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Current Research and Scholarly Interests


The central question behind our work is how the centrosome and primary cilium control cell function and influence development, and how defects in these structures cause a remarkable range of human disease, ranging from cancer, polycystic kidney disease, and obesity, to neurocognitive defects including mental retardation, schizophrenia, and dyslexia.

The centrosome consists of a pair of centrioles and pericentriolar material and organizes the cytoplasmic microtubules of most animal cells. Most importantly, the mother centriole (the older of the two in the pair) nucleates the formation of a primary cilium in most cells in the body. First seen by cell biologists in the 1950's, the primary cilium was ignored for many years until a combination of human and model organism genetics revealed that it is a critical sensory organelle with functions in many important processes. Defects in primary cilium structure and function cause a set of human conditions, called ciliopathies, that share a set of phenotypes that reflect the importance of the cilium in signaling pathways.

There are three main projects in the lab:

1) Ciliary biogenesis and function. In addition to the microtubules making up the interphase array and the mitotic spindle, many animal cells make a specialized microtubule structure, the primary cilium. This is a single, non-motile cilium that is able to act as a transducer of mechanical and chemical signals - sort of a cellular antenna. The microtubules of the ciliary axoneme grow directly from a centriole at their base, this centriole is often called a basal body. Some epithelial cells in the trachea, oviduct and brain produce hundreds of motile cilia on their surface, each with a centriole at their base. We are studying both the primary cilium and multi-ciliated cells for clues into ciliary structure and function, and centriole formation.

2) Cell cycle control of centrosome duplication. We have shown that duplication of the centrosome, the microtubule organizing center of animal cells, is dependent on the cell cycle kinase cdk2, and on cell cycle-specific proteolysis. We are working to determine the molecular mechanisms of centrosome duplication and to understand how centrosome duplication is controlled so that it happens once and only once per cell cycle. Cancer cells often have aberrant centrosome numbers, and we are investigating the relationship between aberrant centrosome number and the genome instability that is common in cancer cells.

3) Microtubule nucleation and organization. Microtubules are polymers of tubulin, which is a heterodimer of alpha-tubulin and beta-tubulin. We have identified a remarkable complex of proteins associated with a third type of tubulin, gamma-tubulin. Gamma-tubulin and its associated proteins are localized to the centrosome and are critical for initiation, or nucleation, of microtubule assembly. The gamma-tubulin complex (gammaTuRC) is a very large, ring-shaped complex and contains at least 6 proteins in addition to gamma-tubulin. We are determining the role of gamma-tubulin and its associated proteins in microtubule nucleation and organization.

Teaching

2013-14 Courses


Postdoctoral Advisees


Graduate and Fellowship Programs


Publications

Journal Articles


  • The Rilp-like proteins Rilpl1 and Rilpl2 regulate ciliary membrane content. Molecular biology of the cell Schaub, J. R., Stearns, T. 2013; 24 (4): 453-464

    Abstract

    The primary cilium is a microtubule-based structure found in most cell types in mammals. Disruption of cilium function causes a diverse set of human diseases collectively known as ciliopathies. We report that Rab effector-related proteins Rab-interacting lysosomal protein-like 1 (Rilpl1) and Rilpl2 regulate protein localization in the primary cilium. Rilpl2 was initially identified as up-regulated in ciliating mouse tracheal epithelial cells. Rilpl1 and Rilpl2 both localize to the primary cilium and centrosome, Rilpl1 specifically to the distal end of the mother centriole. Live-cell microscopy reveals that Rilpl2 primary cilium localization is dynamic and that it is associated with tubulovesicular structures at the base of the cilium. Depletion of Rilpl1 and Rilpl2 results in accumulation of signaling proteins in the ciliary membrane and prevents proper epithelial cell organization in three-dimensional culture. These data suggest that Rilp-like proteins function in regulation of ciliary membrane protein concentration by promoting protein removal from the primary cilium.

    View details for DOI 10.1091/mbc.E12-08-0598

    View details for PubMedID 23264467

  • Supernumerary Centrosomes Nucleate Extra Cilia and Compromise Primary Cilium Signaling CURRENT BIOLOGY Mahjoub, M. R., Stearns, T. 2012; 22 (17): 1628-1634

    Abstract

    The primary cilium is a nexus of cell signaling, and ciliary dysfunction is associated with polycystic kidney disease, retinal degeneration, polydactyly, neural tube defects, and obesity (ciliopathies). Signaling molecules for cilium-associated pathways are concentrated in the cilium, and this is essential for efficient signaling. Cilia are nucleated from centrioles, and aberrant centriole numbers are seen in many cancers and in some ciliopathies. We tested the effect of supernumerary centrioles on cilium function and found that cells with extra centrioles often formed more than one cilium, had reduced ciliary concentration of Smoothened in response to Sonic hedgehog stimulation, and reduced Shh pathway transcriptional activation. This ciliary dilution phenotype was also observed with the serotonin receptor Htr6, fibrocystin PKHD1, and Arl13b. The presence of extra centrioles and cilia disrupted epithelial organization in 3D spheroid culture. Cells mutant for the tuberous sclerosis gene Tsc2 also had extra cilia and diluted ciliary protein. In most cells, extra cilia were clustered and shared the same ciliary pocket, suggesting that the ciliary pocket is the rate-limiting structure for trafficking of ciliary proteins. Thus, extra centrioles and cilia disrupt signaling and may contribute to disease phenotypes.

    View details for DOI 10.1016/j.cub.2012.06.057

    View details for Web of Science ID 000308849900037

    View details for PubMedID 22840514

  • STED Microscopy with Optimized Labeling Density Reveals 9-Fold Arrangement of a Centriole Protein BIOPHYSICAL JOURNAL Lau, L., Lee, Y. L., Sahl, S. J., Stearns, T., Moerner, W. E. 2012; 102 (12): 2926-2935

    Abstract

    Super-resolution fluorescence microscopy can achieve resolution beyond the optical diffraction limit, partially closing the gap between conventional optical imaging and electron microscopy for elucidation of subcellular architecture. The centriole, a key component of the cellular control and division machinery, is 250 nm in diameter, a spatial scale where super-resolution methods such as stimulated emission depletion (STED) microscopy can provide previously unobtainable detail. We use STED with a resolution of 60 nm to demonstrate that the centriole distal appendage protein Cep164 localizes in nine clusters spaced around a ring of ?300 nm in diameter, and quantify the influence of the labeling density in STED immunofluorescence microscopy. We find that the labeling density dramatically influences the observed number, size, and brightness of labeled Cep164 clusters, and estimate the average number of secondary antibody labels per cluster. The arrangements are morphologically similar in centrioles of both proliferating cells and differentiated multiciliated cells, suggesting a relationship of this structure to function. Our STED measurements in single centrioles are consistent with results obtained by electron microscopy, which involve ensemble averaging or very different sample preparation conditions, suggesting that we have arrived at a direct measurement of a centriole protein by careful optimization of the labeling density.

    View details for DOI 10.1016/j.bpj.2012.05.015

    View details for Web of Science ID 000305546500027

    View details for PubMedID 22735543

  • Myb promotes centriole amplification and later steps of the multiciliogenesis program DEVELOPMENT Tan, F. E., Vladar, E. K., Ma, L., Fuentealba, L. C., Hoh, R., Espinoza, F. H., Axelrod, J. D., Alvarez-Buylla, A., Stearns, T., Kintner, C., Krasnow, M. A. 2013; 140 (20): 4277-4286

    Abstract

    The transcriptional control of primary cilium formation and ciliary motility are beginning to be understood, but little is known about the transcriptional programs that control cilium number and other structural and functional specializations. One of the most intriguing ciliary specializations occurs in multiciliated cells (MCCs), which amplify their centrioles to nucleate hundreds of cilia per cell, instead of the usual monocilium. Here we report that the transcription factor MYB, which promotes S phase and drives cycling of a variety of progenitor cells, is expressed in postmitotic epithelial cells of the mouse airways and ependyma destined to become MCCs. MYB is expressed early in multiciliogenesis, as progenitors exit the cell cycle and amplify their centrioles, then switches off as MCCs mature. Conditional inactivation of Myb in the developing airways blocks or delays centriole amplification and expression of FOXJ1, a transcription factor that controls centriole docking and ciliary motility, and airways fail to become fully ciliated. We provide evidence that MYB acts in a conserved pathway downstream of Notch signaling and multicilin, a protein related to the S-phase regulator geminin, and upstream of FOXJ1. MYB can activate endogenous Foxj1 expression and stimulate a cotransfected Foxj1 reporter in heterologous cells, and it can drive the complete multiciliogenesis program in Xenopus embryonic epidermis. We conclude that MYB has an early, crucial and conserved role in multiciliogenesis, and propose that it promotes a novel S-like phase in which centriole amplification occurs uncoupled from DNA synthesis, and then drives later steps of multiciliogenesis through induction of Foxj1.

    View details for DOI 10.1242/dev.094102

    View details for Web of Science ID 000325153200017

    View details for PubMedID 24048590

  • Myb promotes centriole amplification and later steps of the multiciliogenesis program DEVELOPMENT Tan, F. E., Vladar, E. K., Ma, L., Fuentealba, L. C., Hoh, R., Espinoza, F. H., Axelrod, J. D., Alvarez-Buylla, A., Stearns, T., Kintner, C., Krasnow, M. A. 2013; 140 (20): 4277-4286

    Abstract

    The transcriptional control of primary cilium formation and ciliary motility are beginning to be understood, but little is known about the transcriptional programs that control cilium number and other structural and functional specializations. One of the most intriguing ciliary specializations occurs in multiciliated cells (MCCs), which amplify their centrioles to nucleate hundreds of cilia per cell, instead of the usual monocilium. Here we report that the transcription factor MYB, which promotes S phase and drives cycling of a variety of progenitor cells, is expressed in postmitotic epithelial cells of the mouse airways and ependyma destined to become MCCs. MYB is expressed early in multiciliogenesis, as progenitors exit the cell cycle and amplify their centrioles, then switches off as MCCs mature. Conditional inactivation of Myb in the developing airways blocks or delays centriole amplification and expression of FOXJ1, a transcription factor that controls centriole docking and ciliary motility, and airways fail to become fully ciliated. We provide evidence that MYB acts in a conserved pathway downstream of Notch signaling and multicilin, a protein related to the S-phase regulator geminin, and upstream of FOXJ1. MYB can activate endogenous Foxj1 expression and stimulate a cotransfected Foxj1 reporter in heterologous cells, and it can drive the complete multiciliogenesis program in Xenopus embryonic epidermis. We conclude that MYB has an early, crucial and conserved role in multiciliogenesis, and propose that it promotes a novel S-like phase in which centriole amplification occurs uncoupled from DNA synthesis, and then drives later steps of multiciliogenesis through induction of Foxj1.

    View details for DOI 10.1242/dev.094102

    View details for Web of Science ID 000325153200017

    View details for PubMedID 24048590

  • FOP Is a Centriolar Satellite Protein Involved in Ciliogenesis PLOS ONE Lee, J. Y., Stearns, T. 2013; 8 (3)

    Abstract

    Centriolar satellites are proteinaceous granules that are often clustered around the centrosome. Although centriolar satellites have been implicated in protein trafficking in relation to the centrosome and cilium, the details of their function and composition remain unknown. FOP (FGFR1 Oncogene Partner) is a known centrosome protein with homology to the centriolar satellite proteins FOR20 and OFD1. We find that FOP partially co-localizes with the satellite component PCM1 in a cell cycle-dependent manner, similarly to the satellite and cilium component BBS4. As for BBS4, FOP localization to satellites is cell cycle dependent, with few satellites labeled in G1, when FOP protein levels are lowest, and most labeled in G2. FOP-FGFR1, an oncogenic fusion that causes a form of leukemia called myeloproliferative neoplasm, also localizes to centriolar satellites where it increases tyrosine phosphorylation. Depletion of FOP strongly inhibits primary cilium formation in human RPE-1 cells. These results suggest that FOP is a centriolar satellite cargo protein and, as for several other satellite-associated proteins, is involved in ciliogenesis. Localization of the FOP-FGFR1 fusion kinase to centriolar satellites may be relevant to myeloproliferative neoplasm disease progression.

    View details for DOI 10.1371/journal.pone.0058589

    View details for Web of Science ID 000316252500032

    View details for PubMedID 23554904

  • Transcriptional Program of Ciliated Epithelial Cells Reveals New Cilium and Centrosome Components and Links to Human Disease PLOS ONE Hoh, R. A., Stowe, T. R., Turk, E., Stearns, T. 2012; 7 (12)

    Abstract

    Defects in the centrosome and cilium are associated with a set of human diseases having diverse phenotypes. To further characterize the components that define the function of these organelles we determined the transcriptional profile of multiciliated tracheal epithelial cells. Cultures of mouse tracheal epithelial cells undergoing differentiation in vitro were derived from mice expressing GFP from the ciliated-cell specific FOXJ1 promoter (FOXJ1:GFP). The transcriptional profile of ciliating GFP+ cells from these cultures was defined at an early and a late time point during differentiation and was refined by subtraction of the profile of the non-ciliated GFP- cells. We identified 649 genes upregulated early, when most cells were forming basal bodies, and 73 genes genes upregulated late, when most cells were fully ciliated. Most, but not all, of known centrosome proteins are transcriptionally upregulated early, particularly Plk4, a master regulator of centriole formation. We found that three genes associated with human disease states, Mdm1, Mlf1, and Dyx1c1, are upregulated during ciliogenesis and localize to centrioles and cilia. This transcriptome for mammalian multiciliated epithelial cells identifies new candidate centrosome and cilia proteins, highlights similarities between components of motile and primary cilia, and identifies new links between cilia proteins and human disease.

    View details for DOI 10.1371/journal.pone.0052166

    View details for Web of Science ID 000313872600011

    View details for PubMedID 23300604

  • The centriolar satellite proteins Cep72 and Cep290 interact and are required for recruitment of BBS proteins to the cilium MOLECULAR BIOLOGY OF THE CELL Stowe, T. R., Wilkinson, C. J., Iqbal, A., Stearns, T. 2012; 23 (17): 3322-3335

    Abstract

    Defects in centrosome and cilium function are associated with phenotypically related syndromes called ciliopathies. Centriolar satellites are centrosome-associated structures, defined by the protein PCM1, that are implicated in centrosomal protein trafficking. We identify Cep72 as a PCM1-interacting protein required for recruitment of the ciliopathy-associated protein Cep290 to centriolar satellites. Loss of centriolar satellites by depletion of PCM1 causes relocalization of Cep72 and Cep290 from satellites to the centrosome, suggesting that their association with centriolar satellites normally restricts their centrosomal localization. We identify interactions between PCM1, Cep72, and Cep290 and find that disruption of centriolar satellites by overexpression of Cep72 results in specific aggregation of these proteins and the BBSome component BBS4. During ciliogenesis, BBS4 relocalizes from centriolar satellites to the primary cilium. This relocalization occurs normally in the absence of centriolar satellites (PCM1 depletion) but is impaired by depletion of Cep290 or Cep72, resulting in defective ciliary recruitment of the BBSome subunit BBS8. We propose that Cep290 and Cep72 in centriolar satellites regulate the ciliary localization of BBS4, which in turn affects assembly and recruitment of the BBSome. Finally, we show that loss of centriolar satellites in zebrafish leads to phenotypes consistent with cilium dysfunction and analogous to those observed in human ciliopathies.

    View details for DOI 10.1091/mbc.E12-02-0134

    View details for Web of Science ID 000312221100009

    View details for PubMedID 22767577

  • Mechanosensing by the Primary Cilium: Deletion of Kif3A Reduces Bone Formation Due to Loading PLOS ONE Temiyasathit, S., Tang, W. J., Leucht, P., Anderson, C. T., Monica, S. D., Castillo, A. B., Helms, J. A., Stearns, T., Jacobs, C. R. 2012; 7 (3)

    Abstract

    Primary cilia, solitary microtubule-based structures that grow from the centriole and extend into the extracellular space, have increasingly been implicated as sensors of a variety of biochemical and biophysical signals. Mutations in primary cilium-related genes have been linked to a number of rare developmental disorders as well as dysregulation of cell proliferation. We propose that primary cilia are also important in mechanically regulated bone formation in adults and that their malfunction could play a role in complex multi-factorial bone diseases, such as osteoporosis. In this study, we generated mice with an osteoblast- and osteocyte-specific knockout of Kif3a, a subunit of the kinesin II intraflagellar transport (IFT) protein; IFT is required for primary cilia formation, maintenance, and function. These Col?1(I) 2.3-Cre;Kif3a(fl/fl) mice exhibited no obvious morphological skeletal abnormalities. Skeletally mature Col?1(I) 2.3-Cre;Kif3a(fl/fl) and control mice were exposed to 3 consecutive days of cyclic axial ulna loading, which resulted in a significant increase in bone formation in both the conditional knockouts and controls. However, Col?1(I) 2.3-Cre;Kif3a(fl/fl) mice did exhibit decreased formation of new bone in response to mechanical ulnar loading compared to control mice. These results suggest that primary cilia act as cellular mechanosensors in bone and that their function may be critical for the regulation of bone physiology due to mechanical loading in adults.

    View details for DOI 10.1371/journal.pone.0033368

    View details for Web of Science ID 000302381500135

    View details for PubMedID 22428034

  • A crucial requirement for Hedgehog signaling in small cell lung cancer NATURE MEDICINE Park, K., Martelotto, L. G., Peifer, M., Sos, M. L., Karnezis, A. N., Mahjoub, M. R., Bernard, K., Conklin, J. F., Szczepny, A., Yuan, J., Guo, R., Ospina, B., Falzon, J., Bennett, S., Brown, T. J., Markovic, A., Devereux, W. L., Ocasio, C. A., Chen, J. K., Stearns, T., Thomas, R. K., Dorsch, M., Buonamici, S., Watkins, D. N., Peacock, C. D., Sage, J. 2011; 17 (11): 1504-U1506

    Abstract

    Small-cell lung cancer (SCLC) is an aggressive neuroendocrine subtype of lung cancer for which there is no effective treatment. Using a mouse model in which deletion of Rb1 and Trp53 in the lung epithelium of adult mice induces SCLC, we found that the Hedgehog signaling pathway is activated in SCLC cells independently of the lung microenvironment. Constitutive activation of the Hedgehog signaling molecule Smoothened (Smo) promoted the clonogenicity of human SCLC in vitro and the initiation and progression of mouse SCLC in vivo. Reciprocally, deletion of Smo in Rb1 and Trp53-mutant lung epithelial cells strongly suppressed SCLC initiation and progression in mice. Furthermore, pharmacological blockade of Hedgehog signaling inhibited the growth of mouse and human SCLC, most notably following chemotherapy. These findings show a crucial cell-intrinsic role for Hedgehog signaling in the development and maintenance of SCLC and identify Hedgehog pathway inhibition as a therapeutic strategy to slow the progression of disease and delay cancer recurrence in individuals with SCLC.

    View details for DOI 10.1038/nm.2473

    View details for Web of Science ID 000296779300043

    View details for PubMedID 21983857

  • Curcumin Inhibits Growth of Saccharomyces cerevisiae through Iron Chelation EUKARYOTIC CELL Minear, S., O'Donnell, A. F., Ballew, A., Giaever, G., Nislow, C., Stearns, T., Cyert, M. S. 2011; 10 (11): 1574-1581

    Abstract

    Curcumin, a polyphenol derived from turmeric, is an ancient therapeutic used in India for centuries to treat a wide array of ailments. Interest in curcumin has increased recently, with ongoing clinical trials exploring curcumin as an anticancer therapy and as a protectant against neurodegenerative diseases. In vitro, curcumin chelates metal ions. However, although diverse physiological effects have been documented for this compound, curcumin's mechanism of action on mammalian cells remains unclear. This study uses yeast as a model eukaryotic system to dissect the biological activity of curcumin. We found that yeast mutants lacking genes required for iron and copper homeostasis are hypersensitive to curcumin and that iron supplementation rescues this sensitivity. Curcumin penetrates yeast cells, concentrates in the endoplasmic reticulum (ER) membranes, and reduces the intracellular iron pool. Curcumin-treated, iron-starved cultures are enriched in unbudded cells, suggesting that the G(1) phase of the cell cycle is lengthened. A delay in cell cycle progression could, in part, explain the antitumorigenic properties associated with curcumin. We also demonstrate that curcumin causes a growth lag in cultured human cells that is remediated by the addition of exogenous iron. These findings suggest that curcumin-induced iron starvation is conserved from yeast to humans and underlies curcumin's medicinal properties.

    View details for DOI 10.1128/EC.05163-11

    View details for Web of Science ID 000296723600022

    View details for PubMedID 21908599

  • The centrosome cycle: Centriole biogenesis, duplication and inherent asymmetries NATURE CELL BIOLOGY Nigg, E. A., Stearns, T. 2011; 13 (10): 1154-1160

    Abstract

    Centrosomes are microtubule-organizing centres of animal cells. They influence the morphology of the microtubule cytoskeleton, function as the base for the primary cilium and serve as a nexus for important signalling pathways. At the core of a typical centrosome are two cylindrical microtubule-based structures termed centrioles, which recruit a matrix of associated pericentriolar material. Cells begin the cell cycle with exactly one centrosome, and the duplication of centrioles is constrained such that it occurs only once per cell cycle and at a specific site in the cell. As a result of this duplication mechanism, the two centrioles differ in age and maturity, and thus have different functions; for example, the older of the two centrioles can initiate the formation of a ciliary axoneme. We discuss spatial aspects of the centrosome duplication cycle, the mechanism of centriole assembly and the possible consequences of the inherent asymmetry of centrioles and centrosomes.

    View details for DOI 10.1038/ncb2345

    View details for Web of Science ID 000295617900003

    View details for PubMedID 21968988

  • Cep152 interacts with Plk4 and is required for centriole duplication JOURNAL OF CELL BIOLOGY Hatch, E. M., Kulukian, A., Holland, A. J., Cleveland, D. W., Stearns, T. 2010; 191 (4): 721-729

    Abstract

    Centrioles are microtubule-based structures that organize the centrosome and nucleate cilia. Centrioles duplicate once per cell cycle, and duplication requires Plk4, a member of the Polo-like kinase family; however, the mechanism linking Plk4 activity and centriole formation is unknown. In this study, we show in human and frog cells that Plk4 interacts with the centrosome protein Cep152, the orthologue of Drosophila melanogaster Asterless. The interaction requires the N-terminal 217 residues of Cep152 and the crypto Polo-box of Plk4. Cep152 and Plk4 colocalize at the centriole throughout the cell cycle. Overexpression of Cep152 (1-217) mislocalizes Plk4, but both Cep152 and Plk4 are able to localize to the centriole independently of the other. Depletion of Cep152 prevents both normal centriole duplication and Plk4-induced centriole amplification and results in a failure to localize Sas6 to the centriole, an early step in duplication. Cep152 can be phosphorylated by Plk4 in vitro, suggesting that Cep152 acts with Plk4 to initiate centriole formation.

    View details for DOI 10.1083/jcb.201006049

    View details for Web of Science ID 000284737200006

    View details for PubMedID 21059850

  • Cep120 is asymmetrically localized to the daughter centriole and is essential for centriole assembly JOURNAL OF CELL BIOLOGY Mahjoub, M. R., Xie, Z., Stearns, T. 2010; 191 (2): 331-346

    Abstract

    Centrioles form the core of the centrosome in animal cells and function as basal bodies that nucleate and anchor cilia at the plasma membrane. In this paper, we report that Cep120 (Ccdc100), a protein previously shown to be involved in maintaining the neural progenitor pool in mouse brain, is associated with centriole structure and function. Cep120 is up-regulated sevenfold during differentiation of mouse tracheal epithelial cells (MTECs) and localizes to basal bodies. Cep120 localizes preferentially to the daughter centriole in cycling cells, and this asymmetry between mother and daughter centrioles is relieved coincident with new centriole assembly. Photobleaching recovery analysis identifies two pools of Cep120, differing in their halftime at the centriole. We find that Cep120 is required for centriole duplication in cycling cells, centriole amplification in MTECs, and centriole overduplication in S phase-arrested cells. We propose that Cep120 is required for centriole assembly and that the observed defect in neuronal migration might derive from a defect in this process.

    View details for DOI 10.1083/jcb.201003009

    View details for Web of Science ID 000283391500013

    View details for PubMedID 20956381

  • The life cycle of centrioles. Cold Spring Harbor symposia on quantitative biology Hatch, E., Stearns, T. 2010; 75: 425-431

    Abstract

    Centrioles organize the centrosome and nucleate the ciliary axoneme, and the centriole life cycle has many parallels to the chromosome cycle. The centriole cycle in animals begins at fertilization with the contribution of two centrioles by the male gamete. In the ensuing cell cycles, the duplication of centrioles is controlled temporally, spatially, and numerically. As a consequence of the duplication mechanism, the two centrioles in a typical interphase cell are of different ages and have different functions. Here, we discuss how new centrioles are assembled, what mechanisms limit centriole number, and the consequences of the inherent asymmetry of centriole duplication and segregation.

    View details for DOI 10.1101/sqb.2010.75.054

    View details for PubMedID 21502410

  • STEM CELLS A fateful age gap NATURE Stearns, T. 2009; 461 (7266): 891-892

    View details for DOI 10.1038/461891a

    View details for Web of Science ID 000270817700029

    View details for PubMedID 19829363

  • Centriole Age Underlies Asynchronous Primary Cilium Growth in Mammalian Cells CURRENT BIOLOGY Anderson, C. T., Stearns, T. 2009; 19 (17): 1498-1502

    Abstract

    Primary cilia are microtubule-based sensory organelles that play important roles in development and disease . They are required for Sonic hedgehog (Shh) and platelet-derived growth factor (PDGF) signaling. Primary cilia grow from the older of the two centrioles of the centrosome, referred to as the mother centriole. In cycling cells, the cilium typically grows in G1 and is lost before mitosis, but the regulation of its growth is poorly understood. Centriole duplication at G1/S results in two centrosomes, one with an older mother centriole and one with a new mother centriole, that are segregated in mitosis. Here we report that primary cilia grow asynchronously in sister cells resulting from a mitotic division and that the sister cell receiving the older mother centriole usually grows a primary cilium first. We also show that the signaling proteins inversin and PDGFRalpha localize asynchronously to sister cell primary cilia and that sister cells respond asymmetrically to Shh. These results suggest that the segregation of differently aged mother centrioles, an asymmetry inherent to every animal cell division, can influence the ability of sister cells to respond to environmental signals, potentially altering the behavior or fate of one or both sister cells.

    View details for DOI 10.1016/j.cub.2009.07.034

    View details for Web of Science ID 000269920500042

    View details for PubMedID 19682908

  • Polo Kinase and Separase Regulate the Mitotic Licensing of Centriole Duplication in Human Cells DEVELOPMENTAL CELL Tsou, M. B., Wang, W., George, K. A., Uryu, K., Stearns, T., Jallepalli, P. V. 2009; 17 (3): 344-354

    Abstract

    It has been proposed that separase-dependent centriole disengagement at anaphase licenses centrosomes for duplication in the next cell cycle. Here we test whether such a mechanism exists in intact human cells. Loss of separase blocked centriole disengagement during mitotic exit and delayed assembly of new centrioles during the following S phase; however, most engagements were eventually dissolved. We identified Polo-like kinase 1 (Plk1) as a parallel activator of centriole disengagement. Timed inhibition of Plk1 mapped its critical period of action to late G2 or early M phase, i.e., prior to securin destruction and separase activation at anaphase onset. Crucially, when cells exited mitosis after downregulation of both separase and Plk1, centriole disengagement failed completely, and subsequent centriole duplication in interphase was also blocked. Our results indicate that Plk1 and separase act at different times during M phase to license centrosome duplication, reminiscent of their roles in removing cohesin from chromosomes.

    View details for DOI 10.1016/j.devcel.2009.07.015

    View details for Web of Science ID 000270017100008

    View details for PubMedID 19758559

  • Plk1-Dependent Recruitment of gamma-Tubulin Complexes to Mitotic Centrosomes Involves Multiple PCM Components PLOS ONE Haren, L., Stearns, T., Luders, J. 2009; 4 (6)

    Abstract

    The nucleation of microtubules requires protein complexes containing gamma-tubulin, which are present in the cytoplasm and associate with the centrosome and with the mitotic spindle. We have previously shown that these interactions require the gamma-tubulin targeting factor GCP-WD/NEDD1, which has an essential role in spindle formation. The recruitment of additional gamma-tubulin to the centrosomes occurs during centrosome maturation at the G2/M transition and is regulated by the mitotic kinase Plk1. However, the molecular details of this important pathway are unknown and a Plk1 substrate that controls gamma-tubulin recruitment has not been identified. Here we show that Plk1 associates with GCP-WD in mitosis and Plk1 activity contributes to phosphorylation of GCP-WD. Plk1 depletion or inhibition prevents accumulation of GCP-WD at mitotic centrosomes, but GCP-WD mutants that are defective in Plk1-binding and -phosphorylation still accumulate at mitotic centrosomes and recruit gamma-tubulin. Moreover, Plk1 also controls the recruitment of other PCM proteins implicated in centrosomal gamma-tubulin attachment (Cep192/hSPD2, pericentrin, Cep215/Cdk5Rap2). Our results support a model in which Plk1-dependent recruitment of gamma-tubulin to mitotic centrosomes is regulated upstream of GCP-WD, involves multiple PCM proteins and therefore potentially multiple Plk1 substrates.

    View details for DOI 10.1371/journal.pone.0005976

    View details for Web of Science ID 000267237400004

    View details for PubMedID 19543530

  • Primary cilia: Cellular sensors for the skeleton ANATOMICAL RECORD-ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY Anderson, C. T., Castillo, A. B., Brugmann, S. A., Helms, J. A., Jacobs, C. R., Stearns, T. 2008; 291 (9): 1074-1078

    Abstract

    The primary cilium is a solitary, immotile cilium that is present in almost every mammalian cell type. Primary cilia are thought to function as chemosensors, mechanosensors, or both, depending on cell type, and have been linked to several developmental signaling pathways. Primary cilium malfunction has been implicated in several human diseases, the symptoms of which include vision and hearing loss, polydactyly, and polycystic kidneys. Recently, primary cilia have also been implicated in the development and homeostasis of the skeleton. In this review, we discuss the structure and formation of the primary cilium and some of the mechanical and chemical signals to which it could be sensitive, with a focus on skeletal biology. We also raise several unanswered questions regarding the role of primary cilia as mechanosensors and chemosensors and identify potential research avenues to address these questions.

    View details for DOI 10.1002/ar.20754

    View details for Web of Science ID 000259324900004

    View details for PubMedID 18727074

  • Primary cilia mediate mechanosensing in bone cells by a calcium-independent mechanism PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Malone, A. M., Anderson, C. T., Tummala, P., Kwon, R. Y., Johnston, T. R., Stearns, T., Jacobs, C. R. 2007; 104 (33): 13325-13330

    Abstract

    Primary cilia are sensory organelles that translate extracellular chemical and mechanical cues into cellular responses. Bone is an exquisitely mechanosensitive organ, and its homeostasis depends on the ability of bone cells to sense and respond to mechanical stimuli. One such stimulus is dynamic fluid flow, which triggers biochemical and transcriptional changes in bone cells by an unknown mechanism. Here we report that bone cells possess primary cilia that project from the cell surface and deflect during fluid flow and that these primary cilia are required for osteogenic and bone resorptive responses to dynamic fluid flow. We also show that, unlike in kidney cells, primary cilia in bone translate fluid flow into cellular responses in bone cells independently of Ca(2+) flux and stretch-activated ion channels. These results suggest that primary cilia might regulate homeostasis in diverse tissues by allowing mechanical signals to alter cellular activity via tissue-specific pathways. Our identification of a mechanism for mechanotransduction in bone could lead to therapeutic approaches for combating bone loss due to osteoporosis and disuse.

    View details for DOI 10.1073/pnas.0700636104

    View details for Web of Science ID 000248899600022

    View details for PubMedID 17673554

  • Molecular characterization of centriole assembly in ciliated epithelial cells JOURNAL OF CELL BIOLOGY Vladar, E. K., Stearns, T. 2007; 178 (1): 31-42

    Abstract

    Ciliated epithelial cells have the unique ability to generate hundreds of centrioles during differentiation. We used centrosomal proteins as molecular markers in cultured mouse tracheal epithelial cells to understand this process. Most centrosomal proteins were up-regulated early in ciliogenesis, initially appearing in cytoplasmic foci and then incorporated into centrioles. Three candidate proteins were further characterized. The centrosomal component SAS-6 localized to basal bodies and the proximal region of the ciliary axoneme, and depletion of SAS-6 prevented centriole assembly. The intraflagellar transport component polaris localized to nascent centrioles before incorporation into cilia, and depletion of polaris blocked axoneme formation. The centriolar satellite component PCM-1 colocalized with centrosomal components in cytoplasmic granules surrounding nascent centrioles. Interfering with PCM-1 reduced the amount of centrosomal proteins at basal bodies but did not prevent centriole assembly. This system will help determine the mechanism of centriole formation in mammalian cells and how the limitation on centriole duplication is overcome in ciliated epithelial cells.

    View details for Web of Science ID 000247864300005

    View details for PubMedID 17606865

  • Opinion - Microtubule-organizing centres: a re-evaluation NATURE REVIEWS MOLECULAR CELL BIOLOGY Luders, J., Stearns, T. 2007; 8 (2): 161-167

    Abstract

    The number, length, distribution and polarity of microtubules are largely controlled by microtubule-organizing centres, which nucleate and anchor microtubule minus ends in a process that requires gamma-tubulin. Here we discuss recent evidence indicating that gamma-tubulin-dependent formation of new microtubules is not restricted to conventional microtubule-organizing centres. These findings suggest that the spatio-temporal control of microtubule nucleation is more complex than previously thought, leading us to a re-evaluation of the concept of the microtubule-organizing center.

    View details for DOI 10.1038/nrm2100

    View details for Web of Science ID 000247564900005

    View details for PubMedID 17245416

  • Mechanism limiting centrosome duplication to once per cell cycle NATURE Tsou, M. B., Stearns, T. 2006; 442 (7105): 947-951

    Abstract

    The centrosome organizes the microtubule cytoskeleton and consists of a pair of centrioles surrounded by pericentriolar material. Cells begin the cell cycle with a single centrosome, which duplicates once before mitosis. During duplication, new centrioles grow orthogonally to existing ones and remain engaged (tightly opposed) with those centrioles until late mitosis or early G1 phase, when they become disengaged. The relationship between centriole engagement/disengagement and centriole duplication potential is not understood, and the mechanisms that control these processes are not known. Here we show that centriole disengagement requires the protease separase at anaphase, and that this disengagement licences centriole duplication in the next cell cycle. We describe an in vitro system using Xenopus egg extract and purified centrioles in which both centriole disengagement and centriole growth occur. Centriole disengagement at anaphase is independent of mitotic exit and Cdk2/cyclin E activity, but requires the anaphase-promoting complex and separase. In contrast to disengagement, new centriole growth occurs in interphase, is dependent on Cdk2/cyclin E, and requires previously disengaged centrioles. This suggests that re-duplication of centrioles within a cell cycle is prevented by centriole engagement itself. We propose that the 'once-only' control of centrosome duplication is achieved by temporally separating licensing in anaphase from growth of new centrioles during S phase. The involvement of separase in both centriole disengagement and sister chromatid separation would prevent premature centriole disengagement before anaphase onset, which can lead to multipolar spindles and genomic instability.

    View details for DOI 10.1038/nature04985

    View details for Web of Science ID 000239960500044

    View details for PubMedID 16862117

  • Controlling centrosome number: licenses and blocks CURRENT OPINION IN CELL BIOLOGY Tsou, M. F., Stearns, T. 2006; 18 (1): 74-78

    Abstract

    Centrosomes organize microtubule structures in animal cells. The centrosome duplicates once per cell cycle in most dividing cells via a pathway that relies on a pre-existing centrosome. The molecular mechanism of this 'once and only once' control is not understood, and recent results show that centrosomes can also be assembled by a de novo pathway that bypasses this control. These results require a rethinking of how proper centrosome number is maintained. We propose that the engagement of centrioles with each other normally blocks centrosome re-duplication, and that disengagement of centrioles from each other at the end of mitosis licenses them for duplication in the subsequent cell cycle.

    View details for DOI 10.1016/j.ceb.2005.12.008

    View details for Web of Science ID 000235242700012

    View details for PubMedID 16361091

  • GCP-WD is a gamma-tubulin targeting factor required for centrosomal and chromatin-mediated microtubule nucleation NATURE CELL BIOLOGY Luders, J., Patel, U. K., Stearns, T. 2006; 8 (2): 137-U10

    Abstract

    The gamma-tubulin ring complex (gammaTuRC) is a large multi-protein complex that is required for microtubule nucleation from the centrosome. Here, we show that the GCP-WD protein (originally named NEDD1) is the orthologue of the Drosophila Dgrip71WD protein, and is a subunit of the human gammaTuRC. GCP-WD has the properties of an attachment factor for the gammaTuRC: depletion or inhibition of GCP-WD results in loss of the gammaTuRC from the centrosome, abolishing centrosomal microtubule nucleation, although the gammaTuRC is intact and able to bind to microtubules. GCP-WD depletion also blocks mitotic chromatin-mediated microtubule nucleation, resulting in failure of spindle assembly. Mitotic phosphorylation of GCP-WD is required for association of gamma-tubulin with the spindle, separately from association with the centrosome. Our results indicate that GCP-WD broadly mediates targeting of the gammaTuRC to sites of microtubule nucleation and to the mitotic spindle, which is essential for spindle formation.

    View details for DOI 10.1038/ncb1349

    View details for Web of Science ID 000235059800008

    View details for PubMedID 16378099

  • Insights into microtubule nucleation from the crystal structure of human gamma-tubulin NATURE Aldaz, H., Rice, L. M., Stearns, T., Agard, D. A. 2005; 435 (7041): 523-527

    Abstract

    Microtubules are hollow polymers of alphabeta-tubulin that show GTP-dependent assembly dynamics and comprise a critical part of the eukaryotic cytoskeleton. Initiation of new microtubules in vivo requires gamma-tubulin, organized as an oligomer within the 2.2-MDa gamma-tubulin ring complex (gamma-TuRC) of higher eukaryotes. Structural insight is lacking regarding gamma-tubulin, its oligomerization and how it promotes microtubule assembly. Here we report the 2.7-A crystal structure of human gamma-tubulin bound to GTP-gammaS (a non-hydrolysable GTP analogue). We observe a 'curved' conformation for gamma-tubulin-GTPgammaS, similar to that seen for GDP-bound, unpolymerized alphabeta-tubulin. Tubulins are thought to represent a distinct class of GTP-binding proteins, and conformational switching in gamma-tubulin might differ from the nucleotide-dependent switching of signalling GTPases. A crystal packing interaction replicates the lateral contacts between alpha- and beta-tubulins in the microtubule, and this association probably forms the basis for gamma-tubulin oligomerization within the gamma-TuRC. Laterally associated gamma-tubulins in the gamma-TuRC might promote microtubule nucleation by providing a template that enhances the intrinsically weak lateral interaction between alphabeta-tubulin heterodimers. Because they are dimeric, alphabeta-tubulins cannot form microtubule-like lateral associations in the curved conformation. The lateral array of gamma-tubulins we observe in the crystal reveals a unique functional property of a monomeric tubulin.

    View details for DOI 10.1038/nature03586

    View details for Web of Science ID 000229337800060

    View details for PubMedID 15917813

  • Mammalian cells lack checkpoints for tetraploidy, aberrant centrosome number, and cytokinesis failure BMC CELL BIOLOGY Wong, C., Stearns, T. 2005; 6

    Abstract

    Mammalian cells have been reported to have a p53-dependent tetraploidy checkpoint that blocks cell cycle progression in G1 in response to failure of cell division. In most cases where the tetraploidy checkpoint has been observed cell division was perturbed by anti-cytoskeleton drug treatments. However, other evidence argues against the existence of a tetraploidy checkpoint. Cells that have failed to divide differ from normal cells in having two nuclei, two centrosomes, a decreased surface to volume ratio, and having undergone an abortive cytokinesis. We tested each of these to determine which, if any, cause a G1 cell cycle arrest.Primary human diploid fibroblasts with intact cell cycle checkpoints were used in all experiments. Synchronized cells exhibited G1 arrest in response to division failure caused by treatment with either cytochalasin or the myosin II inhibitor blebbistatin. The role of tetraploidy, aberrant centrosome number, and increased cell size were tested by cell/cell and cell/cytoplast fusion experiments; none of these conditions resulted in G1 arrest. Instead we found that various drug treatments of the cells resulted in cellular damage, which was the likely cause of the arrest. When cytokinesis was blocked in the absence of damage-inducing drug treatments no G1 arrest was observed.We show that neither tetraploidy, aberrant centrosome number, cell size, nor failure of cytokinesis lead to G1 arrest, suggesting that there is no tetraploidy checkpoint. Rather, certain standard synchronization treatments cause damage that is the likely cause of G1 arrest. Since tetraploid cells can cycle when created with minimal manipulation, previous reports of a tetraploidy checkpoint can probably be explained by side effects of the drug treatments used to observe them.

    View details for DOI 10.1186/1471-2121-6-6

    View details for Web of Science ID 000227559200001

    View details for PubMedID 15713235

  • Centrosome number is controlled by a centrosome-intrinsic block to reduplication NATURE CELL BIOLOGY Wong, C., Stearns, T. 2003; 5 (6): 539-544

    Abstract

    The centrosome duplicates once in S phase. To determine whether there is a block in centrosome reduplication, we used a cell fusion assay to compare the duplication potential of unduplicated G1 centrosomes and recently duplicated G2 centrosomes. By fusing cells in different cell cycle stages, we found that G2 centrosomes were unable to reduplicate in a cellular environment that supports centrosome duplication. Furthermore, G2 cytoplasm did not inhibit centrosome duplication in fused cells, indicating that the block to reduplication is intrinsic to the centrosomes rather than the cytoplasm. To test the underlying mechanism, we created mononucleate G1 cells with two centrosomes by fusing cells with enucleated cytoplasts. Both centrosomes duplicated, indicating that the block is not controlled by centrosome:nucleus ratio. We also found that human primary cells have tight control over centrosome number during prolonged S-phase arrest and that this control is partially abrogated in transformed cells. This suggests a link between the control of centrosome duplication and maintenance of genomic stability.

    View details for DOI 10.1038/ncb993

    View details for Web of Science ID 000183202800015

    View details for PubMedID 12766773

  • Centrosome biology: A SAS-sy centriole in the cell cycle CURRENT BIOLOGY Wong, C., Stearns, T. 2003; 13 (9): R351-R352

    Abstract

    A novel protein in Caenorhabditis elegans, SAS-4, is a component of centrioles and is required for centriole duplication. Depletion of SAS-4 results in stunted centrioles and a smaller centrosome, suggesting a link to organelle size control.

    View details for DOI 10.1016/S0960-9822(03)00273-2

    View details for Web of Science ID 000182639900009

    View details for PubMedID 12725749

  • epsilon-tubulin is required for centriole duplication and microtubule organization NATURE CELL BIOLOGY Chang, P., Giddings, T. H., Winey, M., Stearns, T. 2003; 5 (1): 71-76

    Abstract

    Centrosomes nucleate microtubules and serve as poles of the mitotic spindle. Centrioles are a core component of centrosomes and duplicate once per cell cycle. We previously identified epsilon-tubulin as a new member of the tubulin superfamily that localizes asymmetrically to the two centrosomes after duplication. We show that recruitment of epsilon-tubulin to the new centrosome can only occur after exit from S phase and that epsilon-tubulin is associated with the sub-distal appendages of mature centrioles. Xenopus laevis epsilon-tubulin was cloned and shown to be similar to human epsilon-tubulin in both sequence and localization. Depletion of epsilon-tubulin from Xenopus egg extracts blocks centriole duplication in S phase and formation of organized centrosome-independent microtubule asters in M phase. We conclude that epsilon-tubulin is a component of the sub-distal appendages of the centriole, explaining its asymmetric localization to old and new centrosomes, and that epsilon-tubulin is required for centriole duplication and organization of the pericentriolar material.

    View details for DOI 10.1038/ncb900

    View details for Web of Science ID 000180223700016

    View details for PubMedID 12510196

  • gamma-tubulin CURRENT BIOLOGY Patel, U., Stearns, T. 2002; 12 (12): R408-R409

    View details for Web of Science ID 000176468000005

    View details for PubMedID 12123586

  • Systematic structure-function analysis of the small GTPase Arf1 in yeast MOLECULAR BIOLOGY OF THE CELL Click, E. S., Stearns, T., Botstein, D. 2002; 13 (5): 1652-1664

    Abstract

    Members of the ADP-ribosylation factor (Arf) family of small GTPases are implicated in vesicle traffic in the secretory pathway, although their precise function remains unclear. We generated a series of 23 clustered charge-to-alanine mutations in the Arf1 protein of Saccharomyces cerevisiae to determine the portions of this protein important for its function in cells. These mutants display a number of phenotypes, including conditional lethality at high or low temperature, defects in glycosylation of invertase, dominant lethality, fluoride sensitivity, and synthetic lethality with the arf2 null mutation. All mutations were mapped onto the available crystal structures for Arf1p: Arf1p bound to GDP, to GTP, and complexed with the regulatory proteins ArfGEF and ArfGAP. From this systematic structure-function analysis we demonstrate that all essential mutations studied map to one hemisphere of the protein and provide strong evidence in support of the proposed ArfGEF contact site on Arf1p but minimal evidence in support of the proposed ArfGAP-binding site. In addition, we describe the isolation of a spatially distant intragenic suppressor of a dominant lethal mutation in the guanine nucleotide-binding region of Arf1p.

    View details for DOI 10.1091/mbc.02-01-0007

    View details for Web of Science ID 000175812900018

    View details for PubMedID 12006660

  • GCP5 and GCP6: Two new members of the human gamma-tubulin complex MOLECULAR BIOLOGY OF THE CELL Murphy, S. M., Preble, A. M., Patel, U. K., O'Connell, K. L., Dias, D. P., Moritz, M., Agard, D., Stults, J. T., Stearns, T. 2001; 12 (11): 3340-3352

    Abstract

    The gamma-tubulin complex is a large multiprotein complex that is required for microtubule nucleation at the centrosome. Here we report the purification and characterization of the human gamma-tubulin complex and the identification of its subunits. The human gamma-tubulin complex is a ring of ~25 nm, has a subunit structure similar to that reported for gamma-tubulin complexes from other species, and is able to nucleate microtubule polymerization in vitro. Mass spectrometry analysis of the human gamma-tubulin complex components confirmed the presence of four previously identified components (gamma-tubulin and gamma-tubulin complex proteins [GCPs] 2, 3, and 4) and led to the identification of two new components, GCP5 and GCP6. Sequence analysis revealed that the GCPs share five regions of sequence similarity and define a novel protein superfamily that is conserved in metazoans. GCP5 and GCP6, like other components of the gamma-tubulin complex, localize to the centrosome and associate with microtubules, suggesting that the entire gamma-tubulin complex takes part in both of these interactions. Stoichiometry experiments revealed that there is a single copy of GCP5 and multiple copies of gamma-tubulin, GCP2, GCP3, and GCP4 within the gamma-tubulin complex. Thus, the gamma-tubulin complex is conserved in structure and function, suggesting that the mechanism of microtubule nucleation is conserved.

    View details for Web of Science ID 000172357200004

    View details for PubMedID 11694571

  • Centrosome duplication: A centriolar pas de deux CELL Stearns, T. 2001; 105 (4): 417-420

    View details for Web of Science ID 000168840700001

    View details for PubMedID 11371338

  • The DNA damage checkpoint signal in budding yeast is nuclear limited MOLECULAR CELL Demeter, J., Lee, S. E., Haber, J. E., Stearns, T. 2000; 6 (2): 487-492

    Abstract

    The nature of the DNA damage-induced checkpoint signal that causes the arrest of cells prior to mitosis is unknown. To determine if this signal is transmitted through the cytoplasm or is confined to the nucleus, we created binucleate heterokaryon yeast cells in which one nucleus suffered an unrepairable double-strand break, and the second nucleus was undamaged. In most of these binucleate cells, the damaged nucleus arrested prior to spindle elongation, while the undamaged nucleus completed mitosis, even when the strength of the damage signal was increased. The arrest of the damaged nucleus was dependent upon the function of the RAD9 checkpoint gene. Thus, the DNA damage checkpoint causing G2/M arrest is regulated by a signal that is nuclear limited.

    View details for Web of Science ID 000089166100025

    View details for PubMedID 10983994

  • delta-Tubulin and epsilon-tubulin: two new human centrosomal tubulins reveal new aspects of centrosome structure and function NATURE CELL BIOLOGY Chang, P., Stearns, T. 2000; 2 (1): 30-35

    Abstract

    The centrosome organizes microtubules, which are made up of alpha-tubulin and beta-tubulin, and contains centrosome-bound gamma-tubulin, which is involved in microtubule nucleation. Here we identify two new human tubulins and show that they are associated with the centrosome. One is a homologue of the Chlamydomonas delta-tubulin Uni3, and the other is a new tubulin, which we have named epsilon-tubulin. Localization of delta-tubulin and epsilon-tubulin to the centrosome is independent of microtubules, and the patterns of localization are distinct from each other and from that of gamma-tubulin. Delta-tubulin is found in association with the centrioles, whereas epsilon-tubulin localizes to the pericentriolar material. epsilon-Tubulin exhibits a cell-cycle-specific pattern of localization, first associating with only the older of the centrosomes in a newly duplicated pair and later associating with both centrosomes. epsilon-Tubulin thus distinguishes the old centrosome from the new at the level of the pericentriolar material, indicating that there may be a centrosomal maturation event that is marked by the recruitment of epsilon-tubulin.

    View details for Web of Science ID 000084843600016

    View details for PubMedID 10620804

  • Arrest, adaptation, and recovery following a chromosome double-strand break in Saccharomyces cerevisiae COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY Lee, S. E., Pellicioli, A., Demeter, J., Vaze, M. P., Gasch, A. P., Malkova, A., Brown, P. O., Botstein, D., Stearns, T., Foiani, M., Haber, J. E. 2000; 65: 303-314

    View details for Web of Science ID 000169676800031

    View details for PubMedID 12760044

  • gamma-Tubulin complexes: size does matter TRENDS IN CELL BIOLOGY Jeng, R., Stearns, T. 1999; 9 (9): 339-342

    Abstract

    gamma-Tubulin is a conserved component of all microtubule-organizing centres and is required for these organelles to nucleate microtubule polymerization. However, the mechanism of nucleation is not known. In addition to its localization to organizing centres, a large pool of gamma-tubulin exists in the cytoplasm in a complex with other proteins. The size of the gamma-tubulin complex and number of associated proteins vary among organisms, and the functional significance of these differences is unknown. Recently, the nature of these gamma-tubulin complexes has been explored in different organisms, and this has led us closer to a molecular understanding of microtubule nucleation.

    View details for Web of Science ID 000082139400002

    View details for PubMedID 10461186

  • Components of an SCE ubiquitin ligase localize to the centrosome and regulate the centrosome duplication cycle GENES & DEVELOPMENT Freed, E., Lacey, K. R., Huie, P., Lyapina, S. A., Deshaies, R. J., Stearns, T., Jackson, P. K. 1999; 13 (17): 2242-2257

    Abstract

    Centrosomes organize the mitotic spindle to ensure accurate segregation of the chromosomes in mitosis. The mechanism that ensures accurate duplication and separation of the centrosomes underlies the fidelity of chromosome segregation, but remains unknown. In Saccharomyces cerevisiae, entry into S phase and separation of spindle pole bodies each require CDC4 and CDC34, which encode components of an SCF (Skp1-cullin-F-box) ubiquitin ligase, but a direct (SCF) connection to the spindle pole body is unknown. Using immunofluorescence microscopy, we show that in mammalian cells the Skp1 protein and the cullin Cul1 are localized to interphase and mitotic centrosomes and to the cytoplasm and nucleus. Deconvolution and immunoelectron microscopy suggest that Skp1 forms an extended pericentriolar structure that may function to organize the centrosome. Purified centrosomes also contain Skp1, and Cul1 modified by the ubiquitin-like molecule NEDD8, suggesting a role for NEDD8 in targeting. Using an in vitro assay for centriole separation in Xenopus extracts, antibodies to Skp1 or Cul1 block separation. Proteasome inhibitors block both centriole separation in vitro and centrosome duplication in Xenopus embryos. We identify candidate centrosomal F-box proteins, suggesting that distinct SCF complexes may direct proteolysis of factors mediating multiple steps in the centrosome cycle.

    View details for Web of Science ID 000082647200006

    View details for PubMedID 10485847

  • Primer - The centrosome CURRENT BIOLOGY Urbani, L., Stearns, T. 1999; 9 (9): R315-R317

    View details for Web of Science ID 000080232900004

    View details for PubMedID 10322119

  • Cyclin-dependent kinase control of centrosome duplication PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lacey, K. R., Jackson, P. K., Stearns, T. 1999; 96 (6): 2817-2822

    Abstract

    Centrosomes nucleate microtubules and duplicate once per cell cycle. This duplication and subsequent segregation in mitosis results in maintenance of the one centrosome/cell ratio. Centrosome duplication occurs during the G1/S transition in somatic cells and must be coupled to the events of the nuclear cell cycle; failure to coordinate duplication and mitosis results in abnormal numbers of centrosomes and aberrant mitoses. Using both in vivo and in vitro assays, we show that centrosome duplication in Xenopus laevis embryos requires cyclin/cdk2 kinase activity. Injection of the cdk (cyclin-dependent kinase) inhibitor p21 into one blastomere of a dividing embryo blocks centrosome duplication in that blastomere; the related cdk inhibitor p27 has a similar effect. An in vitro system using Xenopus extracts carries out separation of the paired centrioles within the centrosome. This centriole separation activity is dependent on cyclin/cdk2 activity; depletion of either cdk2 or of the two activating cyclins, cyclin A and cyclin E, eliminates centriole separation activity. In addition, centriole separation is inhibited by the mitotic state, suggesting a mechanism of linking the cell cycle to periodic duplication of the centrosome.

    View details for Web of Science ID 000079224500048

    View details for PubMedID 10077594

  • Alf1p, a CLIP-170 domain-containing protein, is functionally and physically associated with alpha-tubulin JOURNAL OF CELL BIOLOGY Feierbach, B., Nogales, E., Downing, K. H., Stearns, T. 1999; 144 (1): 113-124

    Abstract

    Tubulin is a heterodimer of alpha- and beta-tubulin polypeptides. Assembly of the tubulin heterodimer in vitro requires the CCT chaperonin complex, and a set of five proteins referred to as the tubulin cofactors (Tian, F., Y. Huang, H. Rommelaere, J. Vandekerckhove, C. Ampe, and N.J. Cowan. 1996. Cell. 86:287-296; Tian, G., S.A. Lewis, B. Feierbach, T. Stearns, H. Rommelaere, C. Ampe, and N.J. Cowan. 1997. J. Cell Biol. 138:821-832). We report the characterization of Alf1p, the yeast ortholog of mammalian cofactor B. Alf1p interacts with alpha-tubulin in both two-hybrid and immunoprecipitation assays. Alf1p and cofactor B contain a single CLIP-170 domain, which is found in several microtubule-associated proteins. Mutation of the CLIP-170 domain in Alf1p disrupts the interaction with alpha-tubulin. Mutations in alpha-tubulin that disrupt the interaction with Alf1p map to a domain on the cytoplasmic face of alpha-tubulin; this domain is distinct from the region of interaction between alpha-tubulin and beta-tubulin. Alf1p-green fluorescent protein (GFP) is able to associate with microtubules in vivo, and this localization is abolished either by mutation of the CLIP-170 domain in Alf1p, or by mutation of the Alf1p-binding domain in alpha-tubulin. Analysis of double mutants constructed between null alleles of ALF1 and PAC2, which encodes the other yeast alpha-tubulin cofactor, suggests that Alf1p and Pac2p act in the same pathway leading to functional alpha-tubulin. The phenotype of overexpression of ALF1 suggests that Alf1p can act to sequester alpha-tubulin from interaction with beta-tubulin, raising the possibility that it plays a regulatory role in the formation of the tubulin heterodimer.

    View details for Web of Science ID 000078084800011

    View details for PubMedID 9885248

  • Cytoskeletal dynamics in yeast METHODS IN CELL BIOLOGY, VOL 58 Carminati, J. L., Stearns, T. 1999; 58: 87-105

    View details for Web of Science ID 000165166500006

    View details for PubMedID 9891376

  • Centrosome reduction during mouse spermiogenesis DEVELOPMENTAL BIOLOGY Manandhar, G., Sutovsky, P., Joshi, H. C., Stearns, T., Schatten, G. 1998; 203 (2): 424-434

    Abstract

    The sperm does not contribute the centrosome during murine fertilization. To determine the manner in which a functional centrosome is reduced, we have studied centrosome degeneration during spermiogenesis of mice. The round spermatids display normal centrosomes consisting of a pair of centrioles along with gamma-tubulin containing foci. However, they do not seem to organize microtubules. Elongating spermatids display gamma-tubulin spots in the neck region, while microtubules are organized from the perinuclear ring as the manchette. Electron microscopic studies using immunogold labeling revealed that gamma-tubulin is mainly localized in the centriolar adjunct from which an aster of microtubules emanates. Microtubules repolymerized randomly in the cytoplasm after nocodazole treatment and reversal. gamma-Tubulin dissociates from the neck region and is discarded in the residual bodies during spermiation. The distal centriole degenerates during testicular stage of spermiogenesis, while the proximal centriole is lost during epididymal stage. Loss of centrosomal protein and centrioles in mouse sperm further confirm the maternal inheritance of centrosome during murine fertilization.

    View details for Web of Science ID 000077138400016

    View details for PubMedID 9808791

  • The mammalian gamma-tubulin complex contains homologues of the yeast spindle pole body components Spc97p and Spc98p JOURNAL OF CELL BIOLOGY Murphy, S. M., Urbani, L., Stearns, T. 1998; 141 (3): 663-674

    Abstract

    gamma-Tubulin is a universal component of microtubule organizing centers where it is believed to play an important role in the nucleation of microtubule polymerization. gamma-Tubulin also exists as part of a cytoplasmic complex whose size and complexity varies in different organisms. To investigate the composition of the cytoplasmic gamma-tubulin complex in mammalian cells, cell lines stably expressing epitope-tagged versions of human gamma-tubulin were made. The epitope-tagged gamma-tubulins expressed in these cells localize to the centrosome and are incorporated into the cytoplasmic gamma-tubulin complex. Immunoprecipitation of this complex identifies at least seven proteins, with calculated molecular weights of 48, 71, 76, 100, 101, 128, and 211 kD. We have identified the 100- and 101-kD components of the gamma-tubulin complex as homologues of the yeast spindle pole body proteins Spc97p and Spc98p, and named the corresponding human proteins hGCP2 and hGCP3. Sequence analysis revealed that these proteins are not only related to their respective homologues, but are also related to each other. GCP2 and GCP3 colocalize with gamma-tubulin at the centrosome, cosediment with gamma-tubulin in sucrose gradients, and coimmunoprecipitate with gamma-tubulin, indicating that they are part of the gamma-tubulin complex. The conservation of a complex involving gamma-tubulin, GCP2, and GCP3 from yeast to mammals suggests that structurally diverse microtubule organizing centers such as the yeast spindle pole body and the animal centrosome share a common molecular mechanism for microtubule nucleation.

    View details for Web of Science ID 000073499300009

    View details for PubMedID 9566967

  • Parallel analysis of genetic selections using whole genome oligonucleotide arrays PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Cho, R. J., Fromont-Racine, M., Wodicka, L., Feierbach, B., Stearns, T., Legrain, P., Lockhart, D. J., Davis, R. W. 1998; 95 (7): 3752-3757

    Abstract

    Thousands of genes have recently been sequenced in organisms ranging from Escherichia coli to human. For the majority of these genes, however, available sequence does not define a biological role. Efficient functional characterization of these genes requires strategies for scaling genetic analyses to the whole genome level. Plasmid-based library selections are an established approach to the functional analysis of uncharacterized genes and can help elucidate biological function by identifying, for example, physical interactors for a gene and genetic enhancers and suppressors of mutant phenotypes. The application of these selections to every gene in a eukaryotic genome, however, is generally limited by the need to manipulate and sequence hundreds of DNA plasmids. We present an alternative approach in which identification of nucleic acids is accomplished by direct hybridization to high-density oligonucleotide arrays. Based on the complete sequence of Saccharomyces cerevisiae, high-density arrays containing oligonucleotides complementary to every gene in the yeast genome have been designed and synthesized. Two-hybrid protein-protein interaction screens were carried out for S. cerevisiae genes implicated in mRNA splicing and microtubule assembly. Hybridization of labeled DNA derived from positive clones is sufficient to characterize the results of a screen in a single experiment, allowing rapid determination of both established and previously unknown biological interactions. These results demonstrate the use of oligonucleotide arrays for the analysis of two-hybrid screens. This approach should be generally applicable to the analysis of a range of genetic selections.

    View details for Web of Science ID 000072848500079

    View details for PubMedID 9520439

  • Expression of amino- and carboxyl-terminal gamma- and alpha-tubulin mutants in cultured epithelial cells JOURNAL OF BIOLOGICAL CHEMISTRY Leask, A., Stearns, T. 1998; 273 (5): 2661-2668

    Abstract

    Three distinct tubulin proteins are essential for microtubule function: alpha-, beta-, and gamma-tubulin. After translation, alpha- and beta-tubulin proteins combine into a soluble, 7 S heterodimer that is multimerized to form the microtubule filament. Conversely, gamma-tubulin combines with several proteins into a soluble, 25 S multi-protein particle, the gammasome that is essential for nucleating microtubule filaments at the centrosome. The proteins that assist tubulins in executing their specific functions are largely unknown. As an initial approach to address this issue, we first decided to identify domains of mammalian alpha- and gamma-tubulin necessary for their function by creating mutant mammalian alpha- and gamma-tubulin (both deletion and hybrid mutants) and assaying their behavior in stably transfected Chinese hamster ovary epithelial cells. First, we demonstrated that addition of a carboxyl-terminal epitope tag had no effect on the subcellular localization of either alpha- and gamma-tubulin. Second, we found that both the amino and carboxyl termini of gamma-tubulin were essential for its incorporation into the gammasome. Third, we found that the amino and carboxyl termini of alpha-tubulin were necessary for incorporation of the alpha-beta-tubulin heterodimer into the microtubule filament network. In general, alpha-tubulin sequences could not replace those of gamma-tubulin and vice versa. Taken together, these results suggest that the amino and carboxyl termini of alpha- and gamma-tubulin and perhaps regions throughout these proteins were necessary for their specific functions.

    View details for Web of Science ID 000071736600025

    View details for PubMedID 9446570

  • Nucleation and capture of large cell surface-associated microtubule arrays that are not located near centrosomes in certain cochlear epithelial cells JOURNAL OF ANATOMY Tucker, J. B., Mogensen, M. M., Henderson, C. G., Doxsey, S. J., Wright, M., Stearns, T. 1998; 192: 119-130

    Abstract

    This report deals with the as yet undetermined issue of whether cell-surface associated microtubules in certain cochlear epithelial cells are centrosomally nucleated and subsequently migrate to microtubule-capturing sites located at the surface regions in question. Alternatively, the cells may possess additional nucleating sites which are noncentrosomal and surface-associated. These alternative possibilities have been investigated for highly polarised epithelial cells called supporting cells in the mouse and guinea pig organ of Corti using antibodies to pericentrin and gamma-tubulin. There is substantial evidence that both proteins are essential components of microtubule-nucleating sites in cells generally. Each mature supporting cell possesses a large microtubule array that is remotely located with respect to its centrosome (more than 10 microns away). The antibodies bind to a cell's centrosome. No binding has been detected at 2 other microtubule-organising centres that are associated with the ends of the centrosomally-remote microtubule array while it is being constructed. Such arrays include thousands of microtubules in some of the cell types that have been examined. If all a cell's microtubules are nucleated by its centrosome then the findings reported above imply that microtubules escape from the centrosomal nucleating site and migrate to a new location. Furthermore capture of the plus and minus ends of the errant microtubules is taking place because both ends of a centrosomally-remote microtubule array are attached to sites that are precisely positioned at certain cell surface locations. Minus ends are locating targets with an exactitude comparable to that which has been demonstrated for plus ends in certain cell types. These cells apparently operate a single control centre strategy for microtubule nucleation that is complemented by precise positioning of plus and minus end-capturing sites at the cell surface.

    View details for Web of Science ID 000072900100012

    View details for PubMedID 9568567

  • Cytoskeleton: Anatomy of an organizing center CURRENT BIOLOGY Marschall, L. G., Stearns, T. 1997; 7 (12): R754-R756

    Abstract

    One component of the yeast spindle pole body, Spc42p, has been found to form a crystalline array within one of the central layers of the structure; the Spc42p crystal might provide a scaffold around which the spindle pole body is assembled, and could be involved in regulating the size of the spindle pole body.

    View details for Web of Science ID A1997YL44000009

    View details for PubMedID 9382821

  • The cell center at 100 CELL Stearns, T., Winey, M. 1997; 91 (3): 303-309

    View details for Web of Science ID A1997YD94100004

    View details for PubMedID 9363939

  • Motoring to the finish: Kinesin and dynein work together to orient the yeast mitotic spindle JOURNAL OF CELL BIOLOGY Stearns, T. 1997; 138 (5): 957-960

    View details for Web of Science ID A1997XW46300002

    View details for PubMedID 9281575

  • Tubulin subunits exist in an activated conformational state generated and maintained by protein cofactors JOURNAL OF CELL BIOLOGY Tian, G. L., Lewis, S. A., Feierbach, B., Stearns, T., Rommelaere, H., Ampe, C., Cowan, N. J. 1997; 138 (4): 821-832

    Abstract

    The production of native alpha/beta tubulin heterodimer in vitro depends on the action of cytosolic chaperonin and several protein cofactors. We previously showed that four such cofactors (termed A, C, D, and E) together with native tubulin act on beta-tubulin folding intermediates generated by the chaperonin to produce polymerizable tubulin heterodimers. However, this set of cofactors generates native heterodimers only very inefficiently from alpha-tubulin folding intermediates produced by the same chaperonin. Here we describe the isolation, characterization, and genetic analysis of a novel tubulin folding cofactor (cofactor B) that greatly enhances the efficiency of alpha-tubulin folding in vitro. This enabled an integrated study of alpha- and beta-tubulin folding: we find that the pathways leading to the formation of native alpha- and beta-tubulin converge in that the folding of the alpha subunit requires the participation of cofactor complexes containing the beta subunit and vice versa. We also show that sequestration of native alpha-or beta-tubulins by complex formation with cofactors results in the destabilization and decay of the remaining free subunit. These data demonstrate that tubulin folding cofactors function by placing and/or maintaining alpha-and beta-tubulin polypeptides in an activated conformational state required for the formation of native alpha/beta heterodimers, and imply that each subunit provides information necessary for the proper folding of the other.

    View details for Web of Science ID A1997XU36000008

    View details for PubMedID 9265649

  • Microtubules orient the mitotic spindle in yeast through dynein-dependent interactions with the cell cortex JOURNAL OF CELL BIOLOGY Carminati, J. L., Stearns, T. 1997; 138 (3): 629-641

    Abstract

    Proper orientation of the mitotic spindle is critical for successful cell division in budding yeast. To investigate the mechanism of spindle orientation, we used a green fluorescent protein (GFP)-tubulin fusion protein to observe microtubules in living yeast cells. GFP-tubulin is incorporated into microtubules, allowing visualization of both cytoplasmic and spindle microtubules, and does not interfere with normal microtubule function. Microtubules in yeast cells exhibit dynamic instability, although they grow and shrink more slowly than microtubules in animal cells. The dynamic properties of yeast microtubules are modulated during the cell cycle. The behavior of cytoplasmic microtubules revealed distinct interactions with the cell cortex that result in associated spindle movement and orientation. Dynein-mutant cells had defects in these cortical interactions, resulting in misoriented spindles. In addition, microtubule dynamics were altered in the absence of dynein. These results indicate that microtubules and dynein interact to produce dynamic cortical interactions, and that these interactions result in the force driving spindle orientation.

    View details for Web of Science ID A1997XR47800013

    View details for PubMedID 9245791

  • Synaptically coupled central nervous system neurons lack centrosomal gamma-tubulin NEUROSCIENCE LETTERS Leask, A., Obrietan, K., Stearns, T. 1997; 229 (1): 17-20

    Abstract

    In cycling cells, microtubule assembly is initiated at the centrosome and requires the centrosomal protein gamma-tubulin. Previously, it was reported that gamma-tubulin is present at the centrosome of cervical ganglion cells undergoing axonal growth, but not in the axons or dendrites. We find that although gamma-tubulin is present at the centrosomes of neurons just beginning to extend processes, it is not associated with centrosomes in hypothalamic and cortical neurons on which functional synaptic connections have formed. In contrast, another centrosomal protein, pericentrin, is associated with the centrosome at all stages. These results suggest that centrosomal microtubule nucleation is required for early stages of neurogenesis to supply sufficient microtubule polymer to support rapid axonal growth, but is not required for maintenance of axonal microtubules in synaptically coupled neurons.

    View details for Web of Science ID A1997XK37000005

    View details for PubMedID 9224791

  • Centrosomes isolated from Spisula solidissima oocytes contain rings and an unusual stoichiometric ratio of alpha/beta tubulin JOURNAL OF CELL BIOLOGY Vogel, J. M., Stearns, T., Rieder, C. L., Palazzo, R. E. 1997; 137 (1): 193-202

    Abstract

    Centrosome-dependent microtubule nucleation involves the interaction of tubulin subunits with pericentriolar material. To study the biochemical and structural basis of centrosome-dependent microtubule nucleation, centrosomes capable of organizing microtubules into astral arrays were isolated from parthenogenetically activated Spisula solidissima oocytes. Intermediate voltage electron microscopy tomography revealed that each centrosome was composed of a single centriole surrounded by pericentriolar material that was studded with ring-shaped structures approximately 25 nm in diameter and <25 nm in length. A number of proteins copurified with centrosomes including: (a) proteins that contained M-phase-specific phosphoepitopes (MPM-2), (b) alpha-, beta-, and gamma-tubulins, (c) actin, and (d) three low molecular weight proteins of <20 kD. gamma-Tubulin was not an MPM-2 phosphoprotein and was the most abundant form of tubulin in centrosomes. Relatively little alpha- or beta-tubulin copurified with centrosomes, and the ratio of alpha- to beta-tubulin in centrosomes was not 1:1 as expected, but rather 1:4.6, suggesting that centrosomes contain beta-tubulin that is not dimerized with alpha-tubulin.

    View details for Web of Science ID A1997WU02800017

    View details for PubMedID 9105047

  • Centrosomal deployment of gamma-tubulin and pericentrin: Evidence for a microtubule-nucleating domain and a minus-end docking domain in certain mouse epithelial cells CELL MOTILITY AND THE CYTOSKELETON Mogensen, M. M., Mackie, J. B., Doxsey, S. J., Stearns, T., Tucker, J. B. 1997; 36 (3): 276-290

    Abstract

    This report provides evidence for two functionally and spatially distinct centrosomal domains in certain mouse cochlear epithelial cells. The vast majority of microtubules elongate from sites associated with the apical cell surface in these cells rather than from pericentriolar material surrounding the immediate environs of their apically situate centrioles. The distribution of gamma-tubulin and pericentrin at cell apices has been examined while microtubule nucleation is progressing because these centrosomal proteins are believed to be essential for microtubule nucleation. Antibodies to both proteins bind to pericentriolar regions but no binding has been detected at the apical cell surface-associated sites where the ends of thousands of recently nucleated microtubules are concentrated. Sparse transient microtubule populations can be detected between pericentriolar regions and surface sites while microtubule assembly advances. A procedure apparently operates in which the pericentriolar region functions as a microtubule-nucleating domain and the cell surface-associated sites operate as docking domains which capture the minus ends of microtubules that migrate to them shortly after nucleation. Docking domains may include some components of the pericentriolar material that have been relocated at the cell apex. A docking element hypothesis for centrosomal control of minus end positioning and dynamics in animal cells generally is proposed. This investigation has also shown that the concentration of gamma-tubulin and pericentrin around centrioles differs spatially and quantitatively in ways that are characteristic for the four cell types studied. Some of these characteristics can be related to differences in control of microtubule number and positioning.

    View details for Web of Science ID A1997WL72400008

    View details for PubMedID 9067623

  • Analysis of Tub4p, a yeast gamma-tubulin-like protein: Implications for microtubule-organizing center function JOURNAL OF CELL BIOLOGY Marschall, L. G., Jeng, R. L., Mulholland, J., Stearns, T. 1996; 134 (2): 443-454

    Abstract

    gamma-Tubulin is a conserved component of microtubule-organizing centers and is thought to be involved in microtubule nucleation. A recently discovered Saccharomyces cerevisiae gene (TUB4) encodes a tubulin that is related to, but divergent from, gamma-tubulins. TUB4 is essential for cell viability, and epitope-tagged Tub4 protein (Tub4p) is localized to the spindle pole body (Sobel, S.G., and M. Snyder. 1995.J. Cell Biol. 131:1775-1788). We have characterized the expression of TUB4, the association of Tub4p with the spindle pole body, and its role in microtubule organization. Tub4p is a minor protein in the cell, and expression of TUB4 is regulated in a cell cycle-dependent manner. Wild-type Tub4p is localized to the spindle pole body, and a Tub4p-green fluorescent protein fusion is able to associate with a preexisting spindle pole body, suggesting that there is dynamic exchange between cytoplasmic and spindle pole body forms of Tub4p. Perturbation of Tub4p function, either by conditional mutation or by depletion of the protein, results in spindle as well as spindle pole body defects, but does not eliminate the ability of microtubules to regrow from, or remain attached to, the spindle pole body. The spindle pole bodies in tub4 mutant cells duplicate but do not separate, resulting in a monopolar spindle. EM revealed that one spindle pole body of the duplicated pair appears to be defective for the nucleation of microtubules. These results offer insight into the role of gamma-tubulin in microtubule-organizing center function.

    View details for Web of Science ID A1996UX94600015

    View details for PubMedID 8707828

  • Cytoskeleton: Microtubule nucleation takes shape CURRENT BIOLOGY Murphy, S. M., Stearns, T. 1996; 6 (6): 642-644

    Abstract

    The centrosomal protein gamma-tubulin is part of a ring-shaped complex that can induce microtubule polymerization. This complex may explain how the centrosome nucleates microtubule polymerization, and thereby organizes the microtubule cytoskeleton.

    View details for Web of Science ID A1996UR92800012

    View details for PubMedID 8793282

  • GREEN FLUORESCENT PROTEIN - THE GREEN-REVOLUTION CURRENT BIOLOGY Stearns, T. 1995; 5 (3): 262-264

    Abstract

    Green fluorescent protein allows gene expression and protein localization to be observed in living cells.

    View details for Web of Science ID A1995QM82300014

    View details for PubMedID 7780736

  • MUTATIONAL ANALYSIS OF SACCHAROMYCES-CEREVISIAE ARF1 JOURNAL OF BIOLOGICAL CHEMISTRY Kahn, R. A., Clark, J., RULKA, C., Stearns, T., Zhang, C. J., Randazzo, P. A., Terui, T., Cavenagh, M. 1995; 270 (1): 143-150

    Abstract

    Wild type and eight point mutants of Saccharomyces cerevisiae ARF1 were expressed in yeast and bacteria to determine the roles of specific residues in in vivo and in vitro activities. Mutations at either Gly2 or Asp26 resulted in recessive loss of function. It was concluded that N-myristoylation is required for Arf action in cells but not for either nucleotide exchange or cofactor activities in vitro. Asp26 (homologous to Gly12 of p21ras) was essential for the binding of the activating nucleotide, guanosine 5'-3-O-(thio)triphosphate. This is in marked contrast to results obtained after mutagenesis of the homologous residue in p21ras or Gs alpha, and suggests a fundamental difference in the guanine nucleotide binding site of Arf with respect to these other GTP-binding proteins. Two dominant alleles were also identified, one activating dominant ([Q71L]Arf1) and the other ([N126I]) a negative dominant. A conditional allele, [W66R]Arf1, was characterized and shown to have approximately 300-fold lower specific activity in an in vitro Arf assay. Two high-copy suppressors of this conditional phenotype were cloned and sequenced. One of these suppressors, SFS4, was found to be identical to PBS2/HOG4, recently shown to encode a microtubule-associated protein kinase kinase in yeast.

    View details for Web of Science ID A1995QA28700026

    View details for PubMedID 7814365

  • THE FORM AND THE SUBSTANCE NATURE MEDICINE Stearns, T. 1995; 1 (1): 19-20

    View details for Web of Science ID A1995QX55700017

    View details for PubMedID 7584942

  • IN-VITRO RECONSTITUTION OF CENTROSOME ASSEMBLY AND FUNCTION - THE CENTRAL ROLE OF GAMMA-TUBULIN CELL Stearns, T., Kirschner, M. 1994; 76 (4): 623-637

    Abstract

    The centrosome nucleates microtubule polymerization, affecting microtubule number, polarity, and structure. We use an in vitro system based on extracts of Xenopus eggs to examine the role of gamma-tubulin in centrosome assembly and function. gamma-Tubulin is present in the cytoplasm of frog eggs and vertebrate somatic cells in a large approximately 25S complex. The egg extracts assemble centrosomes around sperm centrioles. Formation of a centrosome in the extract requires both the gamma-tubulin complex and ATP and can take place in the absence of microtubules. gamma-Tubulin is not present on the sperm prior to incubation in extract, but is recruited from the cytoplasm during centrosome assembly. The gamma-tubulin complex also binds to microtubules, likely the minus end, independent of the centrosome. These results suggest that gamma-tubulin is an essential component of the link between the centrosome and the microtubule, probably playing a direct role in microtubule nucleation.

    View details for Web of Science ID A1994MZ28500006

    View details for PubMedID 8124706

  • SPECIFICITY DOMAINS DISTINGUISH THE RAS-RELATED GTPASES YPT1 AND SEC4 NATURE Dunn, B., Stearns, T., Botstein, D. 1993; 362 (6420): 563-565

    Abstract

    The essential Ras-related GTPases Ypt1 and Sec4 act at distinct stages of the secretion pathway in the yeast Saccharomyces cerevisiae: Ypt1 is required for vesicular transport from the endoplasmic reticulum to the Golgi apparatus, whereas Sec4 is required for fusion of secretory vesicles to the plasma membrane. Here we use chimaeras of the two proteins to identify a 9-residue segment of Ypt1 that, when substituted for the analogous segment of Sec4, allows the chimaera to perform the minimal functions of both proteins in vivo. This segment corresponds to loop L7 of the p21ras crystal structure. Substitution of a 24-residue Ypt1 segment, including the residues just mentioned, together with 12 residues of Ypt1 corresponding to the 'effector region' of p21ras (loop L2; refs 7,8), transforms Sec4 into a fully functional Ypt1 protein without residual Sec4 function.

    View details for Web of Science ID A1993KW45300061

    View details for PubMedID 8464499

  • Spindle positioning and cell polarity. Current biology Hyman, A. A., Stearns, T. 1992; 2 (9): 469-471

    View details for PubMedID 15335895

  • At the heart of the organizing center. Current biology Cande, W. Z., Stearns, T. 1991; 1 (4): 254-256

    View details for PubMedID 15336136

  • GAMMA-TUBULIN IS A HIGHLY CONSERVED COMPONENT OF THE CENTROSOME CELL Stearns, T., Evans, L., Kirschner, M. 1991; 65 (5): 825-836

    Abstract

    We have cloned and characterized gamma-tubulin genes from both X. laevis and S. pombe, and partial genes from maize, diatom, and a budding yeast. The proteins encoded by these genes are very similar to each other and to the original Aspergillus protein, indicating that gamma-tubulins are an ubiquitous and highly conserved subfamily of the tubulin family. A null mutation of the S. pombe gene is lethal. gamma-tubulin is a minor protein, present at less than 1% the level of alpha- and beta-tubulin, and is limited to the centrosome. In particular, gamma-tubulin is associated with the pericentriolar material, the microtubule-nucleating material of the centrosome. gamma-Tubulin remains associated with the centrosome when microtubules are depolymerized, suggesting that it is an integral component that might play a role in microtubule organization.

    View details for Web of Science ID A1991FP51600013

    View details for PubMedID 1840506

  • ADP RIBOSYLATION FACTOR IS AN ESSENTIAL PROTEIN IN SACCHAROMYCES-CEREVISIAE AND IS ENCODED BY 2 GENES MOLECULAR AND CELLULAR BIOLOGY Stearns, T., Kahn, R. A., Botstein, D., Hoyt, M. A. 1990; 10 (12): 6690-6699

    Abstract

    ADP ribosylation factor (ARF) is a ubiquitous 21-kDa GTP-binding protein in eucaryotes. ARF was first identified in animal cells as the protein factor required for the efficient ADP-ribosylation of the mammalian G protein Gs by cholera toxin in vitro. A gene (ARF1) encoding a protein homologous to mammalian ARF was recently cloned from Saccharomyces cerevisiae (Sewell and Kahn, Proc. Natl. Acad. Sci. USA, 85:4620-4624, 1988). We have found a second gene encoding ARF in S. cerevisiae, ARF2. The two ARF genes are within 28 centimorgans of each other on chromosome IV, and the proteins encoded by them are 96% identical. Disruption of ARF1 causes slow growth, cold sensitivity, and sensitivity to normally sublethal concentrations of fluoride ion in the medium. Disruption of ARF2 causes no detectable phenotype. Disruption of both genes is lethal; thus, ARF is essential for mitotic growth. The ARF1 and ARF2 proteins are functionally homologous, and the phenotypic differences between mutations in the two genes can be accounted for by the level of expression; ARF1 produces approximately 90% of total ARF. Among revertants of the fluoride sensitivity of an arf1 null mutation were ARF1-ARF2 fusion genes created by a gene conversion event in which the deleted ARF1 sequences were repaired by recombination with ARF2.

    View details for Web of Science ID A1990EJ60200063

    View details for PubMedID 2123295

  • ADP-RIBOSYLATION FACTOR IS FUNCTIONALLY AND PHYSICALLY ASSOCIATED WITH GOLGI-COMPLEX PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Stearns, T., Willingham, M. C., Botstein, D., Kahn, R. A. 1990; 87 (3): 1238-1242

    Abstract

    ADP-ribosylation factor (ARF) is a ubiquitous, highly conserved 21-kDa GTP-binding protein, first identified in animal cells as the cofactor required for the in vitro ADP-ribosylation of the stimulatory regulatory subunit of adenylate cyclase, Gs, by cholera toxin. As the relevance of this activity to in vivo function is unknown, we have taken advantage of the conserved nature of ARF to study its function in Saccharomyces cerevisiae. Yeast cells bearing an arf1 null mutation display a number of phenotypes suggesting a defect in the secretory pathway. Secreted invertase is only partially glycosylated, and there is a small internal accumulation of invertase. Genetic experiments revealed interactions between ARF1 and other genes known to be involved in the secretory pathway, including YPT1, which encodes a different GTP-binding protein. In accord with these genetic results, immunofluorescence and immunoelectron microscopy show that ARF protein is localized to the Golgi apparatus in mammalian cells, in particular to the cytosolic surface of predominantly cis-Golgi membranes. Together, these results indicate that ARF functions in intracellular protein transport to or within the Golgi apparatus, a role not predicted by the previous in vitro biochemical studies.

    View details for Web of Science ID A1990CM07700080

    View details for PubMedID 2105501

  • YEAST MUTANTS SENSITIVE TO ANTIMICROTUBULE DRUGS DEFINE 3 GENES THAT AFFECT MICROTUBULE FUNCTION GENETICS Stearns, T., Hoyt, M. A., Botstein, D. 1990; 124 (2): 251-262

    Abstract

    Three new genes affecting microtubule function in Saccharomyces cerevisiae were isolated by screening for mutants displaying supersensitivity to the antimicrotubule drug benomyl. Such mutants fall into six complementation groups: TUB1, TUB2 and TUB3, the three tubulin genes of yeast, and three new genes, which we have named CIN1, CIN2 and CIN4. Mutations in each of the CIN genes were also independently isolated by screening for mutants with increased rates of chromosome loss. Strains bearing mutations in the CIN genes are approximately tenfold more sensitive than wild type to both benomyl and to the related antimicrotubule drug, nocodazole. This phenotype is recessive for all alleles isolated. The CIN1, CIN2 and CIN4 genes were cloned by complementation of the benomyl-supersensitive phenotype. Null mutants of each of the genes are viable, and have phenotypes similar to those of the point mutants. Genetic evidence for the involvement of the CIN gene products in microtubule function comes from the observation that some tubulin mutations are suppressed by cin mutations, while other tubulin mutations are lethal in combination with cin mutations. Additional genetic experiments with cin mutants suggest that the three genes act together in the same pathway or structure to affect microtubule function.

    View details for Web of Science ID A1990CM17600005

    View details for PubMedID 2407611

  • The cytoskeleton of Saccharomyces cerevisiae. Current opinion in cell biology BARNES, G., Drubin, D. G., Stearns, T. 1990; 2 (1): 109-115

    View details for PubMedID 2183834

  • CHROMOSOME INSTABILITY MUTANTS OF SACCHAROMYCES-CEREVISIAE THAT ARE DEFECTIVE IN MICROTUBULE-MEDIATED PROCESSES MOLECULAR AND CELLULAR BIOLOGY Hoyt, M. A., Stearns, T., Botstein, D. 1990; 10 (1): 223-234

    Abstract

    By using a multiply marked supernumerary chromosome III as an indicator, we isolated mutants of Saccharomyces cerevisiae that display increased rates of chromosome loss. In addition to mutations in the tubulin-encoding TUB genes, we found mutations in the CIN1, CIN2, and CIN4 genes. These genes have been defined independently by mutations causing benomyl supersensitivity and are distinct from other known yeast genes that affect chromosome segregation. Detailed phenotypic characterization of cin mutants revealed several other phenotypes similar to those of tub mutants. Null alleles of these genes caused cold sensitivity for viability. At 11 degrees C, cin mutants arrest at the mitosis stage of their cell cycle because of loss of most microtubule structure. cin1, cin2, and cin4 mutations also cause defects in two other microtubule-mediated processes, nuclear migration and nuclear fusion (karyogamy). Overproduction of the CIN1 gene product was found to cause the same phenotype as loss of function, supersensitivity to benomyl. Our findings suggest that the CIN1, CIN2, and CIN4 proteins contribute to microtubule stability either by regulating the activity of a yeast microtubule component or as structural components of microtubules.

    View details for Web of Science ID A1990CE81900025

    View details for PubMedID 2403635

  • MANIPULATING YEAST GENOME USING PLASMID VECTORS METHODS IN ENZYMOLOGY Stearns, T., Ma, H., Botstein, D. 1990; 185: 280-297

    Abstract

    The vectors and techniques described here enable one to manipulate the yeast genome to meet specific needs. Genes can be cloned, and the clone used to delete the wild-type gene from the chromosome, or replace it with mutant versions. Mutants derived by classical methods, such as mutagenesis of whole cells, or by reversion of a phenotype, can be cloned and analyzed in vitro. Yeast genes and foreign genes can either be inserted into autonomously replicating plasmid vectors that are reasonably stable or integrated into a yeast chromosome where they are maintained at one copy per genome. The combination of these techniques with the characterized promoter systems available in yeast make it possible to express almost any gene in yeast. Once this is achieved, the entire repertoire of yeast genetics is available to probe the function of the gene, or to engineer the expression in useful ways.

    View details for Web of Science ID A1990MC41900023

    View details for PubMedID 2199782

  • THE YEAST MICROTUBULE CYTOSKELETON - GENETIC APPROACHES TO STRUCTURE AND FUNCTION CELL MOTILITY AND THE CYTOSKELETON Stearns, T. 1990; 15 (1): 1-6

    View details for Web of Science ID A1990CG91300001

    View details for PubMedID 2403845

  • DNA TOPOISOMERASE-II MUST ACT AT MITOSIS TO PREVENT NONDISJUNCTION AND CHROMOSOME BREAKAGE MOLECULAR AND CELLULAR BIOLOGY Holm, C., Stearns, T., Botstein, D. 1989; 9 (1): 159-168

    Abstract

    The hypothesis that DNA topoisomerase II facilitates the separation of replicated sister chromatids was tested by examining the consequences of chromosome segregation in the absence of topoisomerase II activity. We observed a substantial elevation in the rate of nondisjunction in top2/top2 cells incubated at the restrictive temperature for one generation time. In contrast, only a minor increase in the amount of chromosome breakage was observed by either physical or genetic assays. These results suggest that aneuploidy is a major cause of the nonviability observed when top2 cells undergo mitosis at the restrictive temperature. In related experiments, we determined that topoisomerase II must act specifically during mitosis. This latter observation is consistent with the hypothesis that the mitotic spindle is necessary to allow topoisomerase II to complete the untangling of sister chromatids.

    View details for Web of Science ID A1989R643200020

    View details for PubMedID 2538717

  • FLUORESCENCE MICROSCOPY METHODS FOR YEAST METHODS IN CELL BIOLOGY Pringle, J. R., Preston, R. A., Adams, A. E., Stearns, T., Drubin, D. G., Haarer, B. K., Jones, E. W. 1989; 31: 357-435

    View details for Web of Science ID A1989AV40500019

    View details for PubMedID 2476649

  • UNLINKED NONCOMPLEMENTATION - ISOLATION OF NEW CONDITIONAL-LETHAL MUTATIONS IN EACH OF THE TUBULIN GENES OF SACCHAROMYCES-CEREVISIAE GENETICS Stearns, T., Botstein, D. 1988; 119 (2): 249-260

    Abstract

    Mutations in genes of Saccharomyces cerevisiae that code for proteins that interact with beta-tubulin were sought by screening for unlinked mutations that fail to complement mutations in the single beta-tubulin-encoding gene (TUB2). Among the first three noncomplementing mutations examined, two are linked to TUB2 while one is unlinked. The unlinked mutation was shown to be a conditional-lethal allele of the major alpha-tubulin-encoding gene (TUB1) and represents the first such mutation in that gene. The tub1-1 mutation itself causes a cold-sensitive cell-cycle arrest, and confers supersensitivity to the antimicrotubule drug benomyl. These phenotypes occur in the presence of a wild-type copy of the minor alpha-tubulin-encoding gene, TUB3; the combination of tub1-1 and a tub3 null mutation is inviable in haploids. Through further application of this method, new mutations in TUB2 and TUB3 were isolated as unlinked noncomplementers of tub1-1. The noncomplementation between tub1 and tub2 mutations is gene specific and allele specific, suggesting that the phenotype is due to an interaction at the protein level. We conclude that isolation of unlinked noncomplementing mutations is likely to be a generally useful method for isolating mutations in interacting gene products.

    View details for Web of Science ID A1988N663300004

    View details for PubMedID 3294100

  • DIVERSE BIOLOGICAL FUNCTIONS OF SMALL GTP-BINDING PROTEINS IN YEAST COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY Botstein, D., Segev, N., Stearns, T., Hoyt, M. A., Holden, J., Kahn, R. A. 1988; 53: 629-636

    View details for Web of Science ID A1988AP53900009

    View details for PubMedID 3151179

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