Interaction of CDK5RAP2 with EB1 to track growing microtubule tips and to regulate microtubule dynamics.
ABSTRACT Mutations in cdk5rap2 are linked to autosomal recessive primary microcephaly, and attention has been paid to its function at centrosomes. In this report, we demonstrate that CDK5RAP2 localizes to microtubules and concentrates at the distal tips in addition to centrosomal localization. CDK5RAP2 interacts directly with EB1, a prototypic member of microtubule plus-end tracking proteins, and contains the basic and Ser-rich motif responsible for EB1 binding. The EB1-binding motif is conserved in the CDK5RAP2 sequences of chimpanzee, bovine, and dog but not in those of rat and mouse, suggesting a function gained during the evolution of mammals. The mutation of the Ile/Leu-Pro dipeptide within the motif abolishes EB1 interaction and plus-end attachment. In agreement with the mutational analysis, suppression of EB1 expression inhibits microtubule tip-tracking of CDK5RAP2. We have also found that the CDK5RAP2-EB1 complex regulates microtubule dynamics and stability. CDK5RAP2 depletion by RNA interference impacts the dynamic behaviors of microtubules. The CDK5RAP2-EB1 complex induces microtubule bundling and acetylation when expressed in cell cultures and stimulates microtubule assembly and bundle formation in vitro. Collectively, these results show that CDK5RAP2 targets growing microtubule tips in association with EB1 to regulate microtubule dynamics.
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Article: Interaction of CDK5RAP2 with EB1 to track growing microtubule tips and to regulate microtubule dynamics.
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ABSTRACT: As the primary microtubule nucleator in animal cells, the γ-tubulin ring complex (γTuRC) plays a crucial role in microtubule organization, but little is known about how the activity of the γTuRC is regulated. Recently, isolated γTuRC was found to contain NME7, a poorly characterized member of the NME family. Here, we report that NME7 is a γTuRC component that regulates the microtubule-nucleating activity of the γTuRC. NME7 contains 2 putative kinase domains (A and B) and shows autophosphorylating activity. Whereas domain A is involved in the autophosphorylation, domain B is inactive. NME7 interacts with the γTuRC through both A and B domains, with Arg322 in domain B being crucial to the binding. In association with the γTuRC, NME7 localizes to centrosomes throughout the cell cycle and to mitotic spindles during mitosis. Suppression of NME7 expression does not affect γTuRC assembly or localization to centrosomes, but impairs centrosome-based microtubule nucleation. Importantly, wild-type NME7 promotes γTuRC-dependent nucleation of microtubules, but kinase-deficient NME7 does so only poorly. These results suggest that NME7 functions in the γTuRC in a kinase-dependent manner to facilitate microtubule nucleation.Molecular biology of the cell 05/2014; 25(13). DOI:10.1091/mbc.E13-06-0339 · 5.98 Impact Factor
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ABSTRACT: Currently there is intense interest in using mesenchymal stem cells (MSC) for therapeutic interventions in many diseases and conditions. To accelerate the therapeutic use of stem cells we must understand how they sense their environment. Primary cilia are an extracellular sensory organelle present on most growth arrested cells that transduce information about the cellular environment into cells, triggering signalling cascades that have profound effects on development, cell cycle, proliferation, differentiation and migration. Migrating cells likely encounter differing oxygen tensions, therefore we investigated the effect of oxygen tension on cilia. Using bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) we found that oxygen tension significantly affected the length of cilia in primary BMSCs. Chronic exposure to hypoxia specifically down-regulated genes involved in hedgehog signalling and re-localized the Smo and Gli2 proteins to cilia. Investigating the effects of chemotactic migration on cilia, we observed significantly longer cilia in migrating cells which was again, strongly influenced by oxygen tension. Finally, using computational modeling we identified links between migration and ciliation signalling pathways, characterizing the novel role of HSP90 and PI3K signalling in regulating BMSC cilia length. These findings enhance our current understanding of BMSC adaptions to hypoxia and advance our knowledge of BMSC biology and cilia regulation.Stem Cell Research 09/2014; 13(2). DOI:10.1016/j.scr.2014.06.006 · 3.91 Impact Factor
Article: The centrosome–Golgi apparatus nexus[Show abstract] [Hide abstract]
ABSTRACT: A shared feature among all microtubule (MT)-dependent processes is the requirement for MTs to be organized in arrays of defined geometry. At a fundamental level, this is achieved by precisely controlling the timing and localization of the nucleation events that give rise to new MTs. To this end, MT nucleation is restricted to specific subcellular sites called MT-organizing centres. The primary MT-organizing centre in proliferating animal cells is the centrosome. However, the discovery of MT nucleation capacity of the Golgi apparatus (GA) has substantially changed our understanding of MT network organization in interphase cells. Interestingly, MT nucleation at the Golgi apparently relies on multiprotein complexes, similar to those present at the centrosome, that assemble at the cis-face of the organelle. In this process, AKAP450 plays a central role, acting as a scaffold to recruit other centrosomal proteins important for MT generation. MT arrays derived from either the centrosome or the GA differ in their geometry, probably reflecting their different, yet complementary, functions. Here, I review our current understanding of the molecular mechanisms involved in MT nucleation at the GA and how Golgi- and centrosome-based MT arrays work in concert to ensure the formation of a pericentrosomal polarized continuous Golgi ribbon structure, a critical feature for cell polarity in mammalian cells. In addition, I comment on the important role of the Golgi-nucleated MTs in organizing specialized MT arrays that serve specific functions in terminally differentiated cells.Philosophical Transactions of The Royal Society B Biological Sciences 09/2014; 369(1650). DOI:10.1098/rstb.2013.0462 · 6.31 Impact Factor