Mitotic crisis: The unmasking of a novel role for RPA

Mitotic DNA damage is a constant threat to genomic integrity, yet understanding of the cellular responses to this stress remain incomplete. Recent work by Anantha et al. (PNAS 2008; 105:12903–8) has found surprising evidence that RPA, the primary eukaryotic single-stranded DNA-binding protein, can stimulate the ability of cells to exit mitosis into a 2N G₁ phase. Along with providing additional discussion of this study, we review evidence suggesting that DNA replication and repair factors can modulate mitotic transit by acting through Polo-like kinase-1 (Plk1) and the centrosome. “A crisis unmasks everyone.”—Mason Cooley, US aphorist.     

The Phosphatase PP1 Promotes Mitotic Slippage through Mad3 Dephosphorylation

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Chk1-Mad2 interaction

Chk1 is implicated in several checkpoints of the cell cycle acting as a key player in the signal transduction pathway activated in response to DNA damage and crucial for the maintenance of genomic stability. Chk1 also plays a role in the mitotic spindle checkpoint, which ensures the fidelity of mitotic segregation during mitosis, preventing chromosomal instability and aneuploidy. Mad2 is one of the main mitotic checkpoint components and also exerts a role in the cellular response to DNA damage. To investigate a possible crosslink existing between Chk1 and Mad2, we studied Mad2 protein levels after Chk1 inhibition either by specific siRNAs or by a specific and selective Chk1 inhibitor (PF-00477736), and we found that after Chk1 inhibition, Mad2 protein levels decrease only in tumor cells sensitive to Chk1 depletion. We then mapped six Chk1’s phosphorylatable sites on Mad2 protein, and found that Chk1 is able to phosphorylate Mad2 in vitro on more than one site, while it is incapable of phoshorylating the Mad2 form mutated on all six phosphorylatable sites. Moreover our studies demonstrate that Chk1 co-localizes and physically associates with Mad2 in cells both under unstressed conditions and after DNA damage, thus providing new and interesting evidence on Chk1 and Mad2 crosstalk in the DNA damage checkpoint and in the mitotic spindle checkpoint.     

Loss of RPA1 induces Chk2 phosphorylation through a caffeine-sensitive pathway

RPA is an important component of DNA replication, repair and recombination, but its involvement in the signaling of cell-cycle checkpoints is not well understood. In this study, we show that knockdown of RPA1 by siRNA duplexes induces ATM (Ser1981) and Chk2 (Thr68), but not Chk1 (Ser345) phosphorylation and results in p21 upregulation in HeLa cells. However, the induction of Chk2 (Thr68) phosphorylation and p21 expression by RPA1 siRNA transfection can be completely blocked by the ATM inhibitor caffeine. Moreover, transfection of siRNAs targeting ATM dramatically reduces Chk2 (Thr68) phosphorylation in RPA1 knockdown cells. Taken together, these results suggest that loss of RPA1 activates the Chk2 signaling pathway in an ATM-dependent manner.     

Mitotic phosphatase activity is required for MCC maintenance during the spindle checkpoint

The spindle checkpoint prevents activation of the anaphase-promoting complex (APC/C) until all chromosomes are correctly attached to the mitotic spindle. Early in mitosis, the mitotic checkpoint complex (MCC) inactivates the APC/C by binding the APC/C activating protein CDC20 until the chromosomes are properly aligned and attached to the mitotic spindle, at which point MCC disassembly releases CDC20 to activate the APC/C. Once the APC/C is activated, it targets cyclin B and securin for degradation, and the cell progresses into anaphase. While phosphorylation is known to drive many of the events during the checkpoint, the precise molecular mechanisms regulating spindle checkpoint maintenance and inactivation are still poorly understood. We sought to determine the role of mitotic phosphatases during the spindle checkpoint. To address this question, we treated spindle checkpoint-arrested cells with various phosphatase inhibitors and examined the effect on the MCC and APC/C activation. Using this approach we found that 2 phosphatase inhibitors, calyculin A and okadaic acid (1 μM), caused MCC dissociation and APC/C activation leading to cyclin A and B degradation in spindle checkpoint-arrested cells. Although the cells were able to degrade cyclin B, they did not exit mitosis as evidenced by high levels of Cdk1 substrate phosphorylation and chromosome condensation. Our results provide the first evidence that phosphatases are essential for maintenance of the MCC during operation of the spindle checkpoint.     

Let's huddle to prevent a muddle: centrosome declustering as an attractive anticancer strategy

Nearly a century ago, cell biologists postulated that the chromosomal aberrations blighting cancer cells might be caused by a mysterious organelle-the centrosome-that had only just been discovered. For years, however, this enigmatic structure was neglected in oncologic investigations and has only recently reemerged as a key suspect in tumorigenesis. A majority of cancer cells, unlike healthy cells, possess an amplified centrosome complement, which they manage to coalesce neatly at two spindle poles during mitosis. This clustering mechanism permits the cell to form a pseudo-bipolar mitotic spindle for segregation of sister chromatids. On rare occasions this mechanism fails, resulting in declustered centrosomes and the assembly of a multipolar spindle. Spindle multipolarity consigns the cell to an almost certain fate of mitotic arrest or death. The catastrophic nature of multipolarity has attracted efforts to develop drugs that can induce declustering in cancer cells. Such chemotherapeutics would theoretically spare healthy cells, whose normal centrosome complement should preclude multipolar spindle formation. In search of the 'Holy Grail' of nontoxic, cancer cell-selective, and superiorly efficacious chemotherapy, research is underway to elucidate the underpinnings of centrosome clustering mechanisms. Here, we detail the progress made towards that end, highlighting seminal work and suggesting directions for future research, aimed at demystifying this riddling cellular tactic and exploiting it for chemotherapeutic purposes. We also propose a model to highlight the integral role of microtubule dynamicity and the delicate balance of forces on which cancer cells rely for effective centrosome clustering. Finally, we provide insights regarding how perturbation of this balance may pave an inroad for inducing lethal centrosome dispersal and death selectively in cancer cells.     

The spindle assembly checkpoint promotes chromosome bi-orientation: A novel Mad1 role in chromosome alignment

Faithful chromosome segregation relies on dynamic interactions between spindle microtubules and chromosomes. Especially, all chromosomes must be aligned at the equator of the spindle to establish bi-orientation before they start to segregate. The spindle assembly checkpoint (SAC) monitors this process, inhibiting chromosome segregation until all chromosomes achieve bi-orientation. The original concept of ‘checkpoints’ was proposed as an external surveillance system that does not play an active role in the process it monitors. However, accumulating evidence from recent studies suggests that SAC components do play an active role in chromosome bi-orientation. In this review, we highlight a novel Mad1 role in chromosome alignment, which is the first conserved mechanism that links the SAC and kinesin-mediated chromosome gliding.     

癌症高表达蛋白--Hec1在纺锤体组装检查点中的作用

细胞增殖依赖于细胞分裂前染色体的复制及随后的姐妹染色单体分离到达两极。纺锤体组装检查点具有确保染色体信息传递保真性的作用,检查点的缺失可能导致染色体的分离异常和肿瘤形成。癌症高表达蛋白(Hec1)通过与调控G2/M期的蛋白间的相互作用而在染色体的分离中发挥重要作用。Hec1与Nuf2的复合物,在G2/M期与动粒相结合,Hec1的缺失将导致严重的染色体分离错误。Hec1具有召集Mps1和Mad1/Mad2复合物结合到动粒上的作用,这种结合可以激活纺锤体组装检查点途径中非常重要的APCCdc20途径。但是Hec1、Mps1、Mad1三者之间的相互作用仍未明了。Hec1还可以通过与26S蛋白酶复合物的不同亚基结合调控其功能。Hec1是一种丝氨酸磷酸化蛋白,其磷酸化是由Nek2在G2/M期完成的。     

GSI promotes vincristine-induced apoptosis by enhancing multi-polar spindle formation

Gamma secretase inhibitors (GSI), cell-permeable small-molecule inhibitors of gamma secretase activity, had been originally developed for the treatment of Alzheimer disease. In recent years, it has been exploited in cancer research to inhibit Notch signaling that is aberrantly activated in various cancers. We previously found that GSI could synergize with anti-microtubule agent, vincristine (VCR) in a Notch-independent manner. Here, we delineate the underlying cell cycle-related mechanism using HeLa cells, which have strong mitotic checkpoints. GSI enhanced VCR-induced cell death, although GSI alone did not affect cell viability at all. GSI augmented VCR-induced mitotic arrest in a dose-dependent manner, which was preceded by apoptotic cell death, as shown by an increase in Annexin V-positive and caspase-positive cell population. Furthermore, GSI amplified multi-polar spindle formation triggered by VCR. Altogether, we show the evidence that GSI enhances VCR-induced apoptosis in HeLa cells via multi-polar mitotic spindle formation, independent of Notch signaling. These data suggest that one or more GS substrates, yet to be identified, in a post-GS processed form, may play a role in maintaining functional centrosomes/mitotic spindles. More significantly, the synergistic effect of GSI in combination with VCR could be exploited in clinical setting to improve the efficacy of VCR.     

Bld10p, a novel protein essential for basal body assembly in Chlamydomonas: localization to the cartwheel, the first ninefold symmetrical structure appearing during assembly

How centrioles and basal bodies assemble is a long-standing puzzle in cell biology. To address this problem, we analyzed a novel basal body-defective Chlamydomonas reinhardtii mutant isolated from a collection of flagella-less mutants. This mutant, bld10, displayed disorganized mitotic spindles and cytoplasmic microtubules, resulting in abnormal cell division and slow growth. Electron microscopic observation suggested that bld10 cells totally lack basal bodies. The product of the BLD10 gene (Bld10p) was found to be a novel coiled-coil protein of 170 kD. Immunoelectron microscopy localizes Bld10p to the cartwheel, a structure with ninefold rotational symmetry positioned near the proximal end of the basal bodies. Because the cartwheel forms the base from which the triplet microtubules elongate, we suggest that Bld10p plays an essential role in an early stage of basal body assembly. A viable mutant having such a severe basal body defect emphasizes the usefulness of Chlamydomonas in studying the mechanism of basal body/centriole assembly by using a variety of mutants.     

Replication-mediated disassociation of replication protein A-XPA complex upon DNA damage: implications for RPA handing off

RPA (replication protein A), the eukaryotic ssDNA (single-stranded DNA)-binding protein, participates in most cellular processes in response to genotoxic insults, such as NER (nucleotide excision repair), DNA, DSB (double-strand break) repair and activation of cell cycle checkpoint signalling. RPA interacts with XPA (xeroderma pigmentosum A) and functions in early stage of NER. We have shown that in cells the RPA-XPA complex disassociated upon exposure of cells to high dose of UV irradiation. The dissociation required replication stress and was partially attributed to tRPA hyperphosphorylation. Treatment of cells with CPT (camptothecin) and HU (hydroxyurea), which cause DSB DNA damage and replication fork collapse respectively and also leads to the disruption of RPA-XPA complex. Purified RPA and XPA were unable to form complex in vitro in the presence of ssDNA. We propose that the competition-based RPA switch among different DNA metabolic pathways regulates the dissociation of RPA with XPA in cells after DNA damage. The biological significances of RPA-XPA complex disruption in relation with checkpoint activation, DSB repair and RPA hyperphosphorylation are discussed.     

The KinI kinesin Kif2a is required for bipolar spindle assembly through a functional relationship with MCAK

Although the microtubule-depolymerizing KinI motor Kif2a is abundantly expressed in neuronal cells, we now show it localizes to centrosomes and spindle poles during mitosis in cultured cells. RNAi-induced knockdown of Kif2a expression inhibited cell cycle progression because cells assembled monopolar spindles. Bipolar spindle assembly was restored in cells lacking Kif2a by treatments that altered microtubule assembly (nocodazole), eliminated kinetochore-microtubule attachment (loss of Nuf2), or stabilized microtubule plus ends at kinetochores (loss of MCAK). Thus, two KinI motors, MCAK and Kif2a, play distinct roles in mitosis, and MCAK activity at kinetochores must be balanced by Kif2a activity at poles for spindle bipolarity. These treatments failed to restore bipolarity to cells lacking the activity of the kinesin Eg5. Thus, two independent pathways contribute to spindle bipolarity, with the Eg5-dependent pathway using motor force to drive spindle bipolarity and the Kif2a-dependent pathway relying on microtubule polymer dynamics to generate force for spindle bipolarity.     

The centrosomal protein C-Nap1 is required for cell cycle-regulated centrosome cohesion

Duplicating centrosomes are paired during interphase, but are separated at the onset of mitosis. Although the mechanisms controlling centrosome cohesion and separation are important for centrosome function throughout the cell cycle, they remain poorly understood. Recently, we have proposed that C-Nap1, a novel centrosomal protein, is part of a structure linking parental centrioles in a cell cycle-regulated manner. To test this model, we have performed a detailed structure-function analysis on C-Nap1. We demonstrate that antibody-mediated interference with C-Nap1 function causes centrosome splitting, regardless of the cell cycle phase. Splitting occurs between parental centrioles and is not dependent on the presence of an intact microtubule or microfilament network. Centrosome splitting can also be induced by overexpression of truncated C-Nap1 mutants, but not full-length protein. Antibodies raised against different domains of C-Nap1 prove that this protein dissociates from spindle poles during mitosis, but reaccumulates at centrosomes at the end of cell division. Use of the same antibodies in immunoelectron microscopy shows that C-Nap1 is confined to the proximal end domains of centrioles, indicating that a putative linker structure must contain additional proteins. We conclude that C-Nap1 is a key component of a dynamic, cell cycle-regulated structure that mediates centriole-centriole cohesion.     

RUNX3 interactome reveals novel centrosomal targeting of RUNX family of transcription factors

RUNX family proteins are critical regulators of lineage differentiation during development. The high prevalence of RUNX mutation/epigenetic inactivation in human cancer indicates a causative role for dysfunctional RUNX in carcinogenesis. This is supported by well-documented evidence of functional interaction of RUNX with components of major oncogenic or tumor suppressive signaling pathways such as TGFβ and Wnt. Here, we explore the binding partners of RUNX3 proteins to further define the scope of RUNX3 function. Using a mass spectrometry-based approach, we found that RUNX3 binds to centrosomal protein rootletin. This led us to uncover the presence of RUNX proteins at the centrosome. Our findings suggest a potential function for RUNX3 during mitosis.     

Characterization of Cep135, a novel coiled-coil centrosomal protein involved in microtubule organization in mammalian cells.

By using monoclonal antibodies raised against isolated clam centrosomes, we have identified a novel 135-kD centrosomal protein (Cep135), present in a wide range of organisms. Cep135 is located at the centrosome throughout the cell cycle, and localization is independent of the microtubule network. It distributes throughout the centrosomal area in association with the electron-dense material surrounding centrioles. Sequence analysis of cDNA isolated from CHO cells predicted a protein of 1,145-amino acid residues with extensive alpha-helical domains. Expression of a series of deletion constructs revealed the presence of three independent centrosome-targeting domains. Overexpression of Cep135 resulted in the accumulation of unique whorl-like particles in both the centrosome and the cytoplasm. Although their size, shape, and number varied according to the level of protein expression, these whorls were composed of parallel dense lines arranged in a 6-nm space. Altered levels of Cep135 by protein overexpression and/or suppression of endogenous Cep135 by RNA interference caused disorganization of interphase and mitotic spindle microtubules. Thus, Cep135 may play an important role in the centrosomal function of organizing microtubules in mammalian cells.     

AIBp regulates mitotic entry and mitotic spindle assembly by controlling activation of both Aurora-A and Plk1

We previously reported that Aurora-A and the hNinein binding protein AIBp facilitate centrosomal structure maintenance and contribute to spindle formation. Here, we report that AIBp also interacts with Plk1, raising the possibility of functional similarity to Bora, which subsequently promotes Aurora-A–mediated Plk1 activation at Thr210 as well as Aurora-A activation at Thr288. In kinase assays, AIBp acts not only as a substrate but also as a positive regulator of both Aurora-A and Plk1. However, AIBp functions as a negative regulator to block phosphorylation of hNinein mediated by Aurora-A and Plk1. These findings suggest a novel AIBp-dependent regulatory machinery that controls mitotic entry. Additionally, knockdown of hNinein caused failure of AIBp to target the centrosome, whereas depletion of AIBp did not affect the localization of hNinein and microtubule nucleation. Notably, knockdown of AIBp in HeLa cells impaired both Aurora-A and Plk1 kinase, resulting in phenotypes with multiple spindle pole formation and chromosome misalignment. Our data show that depletion of AIBp results in the mis-localization of TACC3 and ch-TOG, but not CEP192 and CEP215, suggesting that loss of AIBp dominantly affects the Aurora-A substrate to cause mitotic aberrations. Collectively, our data demonstrate that AIBp contributes to mitotic entry and bipolar spindle assembly and may partially control localization, phosphorylation, and activation of both Aurora-A and Plk1 via hNinein during mitotic progression.