keyword(s):  
Lipid Bilayer ◽  
Membrane Trafficking ◽  
Membrane Curvature ◽  
Wave System ◽  
Common Belief ◽  
Bar Domain ◽  
Curvature Sensing ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Region

Membrane curvature has emerged as an intriguing physical organization principle underlying biological signaling and membrane trafficking. FBP17 of the CIP4/FBP17/Toca-1 F-BAR family is unique in the BAR family because its structurally folded F-BAR domain does not contain any hydrophobic motifs that insert into lipid bilayer. While it has been widely assumed so, whether the banana-shaped F-BAR domain alone can sense curvature has never been experimentally demonstrated. Using a nanopillar-supported lipid bilayer system, we found that the F-BAR domain of FBP17 displayed minimal curvature sensing in vitro. We further identified an alternatively spliced intrinsically disordered region (IDR) of FBP17 next to its F-BAR domain that is conserved in sequence across species. The IDR senses membrane curvature and its sensing ability greatly exceeds that of F-BAR domain alone. In living cells, presence of the IDR domain changed the dynamics of FBP17 recruitment in a curvature-coupled cortical wave system. Collectively, we propose that FBP17 does sense curvature but contrary to the common belief, its curvature sensing capability largely originates from its disordered region, not F-BAR domain itself.

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 Structure of BrlR reveals a potential pyocyanin binding site

2017 ◽  
Author(s):  
Harikiran Raju ◽  
Rukmini Sundararajan ◽  
Rohan Sharma
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Binding Site ◽  
Transcriptional Regulator ◽  
Drug Tolerance ◽  
Binding Partner ◽  
Direct Binding ◽  
High Level

AbstractThe transcriptional regulator BrlR from Pseudomonas aeruginosa is a member of the MerR family of multidrug transport activators. Studies have shown BrlR plays an important role in high level drug tolerance of P. aeruginosa in biofilm. Its drug tolerance ability can be enhanced by 3′,5′-cyclic diguanylic acid (c-di-GMP). Here, we show the apo structure of BrlR and the direct binding between GyrI-like domain of BrlR and P. aeruginosa toxin pyocyanin. Furthermore, pyocyanin can enhance the binding between BrlR and DNA in vitro. These findings suggest BrlR can serve as the binding partner for both c-di-GMP and pyocyanin.

scholarly journals Lamina-associated polypeptide 2α is required for intranuclear MRTF-A activity

2021 ◽  
Author(s):  
Ekaterina Sidorenko ◽  
Maria Sokolova ◽  
Antti Pennanen ◽  
Salla Kyheroinen ◽  
Guido Posern ◽  
...  
Keyword(s):  
Target Genes ◽  
Serum Response Factor ◽  
Actin Dynamics ◽  
Chromatin Binding ◽  
Binding Partner ◽  
Related Transcription Factor ◽  
Direct Binding ◽  
Efficient Expression ◽  
Serum Response

Myocardin-related transcription factor A (MRTF-A), a coactivator of serum response factor (SRF), regulates the expression of many cytoskeletal genes in response to cytoplasmic and nuclear actin dynamics. Here we describe a novel mechanism to regulate MRTF-A activity within the nucleus by showing that lamina-associated polypeptide 2α (Lap2α), the nucleoplasmic isoform of Lap2, is a direct binding partner of MRTF-A, and required for the efficient expression of MRTF-A/SRF target genes. Mechanistically, Lap2α is not required for MRTF-A nuclear localization, unlike most other MRTF-A regulators, but is required for binding of MRTF-A to its target genes. This regulatory step takes place prior to MRTF-A chromatin binding, because Lap2α neither interacts with, nor specifically influences active histone marks on MRTF-A/SRF target genes. Phenotypically, Lap2α is required for serum-induced cell migration, and deregulated MRTF-A activity may also contribute to muscle and proliferation phenotypes associated with loss of Lap2α. Our studies therefore add another regulatory layer to the control of MRTF-A-SRF-mediated gene expression, and broaden the role of Lap2α in transcriptional regulation.

scholarly journals Multiplexed phospholipid membrane platform for curvature sensitive protein screening

Nanoscale ◽  
2021 ◽  
Author(s):  
Eider Berganza Eguiarte ◽  
Mirsana Ebrahimkutty ◽  
Srivatsan Vasantham ◽  
Chunting Zhong ◽  
Alexander Wunsch ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Lipid Membranes ◽  
Membrane Curvature ◽  
In Vitro Studies ◽  
Biological Processes ◽  
Phospholipid Membrane ◽  
Host Virus Interactions ◽  
Virus Interactions

The curvature of lipid membranes plays a key role in many relevant biological processes such as membrane trafficking, vesicular budding or host-virus interactions. In-vitro studies on membrane curvature of simplified...

scholarly journals Cofilin recruitment and function during actin-mediated endocytosis dictated by actin nucleotide state

2007 ◽  
Vol 178 (7) ◽  
pp. 1251-1264 ◽  
Author(s):  
Voytek Okreglak ◽  
David G. Drubin
Keyword(s):  
Actin Filament ◽  
Membrane Trafficking ◽  
Adenosine Diphosphate ◽  
Living Cells ◽  
Actin Assembly ◽  
Actin Networks ◽  
Actin Turnover ◽  
And Function

Cofilin is the major mediator of actin filament turnover in vivo. However, the molecular mechanism of cofilin recruitment to actin networks during dynamic actin-mediated processes in living cells and cofilin's precise in vivo functions have not been determined. In this study, we analyzed the dynamics of fluorescently tagged cofilin and the role of cofilin-mediated actin turnover during endocytosis in Saccharomyces cerevisiae. In living cells, cofilin is not necessary for actin assembly on endocytic membranes but is recruited to molecularly aged adenosine diphosphate actin filaments and is necessary for their rapid disassembly. Defects in cofilin function alter the morphology of actin networks in vivo and reduce the rate of actin flux through actin networks. The consequences of decreasing actin flux are manifested by decreased but not blocked endocytic internalization at the plasma membrane and defects in late steps of membrane trafficking to the vacuole. These results suggest that cofilin-mediated actin filament flux is required for the multiple steps of endocytic trafficking.

scholarly journals Twinfilin1 controls lamellipodial protrusive activity and actin turnover during vertebrate gastrulation

2020 ◽  
Author(s):  
Caitlin C. Devitt ◽  
Chanjae Lee ◽  
Rachael M. Cox ◽  
Ophelia Papoulas ◽  
José Alvarado ◽  
...  
Keyword(s):  
Animal Cell ◽  
Dynamic Control ◽  
Deep Understanding ◽  
Early Embryo ◽  
Cell Polarization ◽  
Actin Dynamics ◽  
Convergent Extension ◽  
Cell Movements ◽  
Actin Turnover

AbstractThe dynamic control of the actin cytoskeleton is a key aspect of essentially all animal cell movements. Experiments in single migrating cells and in vitro systems have provided an exceptionally deep understanding of actin dynamics. However, we still know relatively little of how these systems are tuned in cell-type specific ways, for example in the context of collective cell movements that sculpt the early embryo. Here, we provide an analysis of the actin severing and depolymerization machinery during vertebrate gastrulation, with a focus on Twinfilin1. We confirm previous results on the role of Twf1 in lamellipodia and extend those findings by linking Twf1, actin turnover, and cell polarization required for convergent extension during vertebrate gastrulation.

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