Proteomic and Functional Characterization of STARD9

An Essential Function of the C. elegans Ortholog of TPX2 Is to Localize Activated Aurora A Kinase to Mitotic Spindles

Several chemotherapeutic drugs interfere with assembly of the mitotic spindle of cancer cells, leading to mitotic arrest, mediated by the spindle assembly checkpoint (SAC). However, cancer cells may be SAC-deficient and survive such treatment, due to mitotic slippage. Sensing of defective spindle assembly by the SAC, causes inhibition of the anaphase-promoting complex or cyclosome (APC/C), and blocks mitotic exit, by stabilizing APC/C substrates, such as cyclin B. Mitotic slippage may occur due to residual APC/C activity and slow cyclin B degradation. Recent preclinical data suggests that targeting mitotic exit by blocking APC/C activity is a much more efficient therapeutic approach than disturbing mitotic spindle assembly.

Accurate mitotic spindle assembly is critical for mitotic fidelity and organismal development. Multiple processes coordinate spindle assembly and chromosome segregation. Two key components are centrosomes and the spindle assembly checkpoint (SAC), and mutations affecting either can cause human microcephaly. In vivo studies in Drosophila melanogaster found that loss of either component alone is well tolerated in the developing brain, in contrast to epithelial tissues of the imaginal discs. In this study, we reveal that one reason for that tolerance is the compensatory relationship between centrosomes and the SAC. In the absence of both centrosomes and the SAC, brain cells, including neural stem cells, experience massive errors in mitosis, leading to increased cell death, which reduces the neural progenitor pool and severely disrupts brain development. However, our data also demonstrate that neural cells are much more tolerant of aneuploidy than epithelial cells. Our data provide novel insights into the mechanisms by which different tissues manage genome stability and parallels with human microcephaly.

The majority of human cancer shows chromosomal instability (CIN). Although the precise mechanism remains largely uncertain, proper progression of mitosis is crucial. B-type lamins were suggested to be components of the spindle matrix of mitotic cells and to be involved in mitotic spindle assembly; thus, B-type lamins may contribute to the maintenance of chromosome integrity. Here, using a proteomic approach, we identified lamin B2 as a novel protein involved in CIN. Lamin B2 expression decreased in colorectal cancer cell lines exhibiting CIN, as compared with colorectal cancer cell lines exhibiting microsatellite instability (MIN), which is mutually exclusive to CIN. Importantly, lamin B2 knockdown in MIN-type colorectal cancer cells induced CIN phenotypes such as aneuploidy, chromosome mis-segregation and aberrant spindle assembly, whereas ectopic expression of lamin B2 in CIN-type colorectal cancer cells prevented their CIN phenotypes. Additionally, immunohistochemical analysis showed a lower expression of lamin B2 in cancer tissues extracted from patients with sporadic colorectal cancer (CIN-type) than that from patients with hereditary non-polyposis colorectal cancer (HNPCC; MIN type). Intriguingly, mitotic lamin B2 in MIN cancer cells was localized outside the spindle poles and mitotic lamin B2 localization was diminished in CIN cancer cells, suggesting an important role of lamin B2 in proper mitotic spindle formation. The obtained results suggest that lamin B2 maintains chromosome integrity by ensuring proper spindle assembly and that its downregulation causes CIN in colorectal cancer.

Myosin II-Dependent Cortical Movement Is Required for Centrosome Separation and Positioning during Mitotic Spindle Assembly

<div>Abstract<p>Recent work has made it clear that pericentriolar material (PCM), the matrix of proteins surrounding centrioles, contributes to most functions of centrosomes. Given the occurrence of centrosome amplification in most solid tumors and the unconventional survival of these tumor cells, it is tempting to hypothesize that gel-like mitotic PCM would cluster extra centrosomes to defend against mitotic errors and increase tumor cell survival. However, because PCM lacks an encompassing membrane, is highly dynamic, and is physically connected to centrioles, few methods can decode the components of this microscale matrix. In this study, we took advantage of differential labeling between two sets of APEX2-centrosome reactions to design a strategy for acquiring the PCM proteome in living undisturbed cells without synchronization treatment, which identified 392 PCM proteins. Localization of ubiquitination promotion proteins away from PCM was a predominant mechanism to maintain the large size of PCM for centrosome clustering during mitosis in cancer cells. Depletion of PCM gene kinesin family member 20A (<i>KIF20A</i>) caused centrosome clustering failure and apoptosis in cancer cells <i>in vitro</i> and <i>in vivo</i>. Thus, our study suggests a strategy for targeting a wide range of tumors exhibiting centrosome amplification and provides a proteomic resource for future mining of PCM proteins.</p>Significance:<p>This study identifies the proteome of pericentriolar material and reveals therapeutic vulnerabilities in tumors bearing centrosome amplification.</p></div>

<div>Abstract<p>Recent work has made it clear that pericentriolar material (PCM), the matrix of proteins surrounding centrioles, contributes to most functions of centrosomes. Given the occurrence of centrosome amplification in most solid tumors and the unconventional survival of these tumor cells, it is tempting to hypothesize that gel-like mitotic PCM would cluster extra centrosomes to defend against mitotic errors and increase tumor cell survival. However, because PCM lacks an encompassing membrane, is highly dynamic, and is physically connected to centrioles, few methods can decode the components of this microscale matrix. In this study, we took advantage of differential labeling between two sets of APEX2-centrosome reactions to design a strategy for acquiring the PCM proteome in living undisturbed cells without synchronization treatment, which identified 392 PCM proteins. Localization of ubiquitination promotion proteins away from PCM was a predominant mechanism to maintain the large size of PCM for centrosome clustering during mitosis in cancer cells. Depletion of PCM gene kinesin family member 20A (<i>KIF20A</i>) caused centrosome clustering failure and apoptosis in cancer cells <i>in vitro</i> and <i>in vivo</i>. Thus, our study suggests a strategy for targeting a wide range of tumors exhibiting centrosome amplification and provides a proteomic resource for future mining of PCM proteins.</p>Significance:<p>This study identifies the proteome of pericentriolar material and reveals therapeutic vulnerabilities in tumors bearing centrosome amplification.</p></div>

Combining targeted therapeutic agents is an attractive cancer treatment strategy associated with high efficacy and low toxicity. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is an essential factor in DNA damage repair. Studies from us and others have revealed that DNA-PKcs also plays an important role in normal mitosis progression. Histone deacetylase (HDACs) inhibitors commonly lead to mitotic aberration and have been approved for treating various cancers in the clinic. We showed that DNA-PKcs depletion or kinase activity inhibition increases cancer cells’ sensitivity to HDACs inhibitors in vitro and in vivo. DNA-PKcs deficiency significantly enhances HDACs inhibitors (HDACi)-induced mitotic arrest and is followed by apoptotic cell death. Mechanistically, we found that DNA-PKcs binds to HDAC6 and facilitates its acetylase activity. HDACi is more likely to impair HDAC6-induced deacetylation of HSP90 and abrogate HSP90’s chaperone function on Aurora A, a critical mitotic kinase that regulates centrosome separation and mitotic spindle assembly in DNA-PKcs-deficient cells. Our current work indicates crosstalk between DNA-PKcs and HDACs signaling pathways, and highlights that the combined targeting of DNA-PKcs and HDACs can be used in cancer therapy. Abbreviations: DNA-PKcs, DNA-dependent protein kinase catalytic subunit, HDACs, Histone deacetylases, DSBs, DNA double-strand breaks, ATM, ataxia telangiectasia mutated, ATR, ATM-Rad3-related

The functional role of centrioles or basal bodies in mitotic spindle assembly and function is currently unclear. Although supernumerary centrioles have been associated with multipolar spindles in cancer cells, suggesting centriole number might dictate spindle polarity, bipolar spindles are able to assemble in the complete absence of centrioles, suggesting a level of centriole-independence in the spindle assembly pathway. In this report we perturb centriole number using mutations in Chlamydomonas reinhardtii, and measure the response of the mitotic spindle to these perturbations in centriole number. Although altered centriole number increased the frequency of monopolar and multipolar spindles, the majority of spindles remained bipolar regardless of the centriole number. But even when spindles were bipolar, abnormal centriole numbers led to asymmetries in tubulin distribution, half-spindle length and spindle pole focus. Half spindle length correlated directly with number of centrioles at a pole, such that an imbalance in centriole number between the two poles of a bipolar spindle correlated with increased asymmetry between half spindle lengths. These results are consistent with centrioles playing an active role in regulating mitotic spindle length. Mutants with centriole number alteration also show increased cytokinesis defects, but these do not correlate with centriole number in the dividing cell and may therefore reflect downstream consequences of defects in preceding cell divisions.

Ran-GTP is Non-Essential to Activate NuMA for Spindle Pole Focusing, But Dynamically Polarizes HURP to Control Mitotic Spindle Length

Bipolar spindle formation is essential for faithful chromosome segregation at mitosis. Because centrosomes define spindle poles, abnormal number and structural organization of centrosomes can lead to loss of spindle bipolarity and genetic integrity. ASAP (aster-associated protein or MAP9) is a centrosome- and spindle-associated protein, the deregulation of which induces severe mitotic defects. Its phosphorylation by Aurora A is required for spindle assembly and mitosis progression. Here, we show that ASAP is localized to the spindle poles by Polo-like kinase 1 (Plk1) (a mitotic kinase that plays an essential role in centrosome regulation and mitotic spindle assembly) through the γ-TuRC-dependent pathway. We also demonstrate that ASAP is a novel substrate of Plk1 phosphorylation and have identified serine 289 as the major phosphorylation site by Plk1 in vivo. ASAP phosphorylated on serine 289 is localized to centrosomes during mitosis, but this phosphorylation is not required for its Plk1-dependent localization at the spindle poles. We show that phosphorylated ASAP on serine 289 contributes to spindle pole stability in a microtubule-dependent manner. These data reveal a novel function of ASAP in centrosome integrity. Our results highlight dual ASAP regulation by Plk1 and further confirm the importance of ASAP for spindle pole organization, bipolar spindle assembly, and mitosis.

In contrast to well-studied fungal and animal cells, plant cells assemble bipolar spindles that exhibit a great deal of plasticity in the absence of structurally defined microtubule-organizing centers like the centrosome. While plants employ some evolutionarily conserved proteins to regulate spindle morphogenesis and remodeling, many essential spindle assembly factors found in vertebrates are either missing or not required for producing the plant bipolar microtubule array. Plants also produce proteins distantly related to their fungal and animal counterparts to regulate critical events such as the spindle assembly checkpoint. Plant spindle assembly initiates with microtubule nucleation on the nuclear envelope followed by bipolarization into the prophase spindle. After nuclear envelope breakdown, kinetochore fibers are assembled and unified into the spindle apparatus with convergent poles. Of note, compared to fungal and animal systems, relatively little is known about how plant cells remodel the spindle microtubule array during anaphase. Uncovering mitotic functions of novel proteins for spindle assembly in plants will illuminate both common and divergent mechanisms employed by different eukaryotic organisms to segregate genetic materials.

Cells with blocked microtubule polymerization are delayed in mitosis, but eventually manage to proliferate despite substantial chromosome missegregation. While several studies have analyzed the first cell division after microtubule depolymerization, we have asked how cells cope long-term with microtubule impairment. We allowed 24 clonal populations of yeast cells with beta-tubulin mutations preventing proper microtubule polymerization, to evolve for ˜150 generations. At the end of the laboratory evolution experiment, cells had regained the ability to form microtubules and were less sensitive to microtubule-depolymerizing drugs. Whole-genome sequencing identified recurrently mutated genes, in particular for tubulins and kinesins, as well as pervasive duplication of chromosome VIII. Recreating these mutations and chromosome VIII disomy prior to evolution confirmed that they allow cells to compensate for the original mutation in beta-tubulin. Most of the identified mutations did not abolish function, but rather restored microtubule functionality. Analysis of the temporal order of resistance development in independent populations repeatedly revealed the same series of events: disomy of chromosome VIII followed by a single additional adaptive mutation in either tubulins or kinesins. Since tubulins are highly conserved among eukaryotes, our results have implications for understanding resistance to microtubule-targeting drugs widely used in cancer therapy.

MTA1 localizes to the mitotic spindle apparatus and interacts with TPR in spindle assembly checkpoint regulation

Efficient assembly of a mitotic spindle and stable attachment of microtubules (k-fibers) to kinetochores are essential for the high fidelity of chromosome segregation. Both spindle assembly and Mitotic spindle mediates the segregation of chromosomes in the cell cycle and the proper function of the spindle is crucial to the high fidelity of chromosome segregation and to the stability of the genome. Nucleation of microtubules (MTs) from centrosomes and chromatin represents two well-characterized pathways essential for the assembly of a dynamic spindle in mitosis. Recently, we identified a third MT nucleation pathway, in which existing MTs in the spindle acts as a template to promote the nucleation and polymerization of MTs, thereby efficiently amplifying MTs in the spindle. We will review here our current understanding on the molecular mechanism, the physiological function and the cell-cycle regulation of MT amplification.k-fiber formation require robust nucleation and polymerization of microtubules mediated by the γ-tubulin ring complex (γ TuRC). It has been well established that centrosomes and chromatin are the two centers for microtubule nucleation. We recently demonstrate a third mechanism for microtubule nucleation and polymerization, in which the existing microtubules in the spindle act as templates to promote the formation of new microtubules. We showed that a novel spindle-associated protein, FAM29A, plays a critical role in this microtubule-dependent microtubule amplification. FAM29A associates with spindle microtubules and directly interacts with and recruits NEDD1, the targeting subunit of γTuRC. Spindle-associated γTuRC then promotes microtubule nucleation required for spindle assembly and k-fiber formation. This novel microtubule amplification pathway provides a powerful mechanism to control the local cytoskeleton structures independent of centrosomes and chromatin. We speculate that microtubule amplification not only functions in mitosis, but may also act in other physiological processes to re-enforce existing cytoskeleton structures.