scholarly journals Control of microtubule dynamics using an optogenetic microtubule plus end–F-actin cross-linker

2017 ◽  
Vol 217 (2) ◽  
pp. 779-793 ◽  
Author(s):  
Rebecca C. Adikes ◽  
Ryan A. Hallett ◽  
Brian F. Saway ◽  
Brian Kuhlman ◽  
Kevin C. Slep
Keyword(s):  
Blue Light ◽  
Actin Binding ◽  
Cross Linking ◽  
Dependent Manner ◽  
Exclusion Zone ◽  
Actin Networks ◽  
Drosophila Melanogaster S2 Cells ◽  
Actin Binding Domain ◽  
Specific Factors ◽  
Insight Into

We developed a novel optogenetic tool, SxIP–improved light-inducible dimer (iLID), to facilitate the reversible recruitment of factors to microtubule (MT) plus ends in an end-binding protein–dependent manner using blue light. We show that SxIP-iLID can track MT plus ends and recruit tgRFP-SspB upon blue light activation. We used this system to investigate the effects of cross-linking MT plus ends and F-actin in Drosophila melanogaster S2 cells to gain insight into spectraplakin function and mechanism. We show that SxIP-iLID can be used to temporally recruit an F-actin binding domain to MT plus ends and cross-link the MT and F-actin networks. Cross-linking decreases MT growth velocities and generates a peripheral MT exclusion zone. SxIP-iLID facilitates the general recruitment of specific factors to MT plus ends with temporal control enabling researchers to systematically regulate MT plus end dynamics and probe MT plus end function in many biological processes.

scholarly journals Control of microtubule dynamics using an optogenetic microtubule plus end–F-actin cross-linker

2017 ◽  
Author(s):  
Rebecca C. Adikes ◽  
Ryan A. Hallett ◽  
Brian F. Saway ◽  
Brian Kuhlman ◽  
Kevin C. Slep
Keyword(s):  
Blue Light ◽  
Microtubule Dynamics ◽  
Actin Binding ◽  
Cross Linking ◽  
Dependent Manner ◽  
Exclusion Zone ◽  
Actin Networks ◽  
Actin Binding Domain ◽  
Specific Factors

AbstractWe developed a novel optogenetic tool, SxIP-iLID, to facilitate the reversible recruitment of factors to microtubule (MT) plus ends in an End Binding (EB) protein-dependent manner using blue light. We show that SxIP-iLID can track MT plus ends and recruit tgRFP-SspB upon blue light activation. We then used this system to investigate the effects of cross-linking MT plus ends and F-actin in Drosophila S2 cells to gain insight into spectraplakin function and mechanism. We show that SxIP-iLID can be used to temporally recruit a F-actin binding domain to MT plus ends and cross-link the MT and F-actin networks. Light-mediated MT-F-actin cross-linking decreases MT growth velocities and generates a MT exclusion zone in the lamella. SxIP-iLID facilitates the general recruitment of specific factors to MT plus ends with temporal control enabling researchers to systematically regulate MT plus end dynamics and probe MT plus end function in many biological processes.SummarySxIP-iLID is a novel optogenetic tool designed to assess the spatiotemporal role of proteins on microtubule dynamics. We establish that optogenetic cross-linking of microtubule and actin networks decreases MT growth velocities and increases the cell area void of microtubules.

scholarly journals Microtubule Actin Cross-Linking Factor (Macf)

1999 ◽  
Vol 147 (6) ◽  
pp. 1275-1286 ◽  
Author(s):  
Conrad L. Leung ◽  
Dongming Sun ◽  
Min Zheng ◽  
David R. Knowles ◽  
Ronald K.H. Liem
Keyword(s):  
Calcium Binding ◽  
Coiled Coil ◽  
Specific Protein ◽  
Binding Domain ◽  
Full Length ◽  
Actin Binding ◽  
Cross Linking ◽  
Actin Binding Domain

We cloned and characterized a full-length cDNA of mouse actin cross-linking family 7 (mACF7) by sequential rapid amplification of cDNA ends–PCR. The completed mACF7 cDNA is 17 kb and codes for a 608-kD protein. The closest relative of mACF7 is the Drosophila protein Kakapo, which shares similar architecture with mACF7. mACF7 contains a putative actin-binding domain and a plakin-like domain that are highly homologous to dystonin (BPAG1-n) at its NH2 terminus. However, unlike dystonin, mACF7 does not contain a coiled–coil rod domain; instead, the rod domain of mACF7 is made up of 23 dystrophin-like spectrin repeats. At its COOH terminus, mACF7 contains two putative EF-hand calcium-binding motifs and a segment homologous to the growth arrest–specific protein, Gas2. In this paper, we demonstrate that the NH2-terminal actin-binding domain of mACF7 is functional both in vivo and in vitro. More importantly, we found that the COOH-terminal domain of mACF7 interacts with and stabilizes microtubules. In transfected cells full-length mACF7 can associate not only with actin but also with microtubules. Hence, we suggest a modified name: MACF (microtubule actin cross-linking factor). The properties of MACF are consistent with the observation that mutations in kakapo cause disorganization of microtubules in epidermal muscle attachment cells and some sensory neurons.

scholarly journals Alpha-actinin of the chlorarchiniophyte Bigelowiella natans

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4288 ◽  
Author(s):  
Lars Backman
Keyword(s):  
Domain Structure ◽  
Calcium Binding ◽  
Actin Filaments ◽  
Actin Binding ◽  
Cross Linking ◽  
Primary Sequence ◽  
Dimer Formation ◽  
Spectrin Repeat ◽  
Actin Binding Domain ◽  
Ef Hand

The genome of the chlorarchiniophyte Bigelowiella natans codes for a protein annotated as an α-actinin-like protein. Analysis of the primary sequence indicate that this protein has the same domain structure as other α-actinins, a N-terminal actin-binding domain and a C-terminal calmodulin-like domain. These two domains are connected by a short rod domain, albeit long enough to form a single spectrin repeat. To analyse the functional properties of this protein, the full-length protein as well as the separate domains were cloned and isolated. Characerisation showed that the protein is capable of cross-linking actin filaments into dense bundles, probably due to dimer formation. Similar to human α-actinin, calcium-binding occurs to the most N-terminal EF-hand motif in the calmodulin-like C-terminal domain. The results indicate that this Bigelowiella protein is a proper α-actinin, with all common characteristics of a typical α-actinin.

scholarly journals Ca2+-dependent Binding and Activation of Dormant Ezrin by Dimeric S100P

2003 ◽  
Vol 14 (6) ◽  
pp. 2372-2384 ◽  
Author(s):  
Max Koltzscher ◽  
Claudia Neumann ◽  
Simone König ◽  
Volker Gerke
Keyword(s):  
Binding Site ◽  
S100 Protein ◽  
Biologically Active ◽  
Actin Binding ◽  
Cross Linking ◽  
Dependent Manner ◽  
Cellular Targets ◽  
Stimulus Response ◽  
Protein Ligands ◽  
Ef Hand

S100 proteins are EF hand type Ca2+ binding proteins thought to function in stimulus-response coupling by binding to and thereby regulating cellular targets in a Ca2+-dependent manner. To isolate such target(s) of the S100P protein we devised an affinity chromatography approach that selects for S100 protein ligands requiring the biologically active S100 dimer for interaction. Hereby we identify ezrin, a membrane/F-actin cross-linking protein, as a dimer-specific S100P ligand. S100P-ezrin complex formation is Ca2+ dependent and most likely occurs within cells because both proteins colocalize at the plasma membrane after growth factor or Ca2+ ionophore stimulation. The S100P binding site is located in the N-terminal domain of ezrin and is accessible for interaction in dormant ezrin, in which binding sites for F-actin and transmembrane proteins are masked through an association between the N- and C-terminal domains. Interestingly, S100P binding unmasks the F-actin binding site, thereby at least partially activating the ezrin molecule. This identifies S100P as a novel activator of ezrin and indicates that activation of ezrin's cross-linking function can occur directly in response to Ca2+ transients.

scholarly journals Structural basis of filamin A functions

2007 ◽  
Vol 179 (5) ◽  
pp. 1011-1025 ◽  
Author(s):  
Fumihiko Nakamura ◽  
Teresia M. Osborn ◽  
Christopher A. Hartemink ◽  
John H. Hartwig ◽  
Thomas P. Stossel
Keyword(s):  
Actin Binding ◽  
Structural Basis ◽  
High Avidity ◽  
Filamin A ◽  
Compact Structure ◽  
Actin Networks ◽  
Binding Domains ◽  
Binding Partners ◽  
Actin Binding Domain ◽  
Flexible Segment

Filamin A (FLNa) can effect orthogonal branching of F-actin and bind many cellular constituents. FLNa dimeric subunits have N-terminal spectrin family F-actin binding domains (ABDs) and an elongated flexible segment of 24 immunoglobulin (Ig) repeats. We generated a library of FLNa fragments to examine their F-actin binding to define the structural properties of FLNa that enable its various functions. We find that Ig repeats 9–15 contain an F-actin–binding domain necessary for high avidity F-actin binding. Ig repeats 16–24, where most FLNa-binding partners interact, do not bind F-actin, and thus F-actin does not compete with Ig repeat 23 ligand, FilGAP. Ig repeats 16–24 have a compact structure that suggests their unfolding may accommodate pre-stress–mediated stiffening of F-actin networks, partner binding, mechanosensing, and mechanoprotection properties of FLNa. Our results also establish the orientation of FLNa dimers in F-actin branching. Dimerization, mediated by FLNa Ig repeat 24, accounts for rigid high-angle FLNa/F-actin branching resistant to bending by thermal forces, and high avidity F-actin binding and cross-linking.

The interaction of plectin with actin: evidence for cross-linking of actin filaments by dimerization of the actin-binding domain of plectin

2001 ◽  
Vol 114 (11) ◽  
pp. 2065-2076 ◽  
Author(s):  
Lionel Fontao ◽  
Dirk Geerts ◽  
Ingrid Kuikman ◽  
Jan Koster ◽  
Duco Kramer ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Cell Types ◽  
Binding Domain ◽  
Actin Binding ◽  
Cross Linking ◽  
Binding Domains ◽  
Cell Matrix ◽  
Vitro Binding ◽  
Actin Binding Domain

Plectin is a major component of the cytoskeleton and is expressed in a wide variety of cell types. It plays an important role in the integrity of the cytoskeleton by cross-linking the three filamentous networks and stabilizing cell-matrix and cell-cell contacts. Sequence analysis showed that plectin contains a highly conserved actin-binding domain, consisting of a pair of calponin-like subdomains. Using yeast two-hybrid assays in combination with in vitro binding experiments, we demonstrate that the actin-binding domain of plectin is fully functional and preferentially binds to polymeric actin. The sequences required for actin binding were identified at the C-terminal end of the first calponin homology domain within the actin-binding domain of plectin. We found that the actin-binding domain of plectin is able to bundle actin filaments and we present evidence that this is mediated by the dimerization of this domain. In addition we also show that plectin and another member of the plakin family, dystonin, can heterodimerize by their actin-binding domains. We propose a new mechanism by which plectin and possibly also other actin-binding proteins can regulate the organization of the F-actin network in the cell.

scholarly journals Microcephaly protein Asp focuses the minus ends of spindle microtubules at the pole and within the spindle

2015 ◽  
Vol 211 (5) ◽  
pp. 999-1009 ◽  
Author(s):  
Ami Ito ◽  
Gohta Goshima
Keyword(s):  
Drosophila Melanogaster ◽  
Molecular Model ◽  
Spindle Pole ◽  
Cross Linking ◽  
Dependent Manner ◽  
Spindle Microtubule ◽  
Cross Links ◽  
Spindle Microtubules ◽  
Insight Into

Depletion of Drosophila melanogaster Asp, an orthologue of microcephaly protein ASPM, causes spindle pole unfocusing during mitosis. However, it remains unclear how Asp contributes to pole focusing, a process that also requires the kinesin-14 motor Ncd. We show that Asp localizes to the minus ends of spindle microtubule (MT) bundles and focuses them to make the pole independent of Ncd. We identified a critical domain in Asp exhibiting MT cross-linking activity in vitro. Asp was also localized to, and focuses the minus ends of, intraspindle MTs that were nucleated in an augmin-dependent manner and translocated toward the poles by spindle MT flux. Ncd, in contrast, functioned as a global spindle coalescence factor not limited to MT ends. We propose a revised molecular model for spindle pole focusing in which Asp at the minus ends cross-links MTs at the pole and within the spindle. Additionally, this study provides new insight into the dynamics of intraspindle MTs by using Asp as a minus end marker.

scholarly journals The actin-microtubule cross-linking activity of Drosophila Short stop is regulated by intramolecular inhibition

2013 ◽  
Vol 24 (18) ◽  
pp. 2885-2893 ◽  
Author(s):  
Derek A. Applewhite ◽  
Kyle D. Grode ◽  
Mara C. Duncan ◽  
Stephen L. Rogers
Keyword(s):  
Actin Binding ◽  
Cell Cortex ◽  
Cross Linking ◽  
Inhibitory Mechanism ◽  
Cross Link ◽  
Inactive Conformation ◽  
Closed Conformation ◽  
Actin Binding Domain ◽  
Ef Hand ◽  
Cytoskeletal Networks

Actin and microtubule dynamics must be precisely coordinated during cell migration, mitosis, and morphogenesis—much of this coordination is mediated by proteins that physically bridge the two cytoskeletal networks. We have investigated the regulation of the Drosophila actin-microtubule cross-linker Short stop (Shot), a member of the spectraplakin family. Our data suggest that Shot's cytoskeletal cross-linking activity is regulated by an intramolecular inhibitory mechanism. In its inactive conformation, Shot adopts a “closed” conformation through interactions between its NH2-terminal actin-binding domain and COOH-terminal EF-hand-GAS2 domain. This inactive conformation is targeted to the growing microtubule plus end by EB1. On activation, Shot binds along the microtubule through its COOH-terminal GAS2 domain and binds to actin with its NH2-terminal tandem CH domains. We propose that this mechanism allows Shot to rapidly cross-link dynamic microtubules in response to localized activating signals at the cell cortex.

scholarly journals Cryo-EM structure of NPF-bound human Arp2/3 complex and activation mechanism

2020 ◽  
Vol 6 (23) ◽  
pp. eaaz7651 ◽  
Author(s):  
Austin Zimmet ◽  
Trevor Van Eeuwen ◽  
Malgorzata Boczkowska ◽  
Grzegorz Rebowski ◽  
Kenji Murakami ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Motility ◽  
Binding Sites ◽  
Activation Mechanism ◽  
Actin Binding ◽  
Cross Linking ◽  
Specific Interactions ◽  
Related Protein ◽  
Actin Networks ◽  
Cryo Electron Microscopy

Actin-related protein (Arp) 2/3 complex nucleates branched actin networks that drive cell motility. It consists of seven proteins, including two actin-related subunits (Arp2 and Arp3). Two nucleation-promoting factors (NPFs) bind Arp2/3 complex during activation, but the order, specific interactions, and contribution of each NPF to activation are unresolved. Here, we report the cryo–electron microscopy structure of recombinantly expressed human Arp2/3 complex with two WASP family NPFs bound and address the mechanism of activation. A cross-linking assay that captures the transition of the Arps into the activated filament-like conformation shows that actin binding to NPFs favors this transition. Actin-NPF binding to Arp2 precedes binding to Arp3 and is sufficient to promote the filament-like conformation but not activation. Structure-guided mutagenesis of the NPF-binding sites reveals their distinct roles in activation and shows that, contrary to budding yeast Arp2/3 complex, NPF-mediated delivery of actin at the barbed end of both Arps is required for activation of human Arp2/3 complex.

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