Critical Role for Cold Shock Protein YB-1 in Cytokinesis.

High levels of the cold shock protein Y-box-binding protein-1, YB-1, are tightly correlated with increased cell proliferation and progression. However, the precise mechanism by which YB-1 regulates proliferation is unknown. Here, we found that YB-1 depletion in several cancer cell lines and in immortalized fibroblasts resulted in cytokinesis failure and consequent multinucleation. Rescue experiments indicated that YB-1 was required for completion of cytokinesis. Using confocal imaging we found that YB-1 was essential for orchestrating the spatio-temporal distribution of the microtubules, ß-actin and the chromosome passenger complex (CPC) to define the cleavage plane. We show that phosphorylation at six serine residues was essential for cytokinesis, of which novel sites were identified using mass spectrometry. Using atomistic modelling we show how phosphorylation at multiple sites alters YB-1 conformation, allowing it to interact with protein partners. Our results establish phosphorylated YB-1 as a critical regulator of cytokinesis, defining precisely how YB-1 regulates cell division.

ALPK1 missense pathogenic variant in five families leads to ROSAH syndrome, an ocular multisystem autosomal dominant disorder.

PURPOSE: To identify the molecular cause in five unrelated families with a distinct autosomal dominant ocular systemic disorder we called ROSAH syndrome due to clinical features of retinal dystrophy, optic nerve edema, splenomegaly, anhidrosis, and migraine headache. METHODS: Independent discovery exome and genome sequencing in families 1, 2, and 3, and confirmation in families 4 and 5. Expression of wild-type messenger RNA and protein in human and mouse tissues and cell lines. Ciliary assays in fibroblasts from affected and unaffected family members. RESULTS: We found the heterozygous missense variant in the ɑ-kinase gene, ALPK1, (c.710C>T, [p.Thr237Met]), segregated with disease in all five families. All patients shared the ROSAH phenotype with additional low-grade ocular inflammation, pancytopenia, recurrent infections, and mild renal impairment in some. ALPK1 was notably expressed in retina, retinal pigment epithelium, and optic nerve, with immunofluorescence indicating localization to the basal body of the connecting cilium of the photoreceptors, and presence in the sweat glands. Immunocytofluorescence revealed expression at the centrioles and spindle poles during metaphase, and at the base of the primary cilium. Affected family member fibroblasts demonstrated defective ciliogenesis. CONCLUSION: Heterozygosity for ALPK1, p.Thr237Met leads to ROSAH syndrome, an autosomal dominant ocular systemic disorder.

Targeting Glioma Stem Cells by Functional Inhibition of Dynamin 2: A Novel Treatment Strategy for Glioblastoma.

Glioma stem cells (GSCs) play major roles in drug resistance, tumour maintenance and recurrence of glioblastoma. We investigated inhibition of the GTPase dynamin 2 as a therapy for glioblastoma. Glioma cell lines and patient-derived GSCs were treated with dynamin inhibitors, Dynole 34-2 and CyDyn 4-36. We studied about cell viability, and GSC neurosphere formation in vitro and orthotopic tumour growth in vivo. Dynamin inhibition reduced glioblastoma cell line viability and suppressed neurosphere formation and migration of GSCs. Tumour growth was reduced by CyDyn 4-36 treatment. Dynamin 2 inhibition therefore represents a novel approach for stem cell-directed Glioblastoma therapy.

Automated classification and characterization of the mitotic spindle following knockdown of a mitosis-related protein.

BACKGROUND: Cell division (mitosis) results in the equal segregation of chromosomes between two daughter cells. The mitotic spindle plays a pivotal role in chromosome alignment and segregation during metaphase and anaphase. Structural or functional errors of this spindle can cause aneuploidy, a hallmark of many cancers. To investigate if a given protein associates with the mitotic spindle and regulates its assembly, stability, or function, fluorescence microscopy can be performed to determine if disruption of that protein induces phenotypes indicative of spindle dysfunction. Importantly, functional disruption of proteins with specific roles during mitosis can lead to cancer cell death by inducing mitotic insult. However, there is a lack of automated computational tools to detect and quantify the effects of such disruption on spindle integrity. RESULTS: We developed the image analysis software tool MatQuantify, which detects both large-scale and subtle structural changes in the spindle or DNA and can be used to statistically compare the effects of different treatments. MatQuantify can quantify various physical properties extracted from fluorescence microscopy images, such as area, lengths of various components, perimeter, eccentricity, fractal dimension, satellite objects and orientation. It can also measure textual properties including entropy, intensities and the standard deviation of intensities. Using MatQuantify, we studied the effect of knocking down the protein clathrin heavy chain (CHC) on the mitotic spindle. We analysed 217 microscopy images of untreated metaphase cells, 172 images of metaphase cells transfected with small interfering RNAs targeting the luciferase gene (as a negative control), and 230 images of metaphase cells depleted of CHC. Using the quantified data, we trained 23 supervised machine learning classification algorithms. The Support Vector Machine learning algorithm was the most accurate method (accuracy: 85.1%; area under the curve: 0.92) for classifying a spindle image. The Kruskal-Wallis and Tukey-Kramer tests demonstrated that solidity, compactness, eccentricity, extent, mean intensity and number of satellite objects (multipolar spindles) significantly differed between CHC-depleted cells and untreated/luciferase-knockdown cells. CONCLUSION: MatQuantify enables automated quantitative analysis of images of mitotic spindles. Using this tool, researchers can unambiguously test if disruption of a protein-of-interest changes metaphase spindle maintenance and thereby affects mitosis.

The Clathrin-dependent Spindle Proteome.

The mitotic spindle is required for chromosome congression and subsequent equal segregation of sister chromatids. These processes involve a complex network of signaling molecules located at the spindle. The endocytic protein, clathrin, has a "moonlighting" role during mitosis, whereby it stabilizes the mitotic spindle. The signaling pathways that clathrin participates in to achieve mitotic spindle stability are unknown. Here, we assessed the mitotic spindle proteome and phosphoproteome in clathrin-depleted cells using quantitative MS/MS (data are available via ProteomeXchange with identifier PXD001603). We report a spindle proteome that consists of 3046 proteins and a spindle phosphoproteome consisting of 5157 phosphosites in 1641 phosphoproteins. Of these, 2908 (95.4%) proteins and 1636 (99.7%) phosphoproteins are known or predicted spindle-associated proteins. Clathrin-depletion from spindles resulted in dysregulation of 121 proteins and perturbed signaling to 47 phosphosites. The majority of these proteins increased in mitotic spindle abundance and six of these were validated by immunofluorescence microscopy. Functional pathway analysis confirmed the reported role of clathrin in mitotic spindle stabilization for chromosome alignment and highlighted possible new mechanisms of clathrin action. The data also revealed a novel second mitotic role for clathrin in bipolar spindle formation.

Pharmacology of novel small-molecule tubulin inhibitors in glioblastoma cells with enhanced EGFR signalling.

We recently reported that CMPD1, originally developed as an inhibitor of MK2 activation, primarily inhibits tubulin polymerisation and induces apoptosis in glioblastoma cells. In the present study we provide detailed pharmacological investigation of CMPD1 analogues with improved molecular properties. We determined their anti-cancer efficacy in glioblastoma cells with enhanced EGFR signalling, as deregulated EGFR often leads to chemoresistance. Eight analogues of CMPD1 with varying lipophilicity and basicity were synthesised and tested for efficacy in the cell viability assay using established glioblastoma cell lines and patient-derived primary glioblastoma cells. The mechanism of action for the most potent analogue 15 was determined using MK2 activation and tubulin polymerisation assays, together with the immunofluorescence analysis of the mitotic spindle formation. Apoptosis was analysed by Annexin V staining, immunoblotting analysis of bcl-2 proteins and PARP cleavage. The apoptotic activity of CMPD1 and analogue 15 was comparable across glioblastoma cell lines regardless of the EGFR status. Primary glioblastoma cells of the classical subtype that are characterized by enhanced EGFR activity were most sensitive to the treatment with CMPD1 and 15. In summary, we present mechanism of action for a novel small molecule tubulin inhibitor, compound 15 that inhibits tubulin polymerisation and mitotic spindle formation, induces degradation of anti-apoptotic bcl-2 proteins and leads to apoptosis of glioblastoma cells. We also demonstrate that the enhanced EGFR activity does not decrease the efficacy of tubulin inhibitors developed in this study.

A Cholesterol-Dependent Endocytic Mechanism Generates Midbody Tubules During Cytokinesis.

Cytokinesis is the final stage of cell division and produces two independent daughter cells. Vesicles derived from internal membrane stores, such as the Golgi, lysosomes, and early and recycling endosomes accumulate at the intracellular bridge (ICB) during cytokinesis. Here, we use electron tomography to show that many ICB vesicles are not independent but connected, forming a newly described ICB vesicular structure - narrow tubules that are often branched. These 'midbody tubules' labelled with horseradish peroxidase (HRP) within 10 min after addition to the surrounding medium demonstrating that they are derived from endocytosis. HRP-labelled vesicles and tubules were observed at the rim of the ICB after only 1 min, suggesting that midbody tubules are likely to be generated by local endocytosis occurring at the ICB rim. Indeed, at least one tubule was open to the extracellular space, indicative of a local origin within the ICB. Inhibition of cholesterol-dependent endocytosis by exposure to methyl-ß-cyclodextrin and filipin reduced formation of HRP-labelled midbody tubules, and induced multinucleation following ICB formation. In contrast, dynamin inhibitors, which block clathrin-mediated endocytosis, induced multinucleation but had no effect on the formation of HRP-labelled midbody tubules. Therefore, our data reveal the existence of a cholesterol-dependent endocytic pathway occurring locally at the ICB, which contributes to the accumulation of vesicles and tubules that contribute to the completion of cytokinesis.

IQGAP1 is associated with nuclear envelope reformation and completion of abscission.

The final stage of mitosis is cytokinesis, which results in 2 independent daughter cells. Cytokinesis has 2 phases: membrane ingression followed by membrane abscission. IQGAP1 is a scaffold protein that interacts with proteins implicated in mitosis, including F-actin, myosin and CaM. IQGAP1 in yeast recruits actin and myosin II filaments to the contractile ring for membrane ingression. In contrast, we show that mammalian IQGAP1 is not required for ingression, but coordinates nuclear pore complex (NPC) reassembly and completion of abscission. Depletion of IQGAP1 disrupts Nup98 and mAb414 nuclear envelope localization and delays abscission timing. IQGAP1 phosphorylation increases 15-fold upon mitotic entry at S86, S330 and T1434, with the latter site being targeted by CDK2/Cyclin A and CDK1/Cyclin A/B in vitro. Expressing the phospho-deficient mutant IQGAP1-S330A impairs NPC reassembly in cells undergoing abscission. Thus, mammalian IQGAP1 functions later in mitosis than its yeast counterpart to regulate nuclear pore assembly in a S330 phosphorylation-dependent manner during the abscission phase of cytokinesis.

Rho GTPases as regulators of mitosis and cytokinesis in mammalian cells.

Rho GTPases regulate a diverse range of cellular functions primarily through their ability to modulate microtubule dynamics and the actin-myosin cytoskeleton. Both of these cytoskeletal structures are crucial for a mitotic cell division. Specifically, their assembly and disassembly is tightly regulated in a temporal manner to ensure that each mitotic stage occurs in the correct sequential order and not prematurely until the previous stage is completed. Thus, it is not surprising that the Rho GTPases, RhoA, and Cdc42, have reported roles in several stages of mitosis: cell cortex stiffening during cell rounding, mitotic spindle formation, and bi-orient attachment of the spindle microtubules to the kinetochore and during cytokinesis play multiple roles in establishing the division plane, assembly, and activation of the contractile ring, membrane ingression, and abscission. Here, I review the molecular mechanisms regulating the spatial and temporal activation of RhoA and Cdc42 during mitosis, and how this is critical for mitotic progression and completion.

Building a better dynasore: the dyngo compounds potently inhibit dynamin and endocytosis.

Dynamin GTPase activity increases when it oligomerizes either into helices in the presence of lipid templates or into rings in the presence of SH3 domain proteins. Dynasore is a dynamin inhibitor of moderate potency (IC50 ~ 15 µM in vitro). We show that dynasore binds stoichiometrically to detergents used for in vitro drug screening, drastically reducing its potency (IC50 = 479 µM) and research tool utility. We synthesized a focused set of dihydroxyl and trihydroxyl dynasore analogs called the Dyngo™ compounds, five of which had improved potency, reduced detergent binding and reduced cytotoxicity, conferred by changes in the position and/or number of hydroxyl substituents. The Dyngo compound 4a was the most potent compound, exhibiting a 37-fold improvement in potency over dynasore for liposome-stimulated helical dynamin activity. In contrast, while dynasore about equally inhibited dynamin assembled in its helical or ring states, 4a and 6a exhibited >36-fold reduced activity against rings, suggesting that they can discriminate between helical or ring oligomerization states. 4a and 6a inhibited dynamin-dependent endocytosis of transferrin in multiple cell types (IC50 of 5.7 and 5.8 µM, respectively), at least sixfold more potently than dynasore, but had no effect on dynamin-independent endocytosis of cholera toxin. 4a also reduced synaptic vesicle endocytosis and activity-dependent bulk endocytosis in cultured neurons and synaptosomes. Overall, 4a and 6a are improved and versatile helical dynamin and endocytosis inhibitors in terms of potency, non-specific binding and cytotoxicity. The data further suggest that the ring oligomerization state of dynamin is not required for clathrin-mediated endocytosis.

Sorting nexin 9 recruits clathrin heavy chain to the mitotic spindle for chromosome alignment and segregation.

Sorting nexin 9 (SNX9) and clathrin heavy chain (CHC) each have roles in mitosis during metaphase. Since the two proteins directly interact for their other cellular function in endocytosis we investigated whether they also interact for metaphase and operate on the same pathway. We report that SNX9 and CHC functionally interact during metaphase in a specific molecular pathway that contributes to stabilization of mitotic spindle kinetochore (K)-fibres for chromosome alignment and segregation. This function is independent of their endocytic role. SNX9 residues in the clathrin-binding low complexity domain are required for CHC association and for targeting both CHC and transforming acidic coiled-coil protein 3 (TACC3) to the mitotic spindle. Mutation of these sites to serine increases the metaphase plate width, indicating inefficient chromosome congression. Therefore SNX9 and CHC function in the same molecular pathway for chromosome alignment and segregation, which is dependent on their direct association.

CDC25B overexpression stabilises centrin 2 and promotes the formation of excess centriolar foci.

CDK-cyclin complexes regulate centriole duplication and microtubule nucleation at specific cell cycle stages, although their exact roles in these processes remain unclear. As the activities of CDK-cyclins are themselves positively regulated by CDC25 phosphatases, we investigated the role of centrosomal CDC25B during interphase. We report that overexpression of CDC25B, as is commonly found in human cancer, results in a significant increase in centrin 2 at the centrosomes of interphase cells. Conversely, CDC25B depletion causes a loss of centrin 2 from the centrosome, which can be rescued by treatment with the proteasome inhibitor MG132. CDC25B overexpression also promotes the formation of excess centrin 2 "foci". These foci can accumulate other centrosome proteins, including γ-tubulin and PCM-1, and can function as microtubule organising centres, indicating that these represent functional centrosomes. Formation of centrin 2 foci can be blocked by specific inhibition of CDK2 but not CDK1. CDK2-mediated phosphorylation of Monopolar spindle 1 (Mps1) at the G1/S transition is essential for the initiation of centrosome duplication, and Mps1 is reported to phosphorylate centrin 2. Overexpression of wild-type or non-degradable Mps1 exacerbated the formation of excess centrin 2 foci induced by CDC25B overexpression, while kinase-dead Mps1 has a protective effect. Together, our data suggest that CDC25B, through activation of a centrosomal pool of CDK2, stabilises the local pool of Mps1 which in turn regulates the level of centrin 2 at the centrosome. Overexpression of CDC25B may therefore contribute to tumourigenesis by perturbing the natural turnover of centrosome proteins such as Mps1 and centrin 2, thus resulting in the de novo assembly of extra-numerary centrosomes and potentiating chromosome instability.

Pyrimidyn compounds: dual-action small molecule pyrimidine-based dynamin inhibitors.

Dynamin is required for clathrin-mediated endocytosis (CME). Its GTPase activity is stimulated by phospholipid binding to its PH domain, which induces helical oligomerization. We have designed a series of novel pyrimidine-based "Pyrimidyn" compounds that inhibit the lipid-stimulated GTPase activity of full length dynamin I and II with similar potency. The most potent analogue, Pyrimidyn 7, has an IC50 of 1.1 µM for dynamin I and 1.8 µM for dynamin II, making it among the most potent dynamin inhibitors identified to date. We investigated the mechanism of action of the Pyrimidyn compounds in detail by examining the kinetics of Pyrimidyn 7 inhibition of dynamin. The compound competitively inhibits both GTP and phospholipid interactions with dynamin I. While both mechanisms of action have been previously observed separately, this is the first inhibitor series to incorporate both and thereby to target two distinct domains of dynamin. Pyrimidyn 6 and 7 reversibly inhibit CME of both transferrin and EGF in a number of non-neuronal cell lines as well as inhibiting synaptic vesicle endocytosis (SVE) in nerve terminals. Therefore, Pyrimidyn compounds block endocytosis by directly competing with GTP and lipid binding to dynamin, limiting both the recruitment of dynamin to membranes and its activation. This dual mode of action provides an important new tool for molecular dissection of dynamin's role in endocytosis.

Inhibition of clathrin by pitstop 2 activates the spindle assembly checkpoint and induces cell death in dividing HeLa cancer cells.

BACKGROUND: During metaphase clathrin stabilises the mitotic spindle kinetochore (K)-fibres. Many anti-mitotic compounds target microtubule dynamics. Pitstop 2™ is the first small molecule inhibitor of clathrin terminal domain and inhibits clathrin-mediated endocytosis. We investigated its effects on a second function for clathrin in mitosis. RESULTS: Pitstop 2 did not impair clathrin recruitment to the spindle but disrupted its function once stationed there. Pitstop 2 trapped HeLa cells in metaphase through loss of mitotic spindle integrity and activation of the spindle assembly checkpoint, phenocopying clathrin depletion and aurora A kinase inhibition. CONCLUSIONS: Pitstop 2 is therefore a new tool for investigating clathrin spindle dynamics. Pitstop 2 reduced viability in dividing HeLa cells, without affecting dividing non-cancerous NIH3T3 cells, suggesting that clathrin is a possible novel anti-mitotic drug target.

Development of second-generation indole-based dynamin GTPase inhibitors.

Focused library development of our lead 2-cyano-3-(1-(3-(dimethylamino)propyl)-2-methyl-1H-indol-3-yl)-N-octylacrylamide (2) confirmed the tertiary dimethylamino-propyl moiety as critical for inhibition of dynamin GTPase. The cyanoamide moiety could be replaced with a thiazole-4(5H)-one isostere (19, IC(50(dyn I)) = 7.7 µM), reduced under flow chemistry conditions (20, IC(50(dyn I)) = 5.2 µM) or replaced by a simple amine. The latter provided a basis for a high yield library of compounds via a reductive amination by flow hydrogenation. Two compounds, 24 (IC(50 (dyn I)) = 0.56 µM) and 25 (IC(50(dyn I)) = 0.76 µM), stood out. Indole 24 is nontoxic and showed increased potency against dynamin I and II in vitro and in cells (IC(50(CME)) = 1.9 µM). It also showed 4.4-fold selectivity for dynamin I. The indole 24 compound has improved isoform selectivity and is the most active in-cell inhibitor of clathrin-mediated endocytosis reported to date.

Clathrin-mediated endocytic proteins are involved in regulating mitotic progression and completion.

A few proteins required for clathrin-mediated endocytosis (CME) are associated with successful completion of mitosis at distinct mitotic stages. Clathrin heavy chain (CHC) and epsin are required for chromosome segregation independent of their CME function and dynamin II (dynII) functions in the abscission stage of cytokinesis. In this study we screened for mitotic roles of eight CME proteins: CHC, α-adaptin, CALM, epsin, eps15, endophilin II (edpnII), syndapin II (sdpnII) and the GTPase dynII using a small interfering RNA targeting approach. All proteins, except for CALM, are associated with completion of the abscission stage of cytokinesis, suggesting that they function in this process in an endocytic-dependent manner. In support of this concept, overexpression of epsin(S357D), which blocks endocytosis, induced multinucleation. Moreover, six of them have a secondary role at earlier mitotic stages that is not dependent on their endocytic function: CHC, epsin and eps15 in chromosome segregation, and sdpnII, α-adaptin and CALM have a role in furrow ingression. Therefore, the role of endocytic proteins in mitosis is much broader than previously recognized.

SNX9, SNX18 and SNX33 are required for progression through and completion of mitosis.

Mitosis involves considerable membrane remodelling and vesicular trafficking to generate two independent cells. Consequently, endocytosis and endocytic proteins are required for efficient mitotic progression and completion. Several endocytic proteins also participate in mitosis in an endocytosis-independent manner. Here, we report that the sorting nexin 9 (SNX9) subfamily members - SNX9, SNX18 and SNX33 - are required for progression and completion of mitosis. Depletion of any one of these proteins using siRNA induces multinucleation, an indicator of cytokinesis failure, as well as an accumulation of cytokinetic cells. Time-lapse microscopy on siRNA-treated cells revealed a role for SNX9 subfamily members in progression through the ingression and abscission stages of cytokinesis. Depletion of these three proteins disrupted MRLC(S19) localization during ingression and recruitment of Rab11-positive recycling endosomes to the intracellular bridge between nascent daughter cells. SNX9 depletion also disrupted the localization of Golgi during cytokinesis. Endocytosis of transferrin was blocked during cytokinesis by depletion of the SNX9 subfamily members, suggesting that these proteins participate in cytokinesis in an endocytosis-dependent manner. In contrast, depletion of SNX9 did not block transferrin uptake during metaphase but did delay chromosome alignment and segregation, suggesting that SNX9 plays an additional non-endocytic role at early mitotic stages. We conclude that SNX9 subfamily members are required for mitosis through both endocytosis-dependent and -independent processes.

Role of the clathrin terminal domain in regulating coated pit dynamics revealed by small molecule inhibition.

Clathrin-mediated endocytosis (CME) regulates many cell physiological processes such as the internalization of growth factors and receptors, entry of pathogens, and synaptic transmission. Within the endocytic network, clathrin functions as a central organizing platform for coated pit assembly and dissociation via its terminal domain (TD). We report the design and synthesis of two compounds named pitstops that selectively block endocytic ligand association with the clathrin TD as confirmed by X-ray crystallography. Pitstop-induced inhibition of clathrin TD function acutely interferes with receptor-mediated endocytosis, entry of HIV, and synaptic vesicle recycling. Endocytosis inhibition is caused by a dramatic increase in the lifetimes of clathrin coat components, including FCHo, clathrin, and dynamin, suggesting that the clathrin TD regulates coated pit dynamics. Pitstops provide new tools to address clathrin function in cell physiology with potential applications as inhibitors of virus and pathogen entry and as modulators of cell signaling.

Inhibition of dynamin by dynole 34-2 induces cell death following cytokinesis failure in cancer cells.

Inhibitors of mitotic proteins such as Aurora kinase and polo-like kinase have shown promise in preclinical or early clinical development for cancer treatment. We have reported that the MiTMAB class of dynamin small molecule inhibitors are new antimitotic agents with a novel mechanism of action, blocking cytokinesis. Here, we examined 5 of the most potent of a new series of dynamin GTPase inhibitors called dynoles. They all induced cytokinesis failure at the point of abscission, consistent with inhibition of dynamin while not affecting other cell cycle stages. All 5 dynoles inhibited cell proliferation (MTT and colony formation assays) in 11 cancer cell lines. The most potent GTPase inhibitor, dynole 34-2, also induced apoptosis, as revealed by cell blebbing, DNA fragmentation, and PARP cleavage. Cell death was induced specifically following cytokinesis failure, suggesting that dynole 34-2 selectively targets dividing cells. Dividing HeLa cells were more sensitive to the antiproliferative properties of all 5 dynoles compared with nondividing cells, and nontumorigenic fibroblasts were less sensitive to cell death induced by dynole 34-2. Thus, the dynoles are a second class of dynamin GTPase inhibitors, with dynole 34-2 as the lead compound, that are novel antimitotic compounds acting specifically at the abscission stage.