Centrosome Separation Research Articles

Structure-guided design of purine-based probes for selective Nek2 inhibition.

Nek2 (NIMA-related kinase 2) is a cell cycle-dependent serine/threonine protein kinase that regulates centrosome separation at the onset of mitosis. Overexpression of Nek2 is common in human cancers and suppression can restrict tumor cell growth and promote apoptosis. Nek2 inhibition with small molecules, therefore, offers the prospect of a new therapy for cancer. To achieve this goal, a better understanding of the requirements for selective-inhibition of Nek2 is required. 6-Alkoxypurines were identified as ATP-competitive inhibitors of Nek2 and CDK2. Comparison with CDK2-inhibitor structures indicated that judicious modification of the 6-alkoxy and 2-arylamino substituents could achieve discrimination between Nek2 and CDK2. In this study, a library of 6-cyclohexylmethoxy-2-arylaminopurines bearing carboxamide, sulfonamide and urea substituents on the 2-arylamino ring was synthesized. Few of these compounds were selective for Nek2 over CDK2, with the best result being obtained for 3-((6-(cyclohexylmethoxy)-9H-purin-2-yl)amino)-N,N-dimethylbenzamide (CDK2 IC50 = 7.0 μM; Nek2 IC50 = 0.62 μM) with >10-fold selectivity. Deletion of the 6-substituent abrogated activity against both Nek2 and CDK2. Nine compounds containing an (E)-dialkylaminovinyl substituent at C-6, all showed selectivity for Nek2, e.g. (E)-6-(2-(azepan-1-yl)vinyl)-N-phenyl-9H-purin-2-amine (CDK2 IC50 = 2.70 μM; Nek2 IC50 = 0.27 μM). Structural biology of selected compounds enabled a partial rationalization of the observed structure activity relationships and mechanism of Nek2 activation. This showed that carboxamide 11 is the first reported inhibitor of Nek2 in the DFG-in conformation.

The role of centrosomes in chemical carcinogenesis: Hexavalent chromium induces aberrant centriole and centrosome separation and centrosome amplification

The role of centrosomes in chemical carcinogenesis: Hexavalent chromium induces aberrant centriole and centrosome separation and centrosome amplification

The sequential activation of the mitotic microtubule assembly pathways favors bipolar spindle formation

Centrosome maturation is the process by which the duplicated centrosomes recruit pericentriolar components and increase their microtubule nucleation activity before mitosis. The role of this process in cells entering mitosis has been mostly related to the separation of the duplicated centrosomes and thereby to the assembly of a bipolar spindle. However, spindles can form without centrosomes. In fact, all cells, whether they have centrosomes or not, rely on chromatin-driven microtubule assembly to form a spindle. To test whether the sequential activation of these microtubule assembly pathways, defined by centrosome maturation and nuclear envelope breakdown, plays any role in spindle assembly, we combined experiments in tissue culture cells and Xenopus laevis egg extracts with a mathematical model. We found that interfering with the sequential activation of the microtubule assembly pathways compromises bipolar spindle assembly in tissue culture cells but not in X. laevis egg extracts. Our data suggest a novel function for centrosome maturation that determines the contribution of the chromosomal microtubule assembly pathway and favors bipolar spindle formation in most animal cells in which tubulin is in limiting amounts.

Dishevelled is a NEK2 kinase substrate controlling dynamics of centrosomal linker proteins

Dishevelled (DVL) is a key scaffolding protein and a branching point in Wnt signaling pathways. Here, we present conclusive evidence that DVL regulates the centrosomal cycle. We demonstrate that DVL dishevelled and axin (DIX) domain, but not DIX domain-mediated multimerization, is essential for DVL's centrosomal localization. DVL accumulates during the cell cycle and associates with NIMA-related kinase 2 (NEK2), which is able to phosphorylate DVL at a multitude of residues, as detected by a set of novel phospho-specific antibodies. This creates interfaces for efficient binding to CDK5 regulatory subunit-associated protein 2 (CDK5RAP2) and centrosomal Nek2-associated protein 1 (C-NAP1), two proteins of the centrosomal linker. Displacement of DVL from the centrosome and its release into the cytoplasm on NEK2 phosphorylation is coupled to the removal of linker proteins, an event necessary for centrosomal separation and proper formation of the mitotic spindle. Lack of DVL prevents NEK2-controlled dissolution of loose centrosomal linker and subsequent centrosomal separation. Increased DVL levels, in contrast, sequester centrosomal NEK2 and mimic monopolar spindle defects induced by a dominant negative version of this kinase. Our study thus uncovers molecular crosstalk between centrosome and Wnt signaling.

Abstract 3032: A novel compound ARN-3236 inhibits SIK2 and sensitizes ovarian cancer to paclitaxel

Abstract Ovarian carcinomas account for 4% of total cancers in women in the United States. Improved outcomes might be attained if sensitivity to primary chemotherapy were enhanced. A recent study discovered that the salt inducible kinase 2 (SIK2) plays a key role in the initiation of mitosis and regulates paclitaxel sensitivity. Here we show that SIK2 is overexpressed in 30% ovarian cancer specimens, associated with a poor prognosis. ARN-3236, a selective, highly potent small molecule SIK2 inhibitor with function similar to SIK2 siRNA, blocks cell proliferation in a panel of ovarian cancer cell lines, where the IC50 of ARN-3236 was inversely correlated with endogenous SIK2 expression. ARN-3236 also enhanced response to paclitaxel in ovarian cancer cells (OC316, SKOv3, A2780, HEY, ES2 and UPN251). Similar to the function of SIK2 siRNA, ARN-3236 effectively inhibits AKT phosphorylation at Ser473 and Tyr308, as well as the expression of the downstream effector survivin. ARN-3236 uncouples the centrosome from the nucleus in interphase, and blocks centrosome separation in mitosis, resulting in the accumulation of cells in prometaphase. In addition, ARN-3236 enhances tumor growth inhibition of paclitaxel in ovarian cancer xenograft models. Thus, functional inhibition of SIK2 with ARN-3236 had the same effects on ovarian cancer cell function as knockdown of SIK2 with siRNA. ARN-3236 deserves further study as a SIK2 inhibitor that significantly improves the sensitivity to paclitaxel for treatment of human ovarian cancer cells and xenografts. Citation Format: Jinhua Zhou, Albandri Alfredi, Shu Zhang, Janice M. Santiago-O’Farrill, Ahmed A. Ahmed, Hailing Yang, Weiqun Mao, Yan Wang, Hariprasad Vankayalapati, Zhen Lu, Robert C. Bast Jr. A novel compound ARN-3236 inhibits SIK2 and sensitizes ovarian cancer to paclitaxel. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3032.

Aurora A, MCAK, and Kif18b promote Eg5-independent spindle formation

Inhibition of the microtubule (MT) motor protein Eg5 results in a mitotic arrest due to the formation of monopolar spindles, making Eg5 an attractive target for anti-cancer therapies. However, Eg5-independent pathways for bipolar spindle formation exist, which might promote resistance to treatment with Eg5 inhibitors. To identify essential components for Eg5-independent bipolar spindle formation, we performed a genome-wide siRNA screen in Eg5-independent cells (EICs). We find that the kinase Aurora A and two kinesins, MCAK and Kif18b, are essential for bipolar spindle assembly in EICs and in cells with reduced Eg5 activity. Aurora A promotes bipolar spindle assembly by phosphorylating Kif15, hereby promoting Kif15 localization to the spindle. In turn, MCAK and Kif18b promote bipolar spindle assembly by destabilizing the astral MTs. One attractive way to interpret our data is that, in the absence of MCAK and Kif18b, excessive astral MTs generate inward pushing forces on centrosomes at the cortex that inhibit centrosome separation. Together, these data suggest a novel function for astral MTs in force generation on spindle poles and how proteins involved in regulating microtubule length can contribute to bipolar spindle assembly.

Axin-1 Regulates Meiotic Spindle Organization in Mouse Oocytes.

Axin-1, a negative regulator of Wnt signaling, is a versatile scaffold protein involved in centrosome separation and spindle assembly in mitosis, but its function in mammalian oogenesis remains unknown. Here we examined the localization and function of Axin-1 during meiotic maturation in mouse oocytes. Immunofluorescence analysis showed that Axin-1 was localized around the spindle. Knockdown of the Axin1 gene by microinjection of specific short interfering (si)RNA into the oocyte cytoplasm resulted in severely defective spindles, misaligned chromosomes, failure of first polar body (PB1) extrusion, and impaired pronuclear formation. However, supplementing the culture medium with the Wnt pathway activator LiCl improved spindle morphology and pronuclear formation. Downregulation of Axin1 gene expression also impaired the spindle pole localization of γ-tubulin/Nek9 and resulted in retention of the spindle assembly checkpoint protein BubR1 at kinetochores after 8.5 h of culture. Our results suggest that Axin-1 is critical for spindle organization and cell cycle progression during meiotic maturation in mouse oocytes.

An update on the pharmacokinetics and pharmacodynamics of alisertib, a selective Aurora kinase A inhibitor.

Human Aurora kinases, including Aurora kinase A (AURKA), B (AURKB), and C (AURKC), play an essential role in mitotic events such as monitoring of the mitotic checkpoint, creation of bipolar mitotic spindle and alignment of centrosomes on it, also regulating centrosome separation, bio-orientation of chromosomes and cytokinesis. AURKA and AURKB are key regulators of mitosis and centrosome via polymerizing microfilaments and controlling chromatid segregation. In particular, AURKA plays critical roles in the regulation of mitotic entry, centrosome function, bipolar spindle assembly, and chromosome segregation. AURKA has been found to be overexpressed in various solid and haematological cancers and has been linked with poor prognosis. Its important role in cancer initiation, growth, and metastasis has brought the focus to search for potent and selective AURKA inhibitors for cancer treatment. MLN8237, also known as alisertib, is one selective AURKA inhibitor that has shown remarkable anticancer effects in preclinical studies. Alisertib exhibits favourable pharmacokinetic properties. Alisertib has generally showed good partial response rates of 4-52% and good safety profiles in Phase I and II trials when it is solely administered as well as combined with cytotoxic chemotherapeutic drugs. Recently, the multicentre, randomized Phase III study of alisertib in patients with relapsed or refractory peripheral T-cell lymphoma has been discontinued due to unsatisfactory efficacy. The low risk of side effects, accessibility, and effectiveness of alisertib makes it a new promising anticancer therapy and further mechanistic and clinical studies are warranted.

Non-centrosomal TPX2-Dependent Regulation of the Aurora A Kinase: Functional Implications for Healthy and Pathological Cell Division.

Aurora A has been extensively characterized as a centrosomal kinase with essential functions during cell division including centrosome maturation and separation and spindle assembly. However, Aurora A localization is not restricted to the centrosomes and compelling evidence support the existence of specific mechanisms of activation and functions for non-centrosomal Aurora A in the dividing cell. It has been now well established that spindle assembly involves an acentrosomal RanGTP-dependent pathway that triggers microtubule assembly and organization in the proximity of the chromosomes whether centrosomes are present or not. The mechanism involves the regulation of a number of NLS-containing proteins, generically called SAFS (Spindle Assembly Factors) that exert their functions upon release from karyopherins by RanGTP. One of them, the nuclear protein TPX2 interacts with and activates Aurora A upon release from importins by RanGTP. This basic mechanism triggers the activation of Aurora A in the proximity of the chromosomes potentially translating the RanGTP signaling gradient centered on the chromosome into an Aurora A phosphorylation network. Here, we will review our current knowledge on the RanGTP-dependent TPX2 activation of Aurora A away from centrosomes: from the mechanism of activation and its functional consequences on the kinase stability and regulation to its roles in spindle assembly and cell division. We will then focus on the substrates of the TPX2-activated Aurora A having a role in microtubule nucleation, stabilization, and organization. Finally, we will briefly discuss the implications of the use of Aurora A inhibitors in anti-tumor therapies in the light of its functional interaction with TPX2.

Dynein Transmits Polarized Actomyosin Cortical Flows to Promote Centrosome Separation.

The two centrosomes present at the onset of mitosis must separate in a timely and accurate fashion to ensure proper bipolar spindle assembly. The minus-end-directed motor dynein plays a pivotal role in centrosome separation, but the underlying mechanisms remain elusive, particularly regarding how dynein coordinates this process in space and time. We addressed these questions in the one-cell C. elegans embryo, using a combination of 3D time-lapse microscopy and computational modeling. Our analysis reveals that centrosome separation is powered by the joint action of dynein at the nuclear envelope and at the cell cortex. Strikingly, we demonstrate that dynein at the cell cortex acts as a force-transmitting device that harnesses polarized actomyosin cortical flows initiated by the centrosomes earlier in the cell cycle. This mechanism elegantly couples cell polarization with centrosome separation, thus ensuring faithful cell division.

LIN-41 inactivation leads to delayed centrosome elimination and abnormal chromosome behavior during female meiosis in Caenorhabditis elegans.

During oogenesis, two successive meiotic cell divisions occur without functional centrosomes because of the inactivation and subsequent elimination of maternal centrosomes during the diplotene stage of meiosis I. Despite being a conserved phenomenon in most metazoans, the means by which this centrosome behavior is controlled during female meiosis remain elusive. Here, we conducted a targeted RNAi screening in the Caenorhabditis elegans gonad to identify novel regulators of centrosome behavior during oogenesis. We screened 513 genes known to be essential for embryo production and directly visualized GFP-γ-tubulin to monitor centrosome behavior at all stages of oogenesis. In the screening, we found that RNAi-mediated inactivation of 33 genes delayed the elimination of GFP-γ-tubulin at centrosomes during oogenesis, whereas inactivation of nine genes accelerated the process. Depletion of the TRIM-NHL protein LIN-41 led to a significant delay in centrosome elimination and to the separation and reactivation of centrosomes during oogenesis. Upon LIN-41 depletion, meiotic chromosomes were abnormally condensed and pulled toward one of the two spindle poles around late pachytene even though the spindle microtubules emanated from both centrosomes. Overall, our work provides new insights into the regulation of centrosome behavior to ensure critical meiotic events and the generation of intact oocytes.

Nuclear pore protein NUP88 activates anaphase-promoting complex to promote aneuploidy.

The nuclear pore complex protein NUP88 is frequently elevated in aggressive human cancers and correlates with reduced patient survival; however, it is unclear whether and how NUP88 overexpression drives tumorigenesis. Here, we show that mice overexpressing NUP88 are cancer prone and form intestinal tumors. To determine whether overexpression of NUP88 drives tumorigenesis, we engineered transgenic mice with doxycycline-inducible expression of Nup88. Surprisingly, NUP88 overexpression did not alter global nuclear transport, but was a potent inducer of aneuploidy and chromosomal instability. We determined that NUP88 and the nuclear transport factors NUP98 and RAE1 comprise a regulatory network that inhibits premitotic activity of the anaphase-promoting complex/cyclosome (APC/C). When overexpressed, NUP88 sequesters NUP98-RAE1 away from APC/CCDH1, triggering proteolysis of polo-like kinase 1 (PLK1), a tumor suppressor and multitasking mitotic kinase. Premitotic destruction of PLK1 disrupts centrosome separation, causing mitotic spindle asymmetry, merotelic microtubule-kinetochore attachments, lagging chromosomes, and aneuploidy. These effects were replicated by PLK1 insufficiency, indicating that PLK1 is responsible for the mitotic defects associated with NUP88 overexpression. These findings demonstrate that the NUP88-NUP98-RAE1-APC/CCDH1 axis contributes to aneuploidy and suggest that it may be deregulated in the initiating stages of a broad spectrum of human cancers.

Ubiquitin, the centrosome, and chromosome segregation.

For over a century, the abnormal movement or number of centrosomes has been linked with errors of chromosomes distribution in mitosis. While not essential for the formation of the mitotic spindle, the presence and location of centrosomes has a major influence on the manner in which microtubules interact with the kinetochores of replicated sister chromatids and the accuracy with which they migrate to resulting daughter cells. A complex network has evolved to ensure that cells contain the proper number of centrosomes and that their location is optimal for effective attachment of emanating spindle fibers with the kinetochores. The components of this network are regulated through a series of post-translational modifications, including ubiquitin and ubiquitin-like modifiers, which coordinate the timing and strength of signaling events key to the centrosome cycle. In this review, we examine the role of the ubiquitin system in the events relating to centriole duplication and centrosome separation, and discuss how the disruption of these functions impacts chromosome segregation.

Mitotic entry: Non-genetic heterogeneity exposes the requirement for Plk1.

The quest to develop novel antimitotic chemotherapy agents has led to the generation of several small molecule inhibitors targeting Plk1, a protein kinase required for multiple aspects of cell division. Previous studies have shown that upon exposure to Plk1 inhibitors, cells enter mitosis, delay briefly in prophase and then arrest in mitosis due to an inability to undergo centrosome separation. Here, we show that four different classes of Plk1 inhibitor block mitotic entry in several cancer cell lines and non-transformed RPE-1 cells. The proportion of cells that arrest in G2 is cell line and concentration dependent, and is subject to non-genetic heterogeneity. Following inhibitor washout, the G2 block is alleviated and cells enter mitosis but then fail to complete cell division indicating that most Plk1 inhibitors are not fully reversible. An exception is CYC140844; in contrast to five other inhibitors examined here, this novel Plk1 inhibitor is fully reversible. We discuss the implications for developing Plk1 inhibitors as chemotherapy agents and research tools.

Centrosome separation; a careful balancing act

Centrosome separation is critical for formation of a bipolar spindle and the subsequent accurate segregation of chromosomes during mammalian cell mitosis. The multiple mechanisms that regulate this important process are still being elucidated, with separate pathways acting before nuclear envelope breakdown (NEBD) in prophase and post-NEBD in prometaphase. These mechanisms include cortical actin dynamics, multiple molecular motors and microtubule (MT) pushing forces from kinetochores. However, the plus-end-directed kinesin Eg5 is irrefutably the most important player identified so far. The MT-sliding activity of Eg5 is essential for centrosome separation in prometaphase across many species,1 and also plays a role in the less-understood prophase pathway in mammalian cells.2 Treatment of cells with Eg5 inhibitors leads to monopolar spindles and mitotic arrest,3 which has led to much interest in these drugs as potential anti-cancer therapies over the last few years.

Structure, function, and evolution of plant NIMA-related kinases: implication for phosphorylation-dependent microtubule regulation.

Microtubules are highly dynamic structures that control the spatiotemporal pattern of cell growth and division. Microtubule dynamics are regulated by reversible protein phosphorylation involving both protein kinases and phosphatases. Never in mitosis A (NIMA)-related kinases (NEKs) are a family of serine/threonine kinases that regulate microtubule-related mitotic events in fungi and animal cells (e.g. centrosome separation and spindle formation). Although plants contain multiple members of the NEK family, their functions remain elusive. Recent studies revealed that NEK6 of Arabidopsis thaliana regulates cell expansion and morphogenesis through β-tubulin phosphorylation and microtubule destabilization. In addition, plant NEK members participate in organ development and stress responses. The present phylogenetic analysis indicates that plant NEK genes are diverged from a single NEK6-like gene, which may share a common ancestor with other kinases involved in the control of microtubule organization. On the contrary, another mitotic kinase, polo-like kinase, might have been lost during the evolution of land plants. We propose that plant NEK members have acquired novel functions to regulate cell growth, microtubule organization, and stress responses.

Filanesib (ARRY-520) Demonstrates Potent and Rapid Activity in Preclinical Models of MM, Dependent on Bcl-2 Family Expression, and Synergistic with Dexamethasone and IMiDs

Abstract Introduction: Multiple myeloma (MM) is characterized by the presence of complex karyotypes and chromosome instability, suggesting that cell cycle checkpoints are defective. Filanesib (ARRY-520) is a highly selective, targeted inhibitor of kinesin spindle proteins (KSP), which are required to establish mitotic spindle bipolarity, driving centrosome separation. Filanesib monotherapy has demonstrated clinical activity in heavily pretreated MM patients (Lonial et al, ASH 2013). In this work we aimed to explore the preclinical activity of filanesib alone and in combination with IMiDs. Methods: In vitro activity of filanesib alone and in combination with IMiDs (thalidomide, lenalidomide and pomalidomide) was evaluated in MM cell lines by MTT assay and Annexin V, Propidium Iodide and DiOC6 analysis by flow cytometry, Western Blot and immunofluorescence. Synergy was quantified with combination indices (CI) by Calcusyn software. In vivo efficacy was assessed in a subcutaneous plasmacytoma model of MM1S in CB17-SCID mice. Results: Filanesib demonstrated significant activity in a broad panel of 11 MM cell lines, with 48-hour IC50 values ranging between 0.3 and 5 nM. Interestingly, the highest activity was observed in drug-resistant cell lines such as OPM-2 and RPMI-LR5. We next evaluated whether the cell death MoA was dependent on apoptosis or blockade of proliferation. Time response experiments performed in three different cell lines with different sensitivity to filanesib (OPM-2, MM1S and U266) showed accumulation of cells in G2/M, followed by loss of the mitochondrial membrane potential and activation of apoptosis. Accordingly, Western blot analysis demonstrated an activation of the mitotic checkpoint indicated by an increase in Cyclin B1, and activation of apoptosis with PARP, and caspase-3 and -7 cleavage. Furthermore, filanesib activity was very rapid as 15 minutes of exposure was sufficient to exert all of the apoptotic and cell cycle effects observed at 48 hours. Immunofluorescence microscopy using alpha-tubulin demonstrated that filanesib induces monopolar spindle formation. The sensitivity of MM to filanesib has been previously correlated with the cell-dependency of the anti-apoptotic protein Mcl-1. We, therefore, studied the basal levels of six Bcl-2 family members (Bcl-2, Bcl-XL, Mcl-1, Bax, Bak, Bad) in the 11 cell lines and observed a correlation between the basal levels of these proteins and drug sensitivity. In particular, we confirmed the relationship with the anti-apoptotic proteins Mcl-1 and Bcl-2 and demonstrated that conversely to what is observed with proteasome inhibitors, cells with high basal levels of pro-apoptotic Bax or Bak were more resistant to filanesib. Moreover, treatment with filanesib induced a clear decrease of Mcl-1 in the 3 cell lines analyzed (U266, MM1S and OPM-2) that coincided with a decrease of Bcl-2 in the most sensitive cell line, OPM-2. Finally, we evaluated the activity of filanesib in combination with IMiDs and dexamethasone. In vitro studies showed a synergistic effect of filanesib with dexamethasone (CI: 0.21), and with all IMiDs, being most pronounced with pomalidomide (CI: 0.09). Of note, this triple combination demonstrated the highest synergistic activity (CI: 0.06). These results were confirmed in vivo where the triple combination of filanesib, dexamethasone, and pomalidomide was also synergistic, with a significant reduction of tumor growth of up to 50 days, which correlated with a statistically significant survival improvement. Mechanistic studies on the combination are ongoing. Conclusions: Our results demonstrate the potent, rapid activity of filanesib which induces a cell cycle blockade through the inhibition of KSP, leading to apoptosis in MM. It was identified that this activity is dependent on the anti-apoptotic protein Mcl-1 and pro-apoptotic proteins Bax and Bak. Furthermore, in animal xenograft studies, filanesib exhibited robust synergism in combination with dexamethasone and any IMiD, pomalidomide being the most synergistic. These data are the rationale for the clinical trial Pomdefil which uses this combination in patients with relapsed refractory MM. The trial will begin soon as a collaboration of the Spanish Myeloma Group (GEM). This work was supported in part by Array BioPharma. Disclosures Humphries: Array Biopharma: Employment. Tunquist: Array Biopharma: Employment. Mateos: Array Biopharma: Honoraria. Ocio: Array Biopharma: Honoraria, Research Funding.

Chronic Exposure to Particulate Chromate Induces Premature Centrosome Separation and Centriole Disengagement in Human Lung Cells.

Particulate hexavalent chromium (Cr(VI)) is a well-established human lung carcinogen. Lung tumors are characterized by structural and numerical chromosome instability. Centrosome amplification is a phenotype commonly found in solid tumors, including lung tumors, which strongly correlates with chromosome instability. Human lung cells exposed to Cr(VI) exhibit centrosome amplification but the underlying phenotypes and mechanisms remain unknown. In this study, we further characterize the phenotypes of Cr(VI)-induced centrosome abnormalities. We show that Cr(VI)-induced centrosome amplification correlates with numerical chromosome instability. We also show chronic exposure to particulate Cr(VI) induces centrosomes with supernumerary centrioles and acentriolar centrosomes in human lung cells. Moreover, chronic exposure to particulate Cr(VI) affects the timing of important centriolar events. Specifically, chronic exposure to particulate Cr(VI) causes premature centriole disengagement in S and G2 phase cells. It also induces premature centrosome separation in interphase. Altogether, our data suggest that chronic exposure to particulate Cr(VI) targets the protein linkers that hold centrioles together. These centriolar linkers are important for key events of the centrosome cycle and their premature disruption might underlie Cr(VI)-induced centrosome amplification.

Measurement of separase proteolytic activity in single living cells by a fluorogenic flow cytometry assay.

ESPL1/Separase, an endopeptidase, is required for centrosome duplication and separation of sister-chromatides in anaphase of mitosis. Overexpression and deregulated proteolytic activity of Separase as frequently observed in human cancers is associated with the occurrence of supernumerary centrosomes, chromosomal missegregation and aneuploidy. Recently, we have hypothesized that increased Separase proteolytic activity in a small subpopulation of tumor cells may serve as driver of tumor heterogeneity and clonal evolution in chronic myeloid leukemia (CML). Currently, there is no quantitative assay to measure Separase activity levels in single cells. Therefore, we have designed a flow cytometry-based assay that utilizes a Cy5- and rhodamine 110 (Rh110)-biconjugated Rad21 cleavage site peptide ([Cy5-D-R-E-I-M-R]2-Rh110) as smart probe and intracellular substrate for detection of Separase enzyme activity in living cells. As measured by Cy5 fluorescence the cellular uptake of the fluorogenic peptide was fast and reached saturation after 210 min of incubation in human histiocytic lymphoma U937 cells. Separase activity was recorded as the intensity of Rh110 fluorescence released after intracellular peptide cleavage providing a linear signal gain within a 90–180 min time slot. Compared to conventional cell extract-based methods the flow cytometric assay delivers equivalent results but is more reliable, bypasses the problem of vague loading controls and unspecific proteolysis associated with whole cell extracts. Especially suited for the investigaton of blood- and bone marrow-derived hematopoietic cells the flow cytometric Separase assay allows generation of Separase activity profiles that tell about the number of Separase positive cells within a sample i.e. cells that currently progress through mitosis and about the range of intercellular variation in Separase activity levels within a cell population. The assay was used to quantify Separase proteolytic activity in leukemic cell lines and peripheral blood samples from leukemia patients.

Lamins position the nuclear pores and centrosomes by modulating dynein.

Lamins, the type V nuclear intermediate filament proteins, are reported to function in both interphase and mitosis. For example, lamin deletion in various cell types can lead to an uneven distribution of the nuclear pore complexes (NPCs) in the interphase nuclear envelope, whereas deletion of B-type lamins results in spindle orientation defects in mitotic neural progenitor cells. How lamins regulate these functions is unknown. Using mouse cells deleted of different combinations or all lamins, we show that lamins are required to prevent the aggregation of NPCs in the nuclear envelope near centrosomes in late G2 and prophase. This asymmetric NPC distribution in the absence of lamins is caused by dynein forces acting on NPCs via the dynein adaptor BICD2. We further show that asymmetric NPC distribution upon lamin depletion disrupts the distribution of BICD2 and p150 dynactin on the nuclear envelope at prophase, which results in inefficient dynein-driven centrosome separation during prophase. Therefore lamins regulate microtubule-based motor forces in vivo to ensure proper NPC distribution in interphase and centrosome separation in the mitotic prophase.