Molecular dissection of CLASPs function in mitosis: A proteomic approach

• Author(s): Maia, Ana Rita Ramada
• Date: 2011
• Persistent ID: http://hdl.handle.net/10216/22096
• Origin: Repositório Aberto da Universidade do Porto
• Subject(s): Medicina e Oncologia Molecular; Porto
• Document Type: Master thesis
• Language: Portuguese

Description

CLASPs are well-conserved microtubule plus-end-tracking proteins that participate in chromosome segregation through their key role at the kinetochore-microtubule interface. In yeast, Drosophila, and Xenopus, a single CLASP orthologue is present, which is required for mitotic spindle assembly by regulating microtubule dynamics at the kinetochore. In mammals, however, only CLASP1 has been directly implicated in cell division, despite the existence of a second paralogue, CLASP2. Here we describe the mitotic localization of human CLASP2 in HeLa cells and show that its localization at kinetochores, centrosomes, and spindle throughout mitosis is remarkably similar to CLASP1. Analysis of Clasp2 KO mouse embryonic fibroblasts revealed that CLASP1 kinetochore localization and Mad2 checkpoint response is not compromised in the absence of CLASP2. To further understand CLASP roles in mitosis, namely to rule out potential redundant functions, we performed single CLASP depletion by RNAi. Remarkably, single CLASP depletion caused no significant impairment of mitosis, while reducing the levels of both CLASPs by RNAi caused severe mitotic spindle defects (mainly cells with multipolar spindles), and abnormal DNA content (aneuploidy). Overall, these results suggest that CLASP1 and CLASP2 play overlapping roles during mitosis. In order to understand the molecular mechanisms underlying the function of human CLASPs during mitosis, next we performed a proteomic study for the identification of CLASP1 interacting proteins during mitosis by mass-spectrometry. Our results confirmed the interactions between CLASP1 CLIP-170 and LL5ß, both described in interphase. Moreover, new interactors were found such as CENP-E, Astrin, GCC185, CENP-J/CPAP, MARK2 and the novel protein KIAA0802. In interphase cells, CLASPs accumulate at the Golgi apparatus and this accumulation is related to the presence of stabilized microtubules. However, there is little information concerning Golgi function during mitosis. Analysis of GCC185 mitotic distribution showed that in early stages GCC185 localizes around centrosomes, and disperses in metaphase and anaphase. In telophase, GCC185 re-localizes to the perinuclear region and centrosomes. Noteworthy, we found an extensive co-localization between GCC185 and CLASP1 (especially in prophase, prometaphase and telophase). The interaction between GCC185 and CLASP1 was also confirmed in interphase cell extracts. Finally, many +TIPs like APC, CLIP-170/Restin and EB1 have previously been implicated in aneuploidy and tumourigenesis, forming a complex protein network with CLASPs. To determine the implications of CLASP1 for the mechanisms of tumourigenesis, we performed a mutational screening in a human cell line derived from cervix carcinoma (HeLa cells). In our mutational analysis we found three deletions: two of them correspond to CLASP1 alternative spliced isoforms (737 1538 and 1125 1164), and the third derives from the partial loss of exon 21 (673 679). These mutations could be related to chromosomal instability leading to random mutations, or may reflect that CLASP1 gene is a main target for mutations. These results encourage a larger survey for CLASP mutations in other tumour cell lines and primary tumours. Overall, we found that CLASP1 and CLASP2 play redundant roles during mitosis, whose absence can originate several mitotic defects, and ultimately lead to aneuploidy. Additionally, we uncovered new molecular interactions with important and novel mitotic proteins and provided the molecular linkage between CLASP function and the still mysterious role of the Golgi apparatus during mitosis.