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 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 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 Coupling of the nucleus and cytoplasm: Role of the LINC complex

2005 ◽  
Vol 172 (1) ◽  
pp. 41-53 ◽  
Author(s):  
Melissa Crisp ◽  
Qian Liu ◽  
Kyle Roux ◽  
J.B. Rattner ◽  
Catherine Shanahan ◽  
...  
Keyword(s):  
Nuclear Membrane ◽  
Integral Membrane Proteins ◽  
Actin Binding ◽  
Linc Complex ◽  
Outer Membranes ◽  
Perinuclear Space ◽  
Actin Binding Domain ◽  
Lumenal Domain ◽  
Integral Proteins

The nuclear envelope defines the barrier between the nucleus and cytoplasm and features inner and outer membranes separated by a perinuclear space (PNS). The inner nuclear membrane contains specific integral proteins that include Sun1 and Sun2. Although the outer nuclear membrane (ONM) is continuous with the endoplasmic reticulum, it is nevertheless enriched in several integral membrane proteins, including nesprin 2 Giant (nesp2G), an 800-kD protein featuring an NH2-terminal actin-binding domain. A recent study (Padmakumar, V.C., T. Libotte, W. Lu, H. Zaim, S. Abraham, A.A. Noegel, J. Gotzmann, R. Foisner, and I. Karakesisoglou. 2005. J. Cell Sci. 118:3419–3430) has shown that localization of nesp2G to the ONM is dependent upon an interaction with Sun1. In this study, we confirm and extend these results by demonstrating that both Sun1 and Sun2 contribute to nesp2G localization. Codepletion of both of these proteins in HeLa cells leads to the loss of ONM-associated nesp2G, as does overexpression of the Sun1 lumenal domain. Both treatments result in the expansion of the PNS. These data, together with those of Padmakumar et al. (2005), support a model in which Sun proteins tether nesprins in the ONM via interactions spanning the PNS. In this way, Sun proteins and nesprins form a complex that links the nucleoskeleton and cytoskeleton (the LINC complex).

scholarly journals The WASP Homologue Las17 Activates the Novel Actin-regulatory Activity of Ysc84 to Promote Endocytosis in Yeast

2009 ◽  
Vol 20 (6) ◽  
pp. 1618-1628 ◽  
Author(s):  
Alastair S. Robertson ◽  
Ellen G. Allwood ◽  
Adam P.C. Smith ◽  
Fiona C. Gardiner ◽  
Rosaria Costa ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Actin Polymerization ◽  
Actin Binding ◽  
The Novel ◽  
Cellular Processes ◽  
New Class ◽  
Actin Binding Domain ◽  
Kinetics Of ◽  
Actin Binding Site

Actin plays an essential role in many eukaryotic cellular processes, including motility, generation of polarity, and membrane trafficking. Actin function in these roles is regulated by association with proteins that affect its polymerization state, dynamics, and organization. Numerous proteins have been shown to localize with cortical patches of yeast actin during endocytosis, but the role of many of these proteins remains poorly understood. Here, we reveal that the yeast protein Ysc84 represents a new class of actin-binding proteins, conserved from yeast to humans. It contains a novel N-terminal actin-binding domain termed Ysc84 actin binding (YAB), which can bind and bundle actin filaments. Intriguingly, full-length Ysc84 alone does not bind to actin, but binding can be activated by a specific motif within the polyproline region of the yeast WASP homologue Las17. We also identify a new monomeric actin-binding site on Las17. Together, the polyproline region of Las17 and Ysc84 can promote actin polymerization. Using live cell imaging, kinetics of assembly and disassembly of proteins at the endocytic site were analyzed and reveal that loss of Ysc84 and its homologue Lsb3 decrease inward movement of vesicles consistent with a role in actin polymerization during endocytosis.

A critical role of lipid rafts in the organization of a key FcγRIIa-mediated signaling pathway in human platelets

2003 ◽  
Vol 89 (02) ◽  
pp. 318-330 ◽  
Author(s):  
Stéphane Bodin ◽  
Cécile Viala ◽  
Ashraf Ragab ◽  
Bernard Payrastre
Keyword(s):  
Lipid Rafts ◽  
Critical Role ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Temporal Organization ◽  
Membrane Microdomains ◽  
Cross Linking ◽  
Dependent Manner ◽  
Signaling Complex

SummaryThe involvement of platelet FcγRIIa in heparin-associated thrombocytopenia (HIT) is now well established. However, the precise sequence of molecular events initiated by FcγRIIa cross-linking in platelets remains partly characterized. We investigated here the role of lipid rafts in the spatio-temporal organization of the FcγRIIa-dependent signaling events. Upon cross-linking, FcγRIIa relocated in rafts where the kinase Lyn and the adapter LAT were among the major phosphotyrosyl proteins. Upon stimulation by HIT sera, the second messenger phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) accumulated in rafts in a P2Y12 adenosine diphosphate (ADP) recep- tor-dependent manner. PtdIns(3,4,5)P3 was then essential to specifically recruit phospholipase Cγ2 (PLCγ2) to these membrane microdomains. Controlled disruption of rafts by methyl γ-cyclodextrin reversibly abolished PtdIns(3,4,5)P3 production, PLC activation and platelet responses induced by FcγRIIa cross-linking without affecting the tyrosine phosphorylation events. This work demonstrates that platelet rafts are essential for the integration of a key signaling complex leading to the rapid production of PtdIns(3,4,5)P3 and in turn PLCγ2 activation during HIT.

scholarly journals The dual role of fission yeast Tbc1/cofactor C orchestrates microtubule homeostasis in tubulin folding and acts as a GAP for GTPase Alp41/Arl2

2013 ◽  
Vol 24 (11) ◽  
pp. 1713-1724 ◽  
Author(s):  
Risa Mori ◽  
Takashi Toda
Keyword(s):  
Fission Yeast ◽  
Molecular Level ◽  
Dual Role ◽  
Small Gtpase ◽  
Microtubule Dynamics ◽  
Regulatory Proteins ◽  
Dependent Manner ◽  
Continuous Cycling ◽  
Β Tubulin

Supplying the appropriate amount of correctly folded α/β-tubulin heterodimers is critical for microtubule dynamics. Formation of assembly-competent heterodimers is remarkably elaborate at the molecular level, in which the α- and β-tubulins are separately processed in a chaperone-dependent manner. This sequential step is performed by the tubulin-folding cofactor pathway, comprising a specific set of regulatory proteins: cofactors A–E. We identified the fission yeast cofactor: the orthologue of cofactor C, Tbc1. In addition to its roles in tubulin folding, Tbc1 acts as a GAP in regulating Alp41/Arl2, a highly conserved small GTPase. Of interest, the expression of GDP- or GTP-bound Alp41 showed the identical microtubule loss phenotype, suggesting that continuous cycling between these forms is important for its functions. In addition, we found that Alp41 interacts with Alp1D, the orthologue of cofactor D, specifically when in the GDP-bound form. Intriguingly, Alp1D colocalizes with microtubules when in excess, eventually leading to depolymerization, which is sequestered by co-overproducing GDP-bound Alp41. We present a model of the final stages of the tubulin cofactor pathway that includes a dual role for both Tbc1 and Alp1D in opposing regulation of the microtubule.

Neutrophil polarity and locomotion are associated with surface redistribution of leukosialin (CD43), an antiadhesive membrane molecule

Blood ◽  
2000 ◽  
Vol 95 (8) ◽  
pp. 2462-2470 ◽  
Author(s):  
Stéphanie Seveau ◽  
Hansuli Keller ◽  
Frederick R. Maxfield ◽  
Friedrich Piller ◽  
Lise Halbwachs-Mecarelli
Keyword(s):  
Confocal Microscopy ◽  
Leading Edge ◽  
Domain Analysis ◽  
Cell Polarization ◽  
Actin Binding ◽  
Cross Linking ◽  
Chemotactic Peptide ◽  
Membrane Molecule ◽  
Stimulation Of

Abstract This study analyzed the behavior of an antiadhesive membrane molecule, CD43, in neutrophil polarization and locomotion. CD43 cross-linking by antibodies induced neutrophil locomotion, with CD43 molecules clustered at the uropod of polarized neutrophils. In contrast, CD11b/CD18 cross-linking by antibodies did not affect either cell polarization or locomotion. Stimulation of suspended or adherent neutrophils with chemotactic peptide results in cell polarization and locomotion and a concomitant redistribution of CD43 to the uropod. This process is entirely reversible. The study also investigated which actin-binding protein could be involved in CD43 lateral redistribution. -Actinin and moesin are preferentially adsorbed on Sepharose beads bearing a recombinant CD43 intracellular domain. Analysis by immunofluorescence confocal microscopy shows a codistribution of moesin during CD43 lateral redistribution. By contrast, -actinin is located at the leading edge, an area devoid of CD43. These results shed new light on the role of CD43 membrane redistribution, which appears to be directly related to neutrophil polarity and locomotion.

scholarly journals Cortactin signalling and dynamic actin networks

2004 ◽  
Vol 382 (1) ◽  
pp. 13-25 ◽  
Author(s):  
Roger J. DALY
Keyword(s):  
Tyrosine Kinase ◽  
Actin Cytoskeleton ◽  
Current Knowledge ◽  
Actin Binding ◽  
Related Protein ◽  
Protein Targets ◽  
Actin Networks ◽  
Multiple Protein ◽  
Initial Characterization

Cortactin was first identified over a decade ago, and its initial characterization as both an F-actin binding protein and v-Src substrate suggested that it was likely to be a key regulator of actin rearrangements in response to tyrosine kinase signalling. The recent discovery that cortactin binds and activates the actin related protein (Arp)2/3 complex, and thus regulates the formation of branched actin networks, together with the identification of multiple protein targets of the cortactin SH3 domain, have revealed diverse cellular roles for this protein. This article reviews current knowledge regarding the role of cortactin in signalling to the actin cytoskeleton in the context of these developments.

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.

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