Biomedical subjects

Stephen J Doxsey

Publications and source records attributed to Stephen J Doxsey.

11 recordsLinked to original sources

PubMed2006

The DNA helicase ChlR1 is required for sister chromatid cohesion in mammalian cells.

It has recently been suggested that the Saccharomyces cerevisiae protein Chl1p plays a role in cohesion establishment. Here, we show that the human ATP-dependent DNA helicase ChlR1 is required for sister chromatid cohesion in mammalian cells. Localization studies show that ChlR1 diffusely coats mitotic chromatin in prophase and then translocates from the chromatids to concentrate at the spindle poles during the transition to metaphase. Depletion of ChlR1 protein by RNA interference results in mitotic failure with replicated chromosomes failing to segregate after a pro-metaphase arrest. We show that depletion also results in abnormal sister chromatid cohesion, determined by increased separation of chromatid pairs at the centromere. Furthermore, biochemical studies show that ChlR1 is in complex with cohesin factors Scc1, Smc1 and Smc3. We conclude that human ChlR1 is required for sister chromatid cohesion and, hence, normal mitotic progression. These functions are important to maintain genetic fidelity.

Animals↗

PubMed2006

Survivin modulates microtubule dynamics and nucleation throughout the cell cycle.

Survivin is a member of the chromosomal passenger complex implicated in kinetochore attachment, bipolar spindle formation, and cytokinesis. However, the mechanism by which survivin modulates these processes is unknown. Here, we show by time-lapse imaging of cells expressing either green fluorescent protein (GFP)-alpha-tubulin or the microtubule plus-end binding protein GFP-EB1 that depletion of survivin by small interfering RNAs (siRNAs) increased both the number of microtubules nucleated by centrosomes and the incidence of microtubule catastrophe, the transition from microtubule growth to shrinking. In contrast, survivin overexpression reduced centrosomal microtubule nucleation and suppressed both microtubule dynamics in mitotic spindles and bidirectional growth of microtubules in midbodies during cytokinesis. siRNA depletion or pharmacologic inhibition of another chromosomal passenger protein Aurora B, had no effect on microtubule dynamics or nucleation in interphase or mitotic cells even though mitosis was impaired. We propose a model in which survivin modulates several mitotic events, including spindle and interphase microtubule organization, the spindle assembly checkpoint and cytokinesis through its ability to modulate microtubule nucleation and dynamics. This pathway may affect the microtubule-dependent generation of aneuploidy and defects in cell polarity in cancer cells, where survivin is commonly up-regulated.

Animals↗

PubMed2006

Microtubule-dependent nuclear-cytoplasmic shuttling of Runx2.

RUNX/AML transcription factors are critical regulators of cell growth and differentiation in multiple lineages and have been linked to human cancers including acute myelogenous leukemia (RUNX1), as well as breast (RUNX2) and gastric cancers (RUNX3). RUNX proteins are targeted to gene regulatory micro-environments within the nucleus via a specific subnuclear targeting signal. However, the dynamics of RUNX distribution and compartmentalization between the cytoplasm and nucleus is minimally understood. Here we show by immunofluorescence microscopy that RUNX2 relocates from the nucleus to the cytoplasm when microtubules are stabilized by the chemotherapeutic agent taxol. The taxol-dependent cytoplasmic accumulation of RUNX2 is inhibited by leptomycin B, which blocks CRM-1 dependent nuclear export, and is not affected by the protein synthesis inhibitor cycloheximide. Using biochemical assays, we show that endogenous RUNX2 associates with stabilized microtubules in a concentration-dependent manner and that the RUNX2 amino terminus mediates the microtubule association. In soluble fractions of cells, RUNX2 co-immunoprecipitates alpha tubulin suggesting that microtubule binding involves the alpha/beta tubulin subunits. We conclude that RUNX2 associates with microtubules and shuttles between the nucleus and the cytoplasm. We propose that nuclear-cytoplasmic shuttling of RUNX2 may modulate its transcriptional activity, as well as its ability to interface with signal transduction pathways that are integrated at RUNX2 containing subnuclear sites. It is possible that taxol-induced acute depletion of the nuclear levels of RUNX2 and/or other cell growth regulatory factors may represent an alternative pathway by which taxol exerts its biological effects during cancer chemotherapies.

Active Transport, Cell Nucleus↗

PubMed2005

Molecular links between centrosome and midbody.

The terminal step in cytokinesis that severs a cell in two-abscission-is poorly understood. In Developmental Cell, Fabbro et al (2005) identify a centrosome protein whose multiple phosphorylations regulate its movement from centrosome to midbody and completion of abscission.

Animals↗

PubMed2005

Centrosomal pericentrin is a direct cleavage target of membrane type-1 matrix metalloproteinase in humans but not in mice: potential implications for tumorigenesis.

Membrane type-1 matrix metalloproteinase (MT1-MMP) exhibits distinctive and important pericellular cleavage functions. Recently, we determined that MT1-MMP was trafficked to the centrosomes in the course of endocytosis. Our data suggested that the functionally important, integral, centrosomal protein, pericentrin-2, was a cleavage target of MT1-MMP in human and in canine cells and that the sequence of the cleavage sites were ALRRLLG1156 downward arrow L1157FG and ALRRLLS2068 downward arrow L2069FG, respectively. The presence of Asp-948 at the P1 position inactivated the corresponding site (ALRRLLD948-L949FGD) in murine pericentrin. To confirm that MT1-MMP itself cleaves pericentrin directly, rather than indirectly, we analyzed the cleavage of the peptides that span the MT1-MMP cleavage site. In addition, we analyzed glioma U251 cells, which co-expressed MT1-MMP with the wild type murine pericentrin and the D948G mutant. We determined that the D948G mutant that exhibited the cleavage sequence of human pericentrin was sensitive to MT1-MMP, whereas unmodified murine pericentrin was resistant to proteolysis. Taken together, our results confirm that MT1-MMP cleaves pericentrin-2 in humans but not in mice and that mouse models of cancer probably cannot be used to critically examine MT1-MMP functionality.

Animals↗

PubMed2005

Centriolin anchoring of exocyst and SNARE complexes at the midbody is required for secretory-vesicle-mediated abscission.

The terminal step in cytokinesis, called abscission, requires resolution of the membrane connection between two prospective daughter cells. Our previous studies demonstrated that the coiled-coil protein centriolin localized to the midbody during cytokinesis and was required for abscission. Here we show that centriolin interacts with proteins of vesicle-targeting exocyst complexes and vesicle-fusion SNARE complexes. These complexes require centriolin for localization to a unique midbody-ring structure, and disruption of either complex inhibits abscission. Exocyst disruption induces accumulation of v-SNARE-containing vesicles at the midbody ring. In control cells, these v-SNARE vesicles colocalize with a GFP-tagged secreted polypeptide. The vesicles move to the midbody ring asymmetrically from one prospective daughter cell; the GFP signal is rapidly lost, suggesting membrane fusion; and subsequently the cell cleaves at the site of vesicle delivery/fusion. We propose that centriolin anchors protein complexes required for vesicle targeting and fusion and integrates membrane-vesicle fusion with abscission.

Cell Cycle Proteins↗

PubMed2005

Membrane type-1 matrix metalloproteinase (MT1-MMP) exhibits an important intracellular cleavage function and causes chromosome instability.

Elevated expression of membrane type-1 matrix metalloproteinase (MT1-MMP) is closely associated with malignancies. There is a consensus among scientists that cell surface-associated MT1-MMP is a key player in pericellular proteolytic events. Now we have identified an intracellular, hitherto unknown, function of MT1-MMP. We demonstrated that MT1-MMP is trafficked along the tubulin cytoskeleton. A fraction of cellular MT1-MMP accumulates in the centrosomal compartment. MT1-MMP targets an integral centrosomal protein, pericentrin. Pericentrin is known to be essential to the normal functioning of centrosomes and to mitotic spindle formation. Expression of MT1-MMP stimulates mitotic spindle aberrations and aneuploidy in non-malignant cells. Volumes of data indicate that chromosome instability is an early event of carcinogenesis. In agreement, the presence of MT1-MMP activity correlates with degraded pericentrin in tumor biopsies, whereas normal tissues exhibit intact pericentrin. We believe that our data show a novel proteolytic pathway to chromatin instability and elucidate the close association of MT1-MMP with malignant transformation.

Animals↗

PubMed2004

Mitosis-specific anchoring of gamma tubulin complexes by pericentrin controls spindle organization and mitotic entry.

Microtubule nucleation is the best known function of centrosomes. Centrosomal microtubule nucleation is mediated primarily by gamma tubulin ring complexes (gamma TuRCs). However, little is known about the molecules that anchor these complexes to centrosomes. In this study, we show that the centrosomal coiled-coil protein pericentrin anchors gamma TuRCs at spindle poles through an interaction with gamma tubulin complex proteins 2 and 3 (GCP2/3). Pericentrin silencing by small interfering RNAs in somatic cells disrupted gamma tubulin localization and spindle organization in mitosis but had no effect on gamma tubulin localization or microtubule organization in interphase cells. Similarly, overexpression of the GCP2/3 binding domain of pericentrin disrupted the endogenous pericentrin-gamma TuRC interaction and perturbed astral microtubules and spindle bipolarity. When added to Xenopus mitotic extracts, this domain uncoupled gamma TuRCs from centrosomes, inhibited microtubule aster assembly, and induced rapid disassembly of preassembled asters. All phenotypes were significantly reduced in a pericentrin mutant with diminished GCP2/3 binding and were specific for mitotic centrosomal asters as we observed little effect on interphase asters or on asters assembled by the Ran-mediated centrosome-independent pathway. Additionally, pericentrin silencing or overexpression induced G2/antephase arrest followed by apoptosis in many but not all cell types. We conclude that pericentrin anchoring of gamma tubulin complexes at centrosomes in mitotic cells is required for proper spindle organization and that loss of this anchoring mechanism elicits a checkpoint response that prevents mitotic entry and triggers apoptotic cell death.

Animals↗

PubMed2003

Centrosomal anchoring of protein kinase C betaII by pericentrin controls microtubule organization, spindle function, and cytokinesis.

Location is a critical determinant in dictating the cellular function of protein kinase C (PKC). Scaffold proteins contribute to localization by poising PKC at specific intracellular sites. Using a yeast two-hybrid screen, we identified the centrosomal protein pericentrin as a scaffold that tethers PKC betaII to centrosomes. Co-immunoprecipitation studies reveal that the native proteins interact in cells. Co-overexpression studies show that the interaction is mediated by the C1A domain of PKC and a segment of pericentrin within residues 494-593. Immunofluorescence analysis reveals that endogenous PKC betaII colocalizes with pericentrin at centrosomes. Disruption of this interaction by expression of the interacting region of pericentrin results in release of PKC from the centrosome, microtubule disorganization, and cytokinesis failure. Overexpression of this disrupting fragment has no effect in cells lacking PKC betaII, indicating a specific regulatory role of this isozyme in centrosome function. These results reveal a novel role for PKC betaII in cytokinesis and indicate that this function is mediated by an interaction with pericentrin at centrosomes.

Animals↗

PubMed2003

Centrosome abnormalities and chromosome instability occur together in pre-invasive carcinomas.

Centrosomes play critical roles in processes that ensure proper segregation of chromosomes and maintain the genetic stability of human cells. They contribute to mitotic spindle organization and regulate aspects of cytokinesis and cell cycle progression. We and others have shown that centrosomes are abnormal in most aggressive carcinomas. Moreover, centrosome defects have been implicated in chromosome instability and loss of cell cycle control in invasive carcinoma. Others have suggested that centrosome defects only occur late in tumorigenesis and may not contribute to early steps of tumor development. To address this issue, we examined pre-invasive human carcinoma in situ lesions for centrosome defects and chromosome instability. We found that a significant fraction of precursor lesions to some of the most common human cancers had centrosome defects, including in situ carcinomas of the uterine cervix, prostate, and female breast. Moreover, centrosome defects occurred together with mitotic spindle defects, chromosome instability, and high cytologic grade. Because most pre-invasive lesions are not uniformly mutant for p53, the development of centrosome defects does not appear to require abrogation of p53 function. Our findings demonstrate that centrosome defects occur concurrently with chromosome instability and cytologic changes in the earliest identifiable step in human cancer. Our results suggest that centrosome defects may contribute to the earliest stages of cancer development through the generation of chromosome instability. This, together with ongoing structural changes in chromosomes, could accelerate accumulation of alleles carrying pro-oncogenic mutations and loss of alleles containing wild-type tumor suppressor genes and thus accelerate the genomic changes characteristic of carcinoma, the most prevalent human cancer.

Breast Neoplasms↗

PubMed2003

Mutations and aneuploidy: co-conspirators in cancer?

The role of intragenic point mutations in human cancer is well established. However, the contribution of massive genomic changes collectively known as aneuploidy is less certain. Recent experimental work suggests that aneuploidy is required for sporadic carcinogenesis in mice and that it may collaborate with intragenic mutations during tumorigenesis. The genomic plasticity afforded by aneuploidy could facilitate emergence of protumorigenic gene dosage changes and accelerate accumulation of oncogenes and loss of tumor suppressor genes. These new findings force us to rethink the pathogenesis of carcinoma in ways that have significant implications for diagnosis and therapy.

Aneuploidy↗