Dynein and Mast/Orbit/CLASP have antagonistic roles in regulating kinetochore-microtubule plus-end dynamics

Reis, Rita M.; Feijão, Tália; Gouveia, Susana Montenegro; Pereira, António J.; Matos, Irina; Sampaio, Paula; Maiato, Hélder; Sunkel, Cláudio E.

doi:10.1242/jcs.044818

Cited by 21 publications

(18 citation statements)

References 69 publications

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“…Notable exceptions included the conserved axon guidance receptor Roundabout (Robo) and the Robo-associated Abl substrate Ena, a conserved actin regulator required for Robo receptor signaling (63,64). to regulate cytoskeletal behavior (7,11,14,23,65). Our extensive genetic modifier screens in the fly retina provided a sensitive means to detect functional interaction with CLASP in vivo (27; this study), revealing an interactome of over 100 genes, but we needed a live-cell time-lapse approach to distinguish interactors that control MT dynamics from other functions downstream of or associated with CLASP.…”

Section: Selection Of Candidates Forward Genetic Screening Approachementioning

confidence: 99%

“…CLASP (cytoplasmic linker protein [CLIP]-associated protein) is a well-conserved MT plus-end interacting protein (ϩTIP), which modulates dynamic instability and facilitates the interaction of MTs with other cellular structures, including the cell cortex (7,8) and kinetochores (9)(10)(11). CLASP functions as an MT-stabilizing factor, promoting MT rescue both in cultured cells and in vivo (12)(13)(14)(15).…”

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Multiparametric Analysis of CLASP-Interacting Protein Functions during Interphase Microtubule Dynamics

Long

Bagonis

Lowery

et al. 2013

Molecular and Cellular Biology

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The microtubule (MT) plus-end tracking protein (؉TIP) CLASP mediates dynamic cellular behaviors and interacts with numerous cytoplasmic proteins. While the influence of some CLASP interactors on MT behavior is known, a comprehensive survey of the proteins in the CLASP interactome as MT regulators is missing. Ultimately, we are interested in understanding how CLASP collaborates with functionally linked proteins to regulate MT dynamics. Here, we utilize multiparametric analysis of time-lapse MT ؉TIP imaging data acquired in Drosophila melanogaster S2R؉ cells to assess the effects on individual microtubule dynamics for RNA interference-mediated depletion of 48 gene products previously identified to be in vivo genetic CLASP interactors. While our analysis corroborates previously described functions of several known CLASP interactors, its multiparametric resolution reveals more detailed functional profiles (fingerprints) that allow us to precisely classify the roles that CLASP-interacting genes play in MT regulation. Using these data, we identify subnetworks of proteins with novel yet overlapping MT-regulatory roles and also uncover subtle distinctions between the functions of proteins previously thought to act via similar mechanisms. The orchestration of cytoskeletal dynamics is critical for a broad range of cellular behaviors, including mitosis, polarity, motility, morphogenesis, and cell-cell interaction (1-3). Microtubule (MT) polymer networks participate in numerous signaling pathways, often helping to assemble and/or deliver effector protein complexes and to define the spatial organization of cellular responses. Many classes of cytoskeletal binding proteins regulate the configuration of MT arrays and often interact with other protein networks. However, our understanding of how these extended effector networks function to control cytoskeletal dynamics is still limited. Large-scale screens for MT regulators have primarily relied on endpoint phenotypes that affect mitosis (4-6). The mitotic spindle is a unique apparatus whose gross architecture can be severely disturbed by accumulated effects of altered MT dynamics and thus offers a simple readout for such studies. However, these readouts report screening hits only on the basis of indirect MT phenotypes in a large complex system without pinpointing the actual role that they play in terms of bona fide MT regulation. Direct detection of altered MT dynamics has been much more challenging. For this reason, we adopted a quantitative live-imaging approach that allowed us to identify with single-MT resolution shifts in MT dynamics induced by RNA interference (RNAi)-mediated depletion of putative MT regulators.CLASP (cytoplasmic linker protein [CLIP]-associated protein) is a well-conserved MT plus-end interacting protein (ϩTIP), which modulates dynamic instability and facilitates the interaction of MTs with other cellular structures, including the cell cortex (7, 8) and kinetochores (9-11). CLASP functions as an MT-stabilizing factor, promoting MT rescue both in cultured ...

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Section: Selection Of Candidates Forward Genetic Screening Approachementioning

confidence: 99%

mentioning

confidence: 99%

Multiparametric Analysis of CLASP-Interacting Protein Functions during Interphase Microtubule Dynamics

Long

Bagonis

Lowery

et al. 2013

Molecular and Cellular Biology

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“…8d the hyperboloid model function with all three parameters isolated, where again the data points combine to collapse to the model surface. The reasons behind the exceptional behavior in Tobacco BY-2 cells and mouse oocytes (MI) are unknown, but we speculate that it might be related with the fact that plants in general are more tolerant to aneuploidy (Matzke et al 2003), while propagation of aneuploidy in the germline is (Goshima et al 2005), and 1.5 (Reis et al 2009) 1.2 Human HeLa 13 16 0.9 SL-14.3 (Cai et al 2009), 12.5 c (Tokai-Nishizumi et al 2005 and 12 (Jorge Ferreira, personal communication); MD-9 d (Meraldi et al 2004), 23.5 (Zhao et al 2008, 17 (Arnaoutov et al 2005), 15.2 (Kallio et al 1998) and14 (Jorge Ferreira, personal communication);and F-0.9 (Amaro et al 2010) 2.2…”

Section: A Relation Between Flux Velocity Spindle Size and Metaphasementioning

confidence: 99%

Maturation of the kinetochore-microtubule interface and the meaning of metaphase

Pereira

Maiato

2012

Chromosome Res

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Chromosome positioning at the equator of the mitotic spindle emerges out of a relatively entropic background. At this moment, termed metaphase, all kinetochores have typically captured microtubules leading to satisfaction of the spindle-assembly checkpoint, but the cell does not enter anaphase immediately. The waiting time in metaphase is related to the kinetics of securin and cyclin B1 degradation, which trigger sisterchromatid separation and promote anaphase processivity, respectively. Yet, as judged by metaphase duration, such kinetics vary widely between cell types and organisms, with no evident correlation to ploidy or cell size. During metaphase, many animal and plant spindles are also characterized by a conspicuous "flux" activity characterized by continuous poleward translocation of spindle microtubules, which maintain steady-state length and position. Whether spindle microtubule flux plays a specific role during metaphase remains arguable. Based on known experimental parameters, we have performed a comparative analysis amongst different cell types from different organisms and show that spindle length, metaphase duration and flux velocity combine within each system to obey a quasi-universal rule. As so, knowledge of two of these parameters is enough to estimate the third. This trend indicates that metaphase duration is tuned to allow approximately one kinetochore-to-pole round of microtubule flux. We propose that the time cells spend in metaphase evolved as a quality enhancement step that allows for the uniform stabilization/correction of kinetochore-microtubule attachments, thereby promoting mitotic fidelity.

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“…Yet, little is known regarding the molecular machinery that governs the integrated coordination of various cytoskeletal components. Evidence suggests that the microtubule (MT) plus-end tracking protein CLASP [cytoplasmic linker protein (CLIP)-associated protein], which has been implicated in mitotic spindle formation (Inoue et al 2000(Inoue et al , 2004 and in linking MT ends to other cell structures such as the cell cortex and kinetochore (Akhmanova et al 2001;Maiato et al 2003;Mimori-Kiyosue et al 2005;Reis et al 2009), may play a pivotal role in the overall coordination of cytoskeletal networks. Not only does it affect MT dynamics, but CLASP may function as an actin-MT crosslinker as well, as it possesses actin-binding activity (Tsvetkov et al 2007) and CLASP-bound microtubules appear to track along F-actin bundles in growth cones (Lee et al 2004).…”

Section: Oordination Of Cytoskeletal Dynamics Is Anmentioning

confidence: 99%

Parallel Genetic and Proteomic Screens Identify Msps as a CLASP–Abl Pathway Interactor in Drosophila

Lowery

Lee²,

Lu³

et al. 2010

Genetics

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Regulation of cytoskeletal structure and dynamics is essential for multiple aspects of cellular behavior, yet there is much to learn about the molecular machinery underlying the coordination between the cytoskeleton and its effector systems. One group of proteins that regulate microtubule behavior and its interaction with other cellular components, such as actin-regulatory proteins and transport machinery, is the plus-end tracking proteins (MT1TIPs). In particular, evidence suggests that the MT1TIP, CLASP, may play a pivotal role in the coordination of microtubules with other cellular structures in multiple contexts, although the molecular mechanism by which it functions is still largely unknown. To gain deeper insight into the functional partners of CLASP, we conducted parallel genetic and proteome-wide screens for CLASP interactors in Drosophila melanogaster. We identified 36 genetic modifiers and 179 candidate physical interactors, including 13 that were identified in both data sets. Grouping interactors according to functional classifications revealed several categories, including cytoskeletal components, signaling proteins, and translation/RNA regulators. We focused our initial investigation on the MT1TIP Minispindles (Msps), identified among the cytoskeletal effectors in both genetic and proteomic screens. Here, we report that Msps is a strong modifier of CLASP and Abl in the retina. Moreover, we show that Msps functions during axon guidance and antagonizes both CLASP and Abl activity. Our data suggest a model in which CLASP and Msps converge in an antagonistic balance in the Abl signaling pathway.

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Dynein and Mast/Orbit/CLASP have antagonistic roles in regulating kinetochore-microtubule plus-end dynamics

Cited by 21 publications

References 69 publications

Multiparametric Analysis of CLASP-Interacting Protein Functions during Interphase Microtubule Dynamics

Multiparametric Analysis of CLASP-Interacting Protein Functions during Interphase Microtubule Dynamics

Maturation of the kinetochore-microtubule interface and the meaning of metaphase

Parallel Genetic and Proteomic Screens Identify Msps as a CLASP–Abl Pathway Interactor in Drosophila

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