Anti-gamma Tubulin Antibody [TU-30] (A86254)

Background: Centrosome amplification (CA) has been reported in nearly all types of human cancer and is associated with deleterious clinical factors such as higher grade and stage. However, previous reports have not shown how CA affects cellular differentiation and clinical outcomes in breast cancer.

Methods: We analyzed centrosomes by immunofluorescence and compared to ploidy and chromosomal instability (CIN) as assessed by 6-chromosome FISH in a cohort of 362 breast cancers with median clinical follow-up of 8.4 years. Centrosomes were recognized by immunofluorescence using antibodies for pericentriolar material (PCM; pericentrin) and centrioles (polyglutamylated tubulin). CA was experimentally induced in cell culture by overexpression of polo-like kinase 4 (PLK4).

Results: CA is associated with reduced all-cause and breast cancer-specific overall survival and recurrence-free survival. CA correlates strongly with high-risk subtypes (e.g. triple negative) and higher stage and grade, and the prognostic nature of CA can be explained largely by these factors. A strong correlation between CA and high tumor ploidy demonstrates that chromosome and centrosome doubling often occur in concert. CA is proposed to be a method of inducing CIN via aberrant mitotic cell divisions; consonant with this, we observed a strong correlation between CA and CIN in breast cancers. However, some CA tumors had low levels of CIN, indicating that protective mechanisms are at play, such as centrosome clustering during mitosis. Intriguingly, some high-risk tumors have more acentriolar centrosomes, suggesting PCM fragmentation as another mechanism of CA. In vitro induction of CA in two non-transformed human cell lines (MCF10A and RPE) demonstrated that CA induces a de-differentiated cellular state and features of high-grade malignancy, supporting the idea that CA intrinsically causes high-grade tumors.

Conclusions: CA is associated with deleterious clinical factors and outcomes in breast cancer. Cell doubling events are the most prevalent causes of CA in cancer, although PCM fragmentation may be a secondary cause. CA promotes high-risk breast cancer in part by inducing high-grade features. These findings highlight the importance of centrosome aberrations in the biology of human breast cancer.

The cytosolic role of ?-tubulin as a microtubule organizer has been studied thoroughly, but its nuclear function is poorly understood. Here, we show that ?-tubulin is located throughout the chromatin of demembranated Xenopus laevis sperm and, as the nucleus is formed, ?-tubulin recruits lamin B3 and nuclear membranes. Immunodepletion of ?-tubulin impairs X. laevis assembly of both the lamina and the nuclear membrane. During nuclear formation in mammalian cell lines, ?-tubulin establishes a cellular protein boundary around chromatin that coordinates nuclear assembly of the daughter nuclei. Furthermore, expression of a ?-tubulin mutant that lacks the DNA-binding domain forms chromatin-empty nuclear like structures and demonstrate that a constant interplay between the chromatin-associated and the cytosolic pools of ?-tubulin is required and, when the balance between pools is impaired, aberrant nuclei are formed. We therefore propose that the nuclear protein meshwork formed by ?-tubulin around chromatin coordinates nuclear formation in eukaryotic cells.

The cell tightly controls histone protein levels in order to achieve proper packaging of the genome into chromatin, while avoiding the deleterious consequences of excess free histones. Our accompanying study has shown that a histone modification that loosens the intrinsic structure of the nucleosome, phosphorylation of histone H3 on threonine 118 (H3 T118ph), exists on centromeres and chromosome arms during mitosis. Here, we show that H3 T118ph localizes to centrosomes in humans, flies, and worms during all stages of mitosis. H3 abundance at the centrosome increased upon proteasome inhibition, suggesting that excess free histone H3 localizes to centrosomes for degradation during mitosis. In agreement, we find ubiquitinated H3 specifically during mitosis and within purified centrosomes. These results suggest that targeting of histone H3 to the centrosome for proteasome-mediated degradation is a novel pathway for controlling histone supply, specifically during mitosis.

CDK10/CycM is a protein kinase deficient in STAR (toe Syndactyly, Telecanthus and Anogenital and Renal malformations) syndrome, which results from mutations in the X-linked FAM58A gene encoding Cyclin M. The biological functions of CDK10/CycM and etiology of STAR syndrome are poorly understood. Here, we report that deficiency of CDK10/Cyclin M promotes assembly and elongation of primary cilia. We establish that this reflects a key role for CDK10/Cyclin M in regulation of actin network organization, which is known to govern ciliogenesis. In an unbiased screen, we identified the RhoA-associated kinase PKN2 as a CDK10/CycM phosphorylation substrate. We establish that PKN2 is a bone fide regulator of ciliogenesis, acting in a similar manner to CDK10/CycM. We discovered that CDK10/Cyclin M binds and phosphorylates PKN2 on threonines 121 and 124, within PKN2's core RhoA-binding domain. Furthermore, we demonstrate that deficiencies in CDK10/CycM or PKN2, or expression of a non-phosphorylatable version of PKN2, destabilize both the RhoA protein and the actin network architecture. Importantly, we established that ectopic expression of RhoA is sufficient to override the induction of ciliogenesis resulting from CDK10/CycM knockdown, indicating that RhoA regulation is critical for CDK10/CycM's negative effect on ciliogenesis. Finally, we show that kidney sections from a STAR patient display dilated renal tubules and abnormal, elongated cilia. Altogether, these results reveal CDK10/CycM as a key regulator of actin dynamics and a suppressor of ciliogenesis through phosphorylation of PKN2 and promotion of RhoA signaling. Moreover, they suggest that STAR syndrome is a ciliopathy.

Phosphorylation of histone H3 threonine 118 (H3 T118ph) weakens histone DNA-contacts, disrupting the nucleosome structure. We show that Aurora-A mediated H3 T118ph occurs at pericentromeres and chromosome arms during prophase and is lost upon chromosome alignment. Expression of H3 T118E or H3 T118I (a SIN mutation that bypasses the need for the ATP-dependent nucleosome remodeler SWI/SNF) leads to mitotic problems including defects in spindle attachment, delayed cytokinesis, reduced chromatin packaging, cohesion loss, cohesin and condensin I loss in human cells. In agreement, overexpression of Aurora-A leads to increased H3 T118ph levels, causing cohesion loss, and reduced levels of cohesin and condensin I on chromatin. Normal levels of H3 T118ph are important because it is required for development in fruit flies. We propose that H3 T118ph alters the chromatin structure during specific phases of mitosis to promote timely condensin I and cohesin disassociation, which is essential for effective chromosome segregation.

A major etiological risk factor for hepatocellular carcinoma (HCC) is infection by Hepatitis viruses, especially hepatitis B virus and hepatitis C virus. Hepatitis B virus and hepatitis C virus do not cause aggressive activation of an oncogenic pathway, but they transactivate a broad array of genes, cause chronic inflammation, and, through interference with mitotic processes, lead to mitotic error-induced chromosome instability (ME-CIN). However, how ME-CIN is involved in the development of HCC remains unclear. Delineating the effect of ME-CIN on HCC development should help in identifying measures to combat HCC. In this study, we used ME-CIN model mice haploinsufficient in Shugoshin 1 (Sgo1(-/+)) to assess the role of ME-CIN in HCC development. Treatment with the carcinogen azoxymethane caused Sgo1(-/+) ME-CIN model mice to develop HCCs within 6 months, whereas control mice developed no HCC (P < 0.003). The HCC development was associated with expression of early HCC markers (glutamine synthetase, glypican 3, heat shock protein 70, and the serum marker alpha fetoprotein), although without fibrosis. ME-CIN preceded the expression of HCC markers, suggesting that ME-CIN is an important early event in HCC development. In 12-month-old untreated Sgo1 mice, persistent DNA damage, altered gene expression, and spontaneous HCCs were observed. Sgo1 protein accumulated in response to DNA damage in vitro. Overall, Sgo1(-/+)-mediated ME-CIN strongly promoted/progressed development of HCC in the presence of an initiator carcinogen, and it had a mild initiator effect by itself. Use of the ME-CIN model mice should help in identifying drugs to counteract the effects of ME-CIN and should accelerate anti-HCC drug development.

Retinal pigment epithelium (RPE) degeneration underpins diseases triggered by disparate genetic lesions, noxious insults, or both. The pleiotropic Ranbp2 controls the expression of intrinsic and extrinsic pathological stressors impinging on cellular viability. However, the physiological targets and mechanisms controlled by Ranbp2 in tissue homeostasis, such as RPE, are ill defined. We show that mice, RPE-cre::Ranbp2(-/-), with selective Ranbp2 ablation in RPE develop pigmentary changes, syncytia, hypoplasia, age-dependent centrifugal and non-apoptotic degeneration of the RPE, and secondary leakage of choriocapillaris. These manifestations are accompanied by the development of F-actin clouds, metalloproteinase-11 activation, deregulation of expression or subcellular localization of critical RPE proteins, atrophic cell extrusions into the subretinal space, and compensatory proliferation of peripheral RPE. To gain mechanistic insights into what Ranbp2 activities are vital to the RPE, we performed genetic complementation analyses of transgenic lines of bacterial artificial chromosomes of Ranbp2 harboring loss of function of selective Ranbp2 domains expressed in a Ranbp2(-/-) background. Among the transgenic lines produced, only Tg(RBD2/3*-HA)::RPE-cre::Ranbp2(-/-)-expressing mutations, which selectively impair binding of RBD2/3 (Ran-binding domains 2 and 3) of Ranbp2 to Ran-GTP, recapitulate RPE degeneration, as observed with RPE-cre::Ranbp2(-/-). By contrast, Tg(RBD2/3*-HA) expression rescues the degeneration of cone photoreceptors lacking Ranbp2. The RPE of RPE-cre::Ranbp2(-/-) and Tg(RBD2/3*-HA)::RPE-cre::Ranbp2(-/-) share proteostatic deregulation of Ran GTPase, serotransferrin, and ?-tubulin and suppression of light-evoked electrophysiological responses. These studies unravel selective roles of Ranbp2 and its RBD2 and RBD3 in RPE survival and functions. We posit that the control of Ran GTPase by Ranbp2 emerges as a novel therapeutic target in diseases promoting RPE degeneration.

Although microtubules (MTs) frequently form highly dynamic networks, subsets of MTs become stabilized in response to environmental cues and function as specialized tracks for vesicle and macromolecular trafficking. MT stabilization is controlled by specialized plus-end tracking proteins (+TIPs) whose accumulation at the MT ends is facilitated by the end-binding protein, EB1, and regulated by various signaling pathways. As cargoes themselves, viruses are dependent on MTs for their intracellular movement. Although many viruses affect MT organization, the potential contribution of MT stabilization by +TIPs to infection remains unknown. Here we show that early in infection of primary human fibroblasts, herpes simplex virus type 1 (HSV-1) disrupts the centrosome, the primary MT organizing center in many cell types. As infection progresses HSV-1 induces the formation of stable MT subsets through inactivation of glycogen synthase kinase 3beta by the viral Ser/Thr kinase, Us3. Stable MT formation is reduced in cells infected with Us3 mutants and those stable MTs that form cluster around the trans-Golgi network. Downstream of glycogen synthase kinase 3beta, cytoplasmic linker-associated proteins (CLASPs), specialized host +TIPs that control MT formation at the trans-Golgi network and cortical capture, are specifically required for virus-induced MT stabilization and HSV-1 spread. Our findings demonstrate the biological importance of +TIPs to viral infection and suggest that HSV-1 has evolved to exploit the trans-Golgi network as an alternate MT organizing center to facilitate virus spread.

We investigated three families whose offspring had extreme microcephaly at birth and profound mental retardation. Brain scans and postmortem data showed that affected individuals had brains less than 10% of expected size (=10 standard deviation) and that in addition to a massive reduction in neuron production they displayed partially deficient cortical lamination (microlissencephaly). Other body systems were apparently unaffected and overall growth was normal. We found two distinct homozygous mutations of NDE1, c.83+1G>T (p.Ala29GlnfsX114) in a Turkish family and c.684_685del (p.Pro229TrpfsX85) in two families of Pakistani origin. Using patient cells, we found that c.83+1G>T led to the use of a novel splice site and to a frameshift after NDE1 exon 2. Transfection of tagged NDE1 constructs showed that the c.684_685del mutation resulted in a NDE1 that was unable to localize to the centrosome. By staining a patient-derived cell line that carried the c.83+1G>T mutation, we found that this endogeneously expressed mutated protein equally failed to localize to the centrosome. By examining human and mouse embryonic brains, we determined that NDE1 is highly expressed in neuroepithelial cells of the developing cerebral cortex, particularly at the centrosome. We show that NDE1 accumulates on the mitotic spindle of apical neural precursors in early neurogenesis. Thus, NDE1 deficiency causes both a severe failure of neurogenesis and a deficiency in cortical lamination. Our data further highlight the importance of the centrosome in multiple aspects of neurodevelopment.

Autosomal recessive primary microcephaly (MCPH) is a disorder of neurodevelopment resulting in a small brain. We identified WDR62 as the second most common cause of MCPH after finding homozygous missense and frame-shifting mutations in seven MCPH families. In human cell lines, we found that WDR62 is a spindle pole protein, as are ASPM and STIL, the MCPH7 and MCHP7 proteins. Mutant WDR62 proteins failed to localize to the mitotic spindle pole. In human and mouse embryonic brain, we found that WDR62 expression was restricted to neural precursors undergoing mitosis. These data lend support to the hypothesis that the exquisite control of the cleavage furrow orientation in mammalian neural precursor cell mitosis, controlled in great part by the centrosomes and spindle poles, is critical both in causing MCPH when perturbed and, when modulated, generating the evolutionarily enlarged human brain.

Genes associated with human microcephaly, a condition characterized by a small brain, include critical regulators of proliferation, cell fate and DNA repair. We describe a syndrome of congenital microcephaly and diverse defects in cerebral cortical architecture. Genome-wide linkage analysis in two families identified a 7.5-Mb locus on chromosome 19q13.12 containing 148 genes. Targeted high throughput sequence analysis of linked genes in each family yielded > 4,000 DNA variants and implicated a single gene, WDR62, as harboring potentially deleterious changes. We subsequently identified additional WDR62 mutations in four other families. Magnetic resonance imaging and postmortem brain analysis supports important roles for WDR62 in the proliferation and migration of neuronal precursors. WDR62 is a WD40 repeat-containing protein expressed in neuronal precursors as well as in postmitotic neurons in the developing brain and localizes to the spindle poles of dividing cells. The diverse phenotypes of WDR62 suggest it has central roles in many aspects of cerebral cortical development.

Centrosomes have recently emerged as key regulators of the cell cycle. The G1/S transition requires a functional centrosome, and centrosomal localization of numerous proteins, including cyclin/Cdk complexes, is important for the G2/M transition. Here we identify a modular centrosomal localization signal (CLS) localizing cyclin A to centrosomes independently of Cdk binding. The cyclin A CLS is located in a distinct part of the molecule compared with the cyclin E CLS and includes the MRAIL hydrophobic patch involved in substrate recognition. The cyclin A CLS interacts with p27(KIP1), and expression of p27(KIP1) removes cyclin A but not cyclin E from centrosomes. Expression of the cyclin A CLS displaces both endogenous cyclin A and E from centrosomes and inhibits DNA replication, supporting an emerging concept that DNA replication is linked to centrosomal events. Structural analysis indicates that differences in surface charge and length of the C-terminal helix explain why the MRAIL region in cyclin E is not a functional CLS. These results indicate that the cyclin A CLS may contribute to targeting and recognition of centrosomal Cdk substrates and is required for specific effects of p27(KIP1) on cyclin A-Cdk2.

We have previously shown that the neuronal-associated class III beta-tubulin isotype and the centrosome-associated gamma-tubulin are aberrantly expressed in astrocytic gliomas (Cell Motil Cytoskeleton 2003, 55:77-96; J Neuropathol Exp Neurol 2006, 65:455-467). Here we determined the expression, distribution and interaction of betaIII-tubulin and gamma-tubulin in diffuse-type astrocytic gliomas (grades II-IV) (n = 17) and the human glioblastoma cell line T98G. By immunohistochemistry and immunofluorescence microscopy, betaIII-tubulin and gamma-tubulin were co-distributed in anaplastic astrocytomas and glioblastomas and to a lesser extent, in low-grade diffuse astrocytomas (P < 0.05). In T98G glioblastoma cells betaIII-tubulin was associated with microtubules whereas gamma-tubulin exhibited striking diffuse cytoplasmic staining in addition to its expectant centrosome-associated pericentriolar distribution. Treatment with different anti-microtubule drugs revealed that betaIII-tubulin was not associated with insoluble gamma-tubulin aggregates. On the other hand, immunoprecipitation experiments unveiled that both tubulins formed complexes in soluble cytoplasmic pools, where substantial amounts of these proteins were located. We suggest that aberrant expression and interactions of betaIII-tubulin and gamma-tubulin may be linked to malignant changes in glial cells.

The mechanism of marginal band (MB) formation in differentiating erythroid cells is not fully understood, and the proteins involved in nucleation of MB microtubules are largely unknown. To gain insights into the function of gamma-tubulin in MB formation, we have followed its distribution in developing chicken erythrocytes and characterized soluble forms of the protein. In early stages of erythroid cells differentiation, gamma-tubulin was present in microtubule-organizing centers, mitotic spindles, as well as on MB. Its subcellular localization changed in the course of differentiation, and in postnatal peripheral erythrocytes gamma-tubulin was found only in soluble forms. After cold-induced depolymerization gamma-tubulin in erythroid cells formed large clusters that were not observed in matured cells, and re-growth experiments demonstrated that gamma-tubulin was not present in distinct nucleation structures at the cell periphery. Soluble gamma-tubulin formed complexes of various size and large complexes were prone to dissociation in the presence of high salt concentration. Interaction of gamma-tubulin with tubulin dimers was revealed by precipitation experiments. gamma-Tubulin occurred in multiple charge variants whose number increased in the course of erythrocyte differentiation and corresponded with decreased binding to MB. The presented data demonstrate for the first time that gamma-tubulin is a substrate for developmentally regulated posttranslational modifications and that the binding properties of gamma-tubulin or its complexes change during differentiation events.

Neither the molecular mechanism by which plant microtubules nucleate in the cytoplasm nor the organization of plant mitotic spindles, which lack centrosomes, is well understood. Here, using immunolocalization and cell fractionation techniques, we provide evidence that gamma-tubulin, a universal component of microtubule organizing centers, is present in both the cytoplasm and the nucleus of plant cells. The amount of gamma-tubulin in nuclei increased during the G(2) phase, when cells are synchronized or sorted for particular phases of the cell cycle. gamma-Tubulin appeared on prekinetochores before preprophase arrest caused by inhibition of the cyclin-dependent kinase and before prekinetochore labeling of the mitosis-specific phosphoepitope MPM2. The association of nuclear gamma-tubulin with chromatin displayed moderately strong affinity, as shown by its release after DNase treatment and by using extraction experiments. Subcellular compartmentalization of gamma-tubulin might be an important factor in the organization of plant-specific microtubule arrays and acentriolar mitotic spindles.

The negative-sense RNA genome of influenza A virus is transcribed and replicated by the viral RNA-dependent RNA polymerase (RdRP). The viral RdRP is an important host range determinant, indicating that its function is affected by interactions with cellular factors. However, the identities and the roles of most of these factors remain unknown. Here, we employed affinity purification followed by mass spectrometry to identify cellular proteins that interact with the influenza A virus RdRP in infected human cells. We purified RdRPs using a recombinant influenza virus in which the PB2 subunit of the RdRP is fused to a Strep-tag. When this tagged subunit was purified from infected cells, copurifying proteins included the other RdRP subunits (PB1 and PA) and the viral nucleoprotein and neuraminidase, as well as 171 cellular proteins. Label-free quantitative mass spectrometry revealed that the most abundant of these host proteins were chaperones, cytoskeletal proteins, importins, proteins involved in ubiquitination, kinases and phosphatases, and mitochondrial and ribosomal proteins. Among the phosphatases, we identified three subunits of the cellular serine/threonine protein phosphatase 6 (PP6), including the catalytic subunit PPP6C and regulatory subunits PPP6R1 and PPP6R3. PP6 was found to interact directly with the PB1 and PB2 subunits of the viral RdRP, and small interfering RNA (siRNA)-mediated knockdown of the catalytic subunit of PP6 in infected cells resulted in the reduction of viral RNA accumulation and the attenuation of virus growth. These results suggest that PP6 interacts with and positively regulates the activity of the influenza virus RdRP.

IMPORTANCE:

Influenza A viruses are serious clinical and veterinary pathogens, causing substantial health and economic impacts. In addition to annual seasonal epidemics, occasional global pandemics occur when viral strains adapt to humans from other species. To replicate efficiently and cause disease, influenza viruses must interact with a large number of host factors. The reliance of the viral RNA-dependent RNA polymerase (RdRP) on host factors makes it a major host range determinant. This study describes and quantifies host proteins that interact, directly or indirectly, with a subunit of the RdRP. It increases our understanding of the role of host proteins in viral replication and identifies a large number of potential barriers to pandemic emergence. Identifying host factors allows their importance for viral replication to be tested. Here, we demonstrate a role for the cellular phosphatase PP6 in promoting viral replication, contributing to our emerging knowledge of regulatory phosphorylation in influenza virus biology.

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