Cobl-like promotes actin filament formation and dendritic branching using only a single WH2 domain

Local actin filament formation powers the development of the signal-receiving arbor of neurons that underlies neuronal network formation. Yet, little is known about the molecules that drive these processes and may functionally connect them to the transient calcium pulses observed in restricted areas in the forming dendritic arbor. Here we demonstrate that Cordon-Bleu (Cobl)–like, an uncharacterized protein suggested to represent a very distantly related, evolutionary ancestor of the actin nucleator Cobl, despite having only a single G-actin–binding Wiskott–Aldrich syndrome protein Homology 2 (WH2) domain, massively promoted the formation of F-actin–rich membrane ruffles of COS-7 cells and of dendritic branches of neurons. Cobl-like hereby integrates WH2 domain functions with those of the F-actin–binding protein Abp1. Cobl-like–mediated dendritic branching is dependent on Abp1 as well as on Ca2+/calmodulin (CaM) signaling and CaM association. Calcium signaling leads to a promotion of complex formation with Cobl-like’s cofactor Abp1. Thus, Ca2+/CaM control of actin dynamics seems to be a much more broadly used principle in cell biology than previously thought.

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Functional interdependence of the actin nucleator Cobl and Cobl-like in dendritic arbor development

eLife ◽

10.7554/elife.67718 ◽

2021 ◽

Vol 10 ◽

Author(s):

Maryam Izadi ◽

Eric Seemann ◽

Dirk Schlobinski ◽

Lukas Schwintzer ◽

Britta Qualmann ◽

...

Keyword(s):

Plasma Membrane ◽

Actin Filament ◽

Network Formation ◽

Actin Dynamics ◽

Binding Motif ◽

Dendritic Arbor ◽

Filament Formation ◽

Calmodulin Binding ◽

Physical Linkage ◽

Actin Nucleator

Local actin filament formation is indispensable for development of the dendritic arbor of neurons. We show that, surprisingly, the action of single actin filament-promoting factors was insufficient for powering dendritogenesis. Instead, this required the actin nucleator Cobl and its only evolutionary distant ancestor Cobl-like acting interdependently. This coordination between Cobl-like and Cobl was achieved by physical linkage by syndapins. Syndapin I formed nanodomains at convex plasma membrane areas at the base of protrusive structures and interacted with three motifs in Cobl-like, one of which was Ca2+/calmodulin-regulated. Consistently, syndapin I, Cobl-like’s newly identified N terminal calmodulin-binding site and the single Ca2+/calmodulin-responsive syndapin-binding motif all were critical for Cobl-like’s functions. In dendritic arbor development, local Ca2+/CaM-controlled actin dynamics thus relies on regulated and physically coordinated interactions of different F-actin formation-promoting factors and only together they have the power to bring about the sophisticated neuronal morphologies required for neuronal network formation in mammals.

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Functional interdependence of the actin nucleator Cobl and Cobl-like in dendritic arbor development

10.1101/2021.03.03.433715 ◽

2021 ◽

Author(s):

Maryam Izadi ◽

Eric Seemann ◽

Dirk Schlobinski ◽

Lukas Schwintzer ◽

Britta Qualmann ◽

...

Keyword(s):

Plasma Membrane ◽

Actin Filament ◽

Network Formation ◽

Actin Dynamics ◽

Binding Motif ◽

Dendritic Arbor ◽

Filament Formation ◽

Calmodulin Binding ◽

Physical Linkage ◽

Actin Nucleator

AbstractLocal actin filament formation is indispensable for development of the dendritic arbor of neurons. We show that, surprisingly, the action of single actin filament-promoting factors was insufficient for powering dendritogenesis. Instead, this process required the actin nucleator Cobl and its only evolutionary distant ancestor Cobl-like acting interdependently. This coordination between Cobl-like and Cobl was achieved by physical linkage by syndapin I. Syndapin I formed nanodomains at convex plasma membrane areas at the base of protrusive structures and interacted with three motifs in Cobl-like, one of which was Ca2+/calmodulin-regulated. Consistently, syndapin I, Cobl-like’s newly identified N terminal calmodulin-binding site and the single Ca2+/calmodulin-responsive syndapin-binding motif all were critical for Cobl-like’s functions. In dendritic arbor development, local Ca2+/CaM-controlled actin dynamics thus relies on regulated and physically coordinated interactions of different F-actin formation-promoting factors and only together they have the power to bring about the sophisticated neuronal morphologies required for neuronal network formation.

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Actin-depolymerizing Factor and Cofilin-1 Play Overlapping Roles in Promoting Rapid F-Actin Depolymerization in Mammalian Nonmuscle Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e04-07-0555 ◽

2005 ◽

Vol 16 (2) ◽

pp. 649-664 ◽

Cited By ~ 240

Author(s):

Pirta Hotulainen ◽

Eija Paunola ◽

Maria K. Vartiainen ◽

Pekka Lappalainen

Keyword(s):

Cell Motility ◽

Actin Filament ◽

Actin Filaments ◽

Actin Dynamics ◽

Actin Binding ◽

Actin Depolymerizing Factor ◽

Cytoskeletal Dynamics ◽

Nonmuscle Cells ◽

In Cells

Actin-depolymerizing factor (ADF)/cofilins are small actin-binding proteins found in all eukaryotes. In vitro, ADF/cofilins promote actin dynamics by depolymerizing and severing actin filaments. However, whether ADF/cofilins contribute to actin dynamics in cells by disassembling “old” actin filaments or by promoting actin filament assembly through their severing activity is a matter of controversy. Analysis of mammalian ADF/cofilins is further complicated by the presence of multiple isoforms, which may contribute to actin dynamics by different mechanisms. We show that two isoforms, ADF and cofilin-1, are expressed in mouse NIH 3T3, B16F1, and Neuro 2A cells. Depleting cofilin-1 and/or ADF by siRNA leads to an accumulation of F-actin and to an increase in cell size. Cofilin-1 and ADF seem to play overlapping roles in cells, because the knockdown phenotype of either protein could be rescued by overexpression of the other one. Cofilin-1 and ADF knockdown cells also had defects in cell motility and cytokinesis, and these defects were most pronounced when both ADF and cofilin-1 were depleted. Fluorescence recovery after photobleaching analysis and studies with an actin monomer-sequestering drug, latrunculin-A, demonstrated that these phenotypes arose from diminished actin filament depolymerization rates. These data suggest that mammalian ADF and cofilin-1 promote cytoskeletal dynamics by depolymerizing actin filaments and that this activity is critical for several processes such as cytokinesis and cell motility.

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EPLIN regulates actin dynamics by cross-linking and stabilizing filaments

The Journal of Cell Biology ◽

10.1083/jcb.200212057 ◽

2003 ◽

Vol 160 (3) ◽

pp. 399-407 ◽

Cited By ~ 92

Author(s):

Raymond S. Maul ◽

Yuhong Song ◽

Kurt J. Amann ◽

Sachi C. Gerbin ◽

Thomas D. Pollard ◽

...

Keyword(s):

Actin Filament ◽

Actin Polymerization ◽

Binding Activity ◽

Stress Fibers ◽

Actin Dynamics ◽

Actin Binding ◽

Membrane Ruffling ◽

Cross Links ◽

As Stress ◽

Kinetics Of

Epithelial protein lost in neoplasm (EPLIN) is a cytoskeleton-associated protein encoded by a gene that is down-regulated in transformed cells. EPLIN increases the number and size of actin stress fibers and inhibits membrane ruffling induced by Rac. EPLIN has at least two actin binding sites. Purified recombinant EPLIN inhibits actin filament depolymerization and cross-links filaments in bundles. EPLIN does not affect the kinetics of spontaneous actin polymerization or elongation at the barbed end, but inhibits branching nucleation of actin filaments by Arp2/3 complex. Side binding activity may stabilize filaments and account for the inhibition of nucleation mediated by Arp2/3 complex. We propose that EPLIN promotes the formation of stable actin filament structures such as stress fibers at the expense of more dynamic actin filament structures such as membrane ruffles. Reduced expression of EPLIN may contribute to the motility of invasive tumor cells.

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Rickettsia Sca2 Recruits Two Actin Subunits for Nucleation but Lacks WH2 Domains

10.1101/467431 ◽

2018 ◽

Author(s):

SS Alqassim ◽

IG Lee ◽

R Dominguez

Keyword(s):

Amino Acid ◽

Actin Filament ◽

Actin Polymerization ◽

Intramolecular Interactions ◽

Actin Binding ◽

Globular Domain ◽

Comet Tail ◽

Capping Protein ◽

High Affinity ◽

Actin Nucleator

AbstractThe Rickettsia ~1,800 amino acid autotransporter protein Sca2 promotes actin polymerization on the surface of the bacterium to drive its movement using an actin comet tail mechanism. Sca2 mimics eukaryotic formins in that it promotes both actin filament nucleation and elongation and competes with capping protein to generate filaments that are long and unbranched. However, despite these functional similarities, Sca2 is structurally unrelated to eukaryotic formins and achieves these functions through an entirely different mechanism. Thus, while formins are dimeric, Sca2 functions as a monomer. However, Sca2 displays intramolecular interactions and functional cooperativity between its N- and C-terminal domains that are crucial for actin nucleation and elongation. Here, we map the interaction of N- and C-terminal fragments of Sca2 and their contributions to actin binding and nucleation. We find that both the N- and C-terminal regions of Sca2 interact with actin monomers, but only weakly, whereas the full-length protein binds two actin monomers with high affinity. Moreover, deletions at both ends of the N- and C-terminal regions disrupt their ability to interact with each other, suggesting that they form a contiguous ring-like structure that wraps around two actin subunits, analogous to the formin homology-2 (FH2) domain. The discovery of Sca2 as an actin nucleator followed the identification of what appeared to be a repeat of three WH2 domains in the middle of the molecule, consistent with the presence of WH2 domains in most actin nucleators. However, we show here that contrary to previous assumptions Sca2 does not contain WH2 domains, and that the corresponding region is folded as a globular domain that cooperates with other parts of the Sca2 molecule for actin binding and nucleation.

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Single-molecule imaging of IQGAP1 regulating actin filament dynamics

Molecular Biology of the Cell ◽

10.1091/mbc.e21-04-0211 ◽

2021 ◽

Author(s):

Gregory J. Hoeprich ◽

Amy N. Sinclair ◽

Shashank Shekhar ◽

Bruce L. Goode

Keyword(s):

Cell Adhesion ◽

Actin Filament ◽

Single Molecule ◽

Actin Dynamics ◽

Actin Binding ◽

Functional Effect ◽

Single Filament ◽

Filament Dynamics ◽

Do So

IQGAP is a conserved family of actin-binding proteins with essential roles in cell motility, cytokinesis, and cell adhesion, yet there remains a limited understanding of how IQGAP proteins directly influence actin filament dynamics. To close this gap, we used single-molecule and single-filament TIRF microscopy to observe IQGAP regulating actin dynamics in real time. To our knowledge, this is the first study to do so. Our results demonstrate that full-length human IQGAP1 forms dimers that stably bind to actin filament sides and transiently cap barbed ends. These interactions organize filaments into thin bundles, suppress barbed end growth, and inhibit filament disassembly. Surprisingly, each activity depends on distinct combinations of IQGAP1 domains and/or dimerization, suggesting that different mechanisms underlie each functional effect on actin. These observations have important implications for how IQGAP functions as an actin regulator in vivo, and how it may be regulated in different biological settings. [Media: see text] [Media: see text] [Media: see text]

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Actin Depolymerizing Factor (ADF/Cofilin) Enhances the Rate of Filament Turnover: Implication in Actin-based Motility

The Journal of Cell Biology ◽

10.1083/jcb.136.6.1307 ◽

1997 ◽

Vol 136 (6) ◽

pp. 1307-1322 ◽

Cited By ~ 703

Author(s):

Marie-France Carlier ◽

Valérie Laurent ◽

Jérôme Santolini ◽

Ronald Melki ◽

Dominique Didry ◽

...

Keyword(s):

Actin Filament ◽

Atp Hydrolysis ◽

Fold Increase ◽

Actin Dynamics ◽

Actin Binding ◽

Actin Depolymerizing Factor ◽

Filament Dynamics ◽

Relevant Effect

Actin-binding proteins of the actin depolymerizing factor (ADF)/cofilin family are thought to control actin-based motile processes. ADF1 from Arabidopsis thaliana appears to be a good model that is functionally similar to other members of the family. The function of ADF in actin dynamics has been examined using a combination of physical–chemical methods and actin-based motility assays, under physiological ionic conditions and at pH 7.8. ADF binds the ADPbound forms of G- or F-actin with an affinity two orders of magnitude higher than the ATP- or ADP-Pi– bound forms. A major property of ADF is its ability to enhance the in vitro turnover rate (treadmilling) of actin filaments to a value comparable to that observed in vivo in motile lamellipodia. ADF increases the rate of propulsion of Listeria monocytogenes in highly diluted, ADF-limited platelet extracts and shortens the actin tails. These effects are mediated by the participation of ADF in actin filament assembly, which results in a change in the kinetic parameters at the two ends of the actin filament. The kinetic effects of ADF are end specific and cannot be accounted for by filament severing. The main functionally relevant effect is a 25-fold increase in the rate of actin dissociation from the pointed ends, while the rate of dissociation from the barbed ends is unchanged. This large increase in the rate-limiting step of the monomer-polymer cycle at steady state is responsible for the increase in the rate of actin-based motile processes. In conclusion, the function of ADF is not to sequester G-actin. ADF uses ATP hydrolysis in actin assembly to enhance filament dynamics.

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TetraThymosinβ Is Required for Actin Dynamics in Caenorhabditis elegans and Acts via Functionally Different Actin-binding Repeats

Molecular Biology of the Cell ◽

10.1091/mbc.e04-03-0225 ◽

2004 ◽

Vol 15 (10) ◽

pp. 4735-4748 ◽

Cited By ~ 28

Author(s):

Marleen Van Troys ◽

Kanako Ono ◽

Daisy Dewitte ◽

Veronique Jonckheere ◽

Natalie De Ruyck ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Actin Filament ◽

Actin Polymerization ◽

Actin Dynamics ◽

Actin Binding ◽

In Vitro Assays ◽

Filamentous Actin ◽

Lethal Mutant ◽

Interaction Sites

Generating specific actin structures via controlled actin polymerization is a prerequisite for eukaryote development and reproduction. We here report on an essential Caenorhabditis elegans protein tetraThymosinβ expressed in developing neurons and crucial during oocyte maturation in adults. TetraThymosinβ has four repeats, each related to the actin monomer-sequestering protein thymosinβ 4 and assists in actin filament elongation. For homologues with similar multirepeat structures, a profilin-like mechanism of ushering actin onto filament barbed ends, based on the formation of a 1:1 complex, is proposed to underlie this activity. We, however, demonstrate that tetraThymosinβ binds multiple actin monomers via different repeats and in addition also interacts with filamentous actin. All repeats need to be functional for attaining full activity in various in vitro assays. The activities on actin are thus a direct consequence of the repeated structure. In containing both G- and F-actin interaction sites, tetraThymosinβ may be reminiscent of nonhomologous multimodular actin regulatory proteins implicated in actin filament dynamics. A mutation that suppresses expression of tetraThymosinβ is homozygous lethal. Mutant organisms develop into adults but display a dumpy phenotype and fail to reproduce as their oocytes lack essential actin structures. This strongly suggests that the activity of tetraThymosinβ is of crucial importance at specific developmental stages requiring actin polymerization.

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Single-molecule imaging of IQGAP1 regulating actin filament dynamics in real time

10.1101/2021.04.18.440338 ◽

2021 ◽

Author(s):

Gregory J Hoeprich ◽

Shashank Shekhar ◽

Bruce L Goode

Keyword(s):

Real Time ◽

Actin Filament ◽

Single Molecule ◽

Actin Dynamics ◽

Actin Binding ◽

Functional Effect ◽

Single Filament ◽

Filament Dynamics ◽

Do So

IQGAP is a conserved family of actin-binding proteins with essential roles in cell motility, cytokinesis, and cell adhesion, yet it has remained poorly understood how IQGAP proteins directly regulate actin filament dynamics. To close this gap, we used single-molecule and single-filament TIRF microscopy to directly visualize IQGAP regulating actin dynamics in real time. To our knowledge, this is the first study to do so. Our results show that full-length human IQGAP1 forms dimers that stably bind to filament sides and transiently cap barbed ends. These interactions organize actin filaments into thin bundles, suppress barbed end growth, and inhibit filament disassembly. Surprisingly, each activity depends on distinct combinations of IQGAP1 domains and/or dimerization, suggesting that different mechanisms underlie each functional effect on actin. These observations have important implications for how IQGAP functions as a direct actin regulator in vivo, and how it is deployed and regulated in different biological settings.

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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.

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