Gregory C Rogers
- Associate Professor, Cellular and Molecular Medicine
- Associate Professor, Molecular and Cellular Biology
- Associate Professor, BIO5 Institute
- Associate Head, Faculty Development
- Associate Professor, Cancer Biology - GIDP
- Associate Professor, Genetics - GIDP
Primary Research Interests:
I. Regulation of Centriole Duplication
Errors in chromosome segregation during cell division can result in the production of aneuploid daughter cells. This is particularly devastating during development, as aneuploidy is an underlying cause of miscarriage, birth defects, and cancers. During cell division, the accurate transmission of replicated chromosomes depends on the assembly of a bipolar spindle which is facilitated by the presence of centrioles, tiny organelles that help generate and organize spindle microtubules. Normally cells contain a single centriole pair, each duplicating only once prior to entering cell division. However, these mother centrioles have the capacity to assemble multiple daughters simultaneously. If cells assemble excess daughter centrioles (known as centriole amplification), then multipolar spindle assembly can ensue, leading to aneuploidy and increased risk for miscarriage/birth defects and cancer. In normal cells, what limits mother centrioles to assemble only a single daughter is unknown. It is known, however, that Polo-like kinase 4 (Plk4), the conserved master-initiator of centriole assembly, plays a key role in the duplication process. Therefore, a major goal of the Rogers lab is discovering how Plk4 works and how Plk4's activity is regulated.
II. Chromatin Organization during Interphase
During interphase, a cell must organize its chromatin to accommodate the active transcription of genes and -- for those cells progressing through the cell cycle -- the replication of the cell's genome. One aspect of chromatin organization is its degree of compaction: while chromatin clearly undergoes extreme compaction during mitosis, the chromatin within interphase cells is also compacted but at a more moderate level. Importantly, interphase chromatin compaction is required for normal cellular function. In collaboration with Gio Bosco (Dartmouth), my lab is interested in understanding the regulation and physiological significance of interphase chromatin organization, particularly at the level of chromatin compaction.
- Ph.D. Cellular Biology
- University of California at Davis, Davis, California, United States
- The functional coordination of three different microtubule-based motors in positioning centrosomes during sea urchin embryogenesis
- Associate Professor, University of Arizona, Tucson, Arizona (2014 - Ongoing)
Cancer biology, cell cycle, cell division, cell signaling, chromosomal instability
DissertationCBIO 920 (Spring 2018)
DissertationCMM 920 (Spring 2018)
Honors Independent StudyMCB 499H (Spring 2018)
Honors ThesisMCB 498H (Spring 2018)
Journal ClubCMM 595A (Spring 2018)
Research ConferenceCBIO 695A (Spring 2018)
Cancer BiologyCBIO 552 (Fall 2017)
Directed RsrchMCB 492 (Fall 2017)
DissertationCBIO 920 (Fall 2017)
DissertationCMM 920 (Fall 2017)
Honors ThesisMCB 498H (Fall 2017)
Introduction to ResearchMCB 795A (Fall 2017)
Journal ClubCMM 595A (Fall 2017)
Prin of Cell BiologyCMM 577 (Fall 2017)
Prin of Cell BiologyMCB 577 (Fall 2017)
Research ConferenceCBIO 695A (Fall 2017)
Cell Biology of DiseaseCMM 404 (Summer I 2017)
Cell Biology of DiseaseCMM 504 (Summer I 2017)
DissertationCBIO 920 (Spring 2017)
DissertationCMM 920 (Spring 2017)
Honors Independent StudyMCB 499H (Spring 2017)
Journal ClubCMM 595A (Spring 2017)
Research ConferenceCBIO 695A (Spring 2017)
Cancer BiologyCBIO 552 (Fall 2016)
Directed RsrchMCB 492 (Fall 2016)
DissertationCBIO 920 (Fall 2016)
DissertationCMM 920 (Fall 2016)
Honors Independent StudyMCB 399H (Fall 2016)
Journal ClubCMM 595A (Fall 2016)
Prin of Cell BiologyCMM 577 (Fall 2016)
Prin of Cell BiologyMCB 577 (Fall 2016)
ResearchCBIO 900 (Fall 2016)
Research ConferenceCBIO 695A (Fall 2016)
Cell Biology of DiseaseCMM 404 (Summer I 2016)
Cell Biology of DiseaseCMM 504 (Summer I 2016)
- Galletta, B. J., Fagerstrom, C. J., Schoborg, T. A., McLamarrah, T. A., Ryniawec, J. M., Buster, D. W., Slep, K. C., Rogers, G. C., & Rusan, N. M. (2016). A centrosome interactome provides insight into organelle assembly and reveals a non-duplication role for Plk4. Nature communications, 7, 12476.More infoThe centrosome is the major microtubule-organizing centre of many cells, best known for its role in mitotic spindle organization. How the proteins of the centrosome are accurately assembled to carry out its many functions remains poorly understood. The non-membrane-bound nature of the centrosome dictates that protein-protein interactions drive its assembly and functions. To investigate this massive macromolecular organelle, we generated a 'domain-level' centrosome interactome using direct protein-protein interaction data from a focused yeast two-hybrid screen. We then used biochemistry, cell biology and the model organism Drosophila to provide insight into the protein organization and kinase regulatory machinery required for centrosome assembly. Finally, we identified a novel role for Plk4, the master regulator of centriole duplication. We show that Plk4 phosphorylates Cep135 to properly position the essential centriole component Asterless. This interaction landscape affords a critical framework for research of normal and aberrant centrosomes.
- Wang, M., Nagle, R. B., Knudsen, B. S., Rogers, G. C., & Cress, A. E. (2017). A basal cell defect promotes budding of prostatic intraepithelial neoplasia. Journal of cell science, 130(1), 104-110.More infoBasal cells in a simple secretory epithelium adhere to the extracellular matrix (ECM), providing contextual cues for ordered repopulation of the luminal cell layer. Early high-grade prostatic intraepithelial neoplasia (HG-PIN) tissue has enlarged nuclei and nucleoli, luminal layer expansion and genomic instability. Additional HG-PIN markers include loss of α6β4 integrin or its ligand laminin-332, and budding of tumor clusters into laminin-511-rich stroma. We modeled the invasive budding phenotype by reducing expression of α6β4 integrin in spheroids formed from two normal human stable isogenic prostate epithelial cell lines (RWPE-1 and PrEC 11220). These normal cells continuously spun in culture, forming multicellular spheroids containing an outer laminin-332 layer, basal cells (expressing α6β4 integrin, high-molecular-weight cytokeratin and p63, also known as TP63) and luminal cells that secrete PSA (also known as KLK3). Basal cells were optimally positioned relative to the laminin-332 layer as determined by spindle orientation. β4-integrin-defective spheroids contained a discontinuous laminin-332 layer corresponding to regions of abnormal budding. This 3D model can be readily used to study mechanisms that disrupt laminin-332 continuity, for example, defects in the essential adhesion receptor (β4 integrin), laminin-332 or abnormal luminal expansion during HG-PIN progression.
- Bozler, J., Nguyen, H. Q., Rogers, G. C., & Bosco, G. (2015). Condensins exert force on chromatin-nuclear envelope tethers to mediate nucleoplasmic reticulum formation in Drosophila melanogaster. G3 (Bethesda, Md.), 5(3), 341-52.More infoAlthough the nuclear envelope is known primarily for its role as a boundary between the nucleus and cytoplasm in eukaryotes, it plays a vital and dynamic role in many cellular processes. Studies of nuclear structure have revealed tissue-specific changes in nuclear envelope architecture, suggesting that its three-dimensional structure contributes to its functionality. Despite the importance of the nuclear envelope, the factors that regulate and maintain nuclear envelope shape remain largely unexplored. The nuclear envelope makes extensive and dynamic interactions with the underlying chromatin. Given this inexorable link between chromatin and the nuclear envelope, it is possible that local and global chromatin organization reciprocally impact nuclear envelope form and function. In this study, we use Drosophila salivary glands to show that the three-dimensional structure of the nuclear envelope can be altered with condensin II-mediated chromatin condensation. Both naturally occurring and engineered chromatin-envelope interactions are sufficient to allow chromatin compaction forces to drive distortions of the nuclear envelope. Weakening of the nuclear lamina further enhanced envelope remodeling, suggesting that envelope structure is capable of counterbalancing chromatin compaction forces. Our experiments reveal that the nucleoplasmic reticulum is born of the nuclear envelope and remains dynamic in that they can be reabsorbed into the nuclear envelope. We propose a model where inner nuclear envelope-chromatin tethers allow interphase chromosome movements to change nuclear envelope morphology. Therefore, interphase chromatin compaction may be a normal mechanism that reorganizes nuclear architecture, while under pathological conditions, such as laminopathies, compaction forces may contribute to defects in nuclear morphology.
- Klebba, J. E., Buster, D. W., McLamarrah, T. A., Rusan, N. M., & Rogers, G. C. (2015). Autoinhibition and relief mechanism for Polo-like kinase 4. Proceedings of the National Academy of Sciences of the United States of America, 112(7), E657-66.More infoPolo-like kinase 4 (Plk4) is a master regulator of centriole duplication, and its hyperactivity induces centriole amplification. Homodimeric Plk4 has been shown to be ubiquitinated as a result of autophosphorylation, thus promoting its own degradation and preventing centriole amplification. Unlike other Plks, Plk4 contains three rather than two Polo box domains, and the function of its third Polo box (PB3) is unclear. Here, we performed a functional analysis of Plk4's structural domains. Like other Plks, Plk4 possesses a previously unidentified autoinhibitory mechanism mediated by a linker (L1) near the kinase domain. Thus, autoinhibition is a conserved feature of Plks. In the case of Plk4, autoinhibition is relieved after homodimerization and is accomplished by PB3 and by autophosphorylation of L1. In contrast, autophosphorylation of the second linker promotes separation of the Plk4 homodimer. Therefore, autoinhibition delays the multiple consequences of activation until Plk4 dimerizes. These findings reveal a complex mechanism of Plk4 regulation and activation which govern the process of centriole duplication.
- Klebba, J. E., Galletta, B. J., Nye, J., Plevock, K. M., Buster, D. W., Hollingsworth, N. A., Slep, K. C., Rusan, N. M., & Rogers, G. C. (2015). Two Polo-like kinase 4 binding domains in Asterless perform distinct roles in regulating kinase stability. The Journal of cell biology, 208(4), 401-14.More infoPlk4 (Polo-like kinase 4) and its binding partner Asterless (Asl) are essential, conserved centriole assembly factors that induce centriole amplification when overexpressed. Previous studies found that Asl acts as a scaffolding protein; its N terminus binds Plk4's tandem Polo box cassette (PB1-PB2) and targets Plk4 to centrioles to initiate centriole duplication. However, how Asl overexpression drives centriole amplification is unknown. In this paper, we investigated the Asl-Plk4 interaction in Drosophila melanogaster cells. Surprisingly, the N-terminal region of Asl is not required for centriole duplication, but a previously unidentified Plk4-binding domain in the C terminus is required. Mechanistic analyses of the different Asl regions revealed that they act uniquely during the cell cycle: the Asl N terminus promotes Plk4 homodimerization and autophosphorylation during interphase, whereas the Asl C terminus stabilizes Plk4 during mitosis. Therefore, Asl affects Plk4 in multiple ways to regulate centriole duplication. Asl not only targets Plk4 to centrioles but also modulates Plk4 stability and activity, explaining the ability of overexpressed Asl to drive centriole amplification.
- Nguyen, H. Q., Nye, J., Buster, D. W., Klebba, J. E., Rogers, G. C., & Bosco, G. (2015). Drosophila casein kinase I alpha regulates homolog pairing and genome organization by modulating condensin II subunit Cap-H2 levels. PLoS genetics, 11(2), e1005014.More infoThe spatial organization of chromosomes within interphase nuclei is important for gene expression and epigenetic inheritance. Although the extent of physical interaction between chromosomes and their degree of compaction varies during development and between different cell-types, it is unclear how regulation of chromosome interactions and compaction relate to spatial organization of genomes. Drosophila is an excellent model system for studying chromosomal interactions including homolog pairing. Recent work has shown that condensin II governs both interphase chromosome compaction and homolog pairing and condensin II activity is controlled by the turnover of its regulatory subunit Cap-H2. Specifically, Cap-H2 is a target of the SCFSlimb E3 ubiquitin-ligase which down-regulates Cap-H2 in order to maintain homologous chromosome pairing, chromosome length and proper nuclear organization. Here, we identify Casein Kinase I alpha (CK1α) as an additional negative-regulator of Cap-H2. CK1α-depletion stabilizes Cap-H2 protein and results in an accumulation of Cap-H2 on chromosomes. Similar to Slimb mutation, CK1α depletion in cultured cells, larval salivary gland, and nurse cells results in several condensin II-dependent phenotypes including dispersal of centromeres, interphase chromosome compaction, and chromosome unpairing. Moreover, CK1α loss-of-function mutations dominantly suppress condensin II mutant phenotypes in vivo. Thus, CK1α facilitates Cap-H2 destruction and modulates nuclear organization by attenuating chromatin localized Cap-H2 protein.
- Wallace, H. A., Klebba, J. E., Kusch, T., Rogers, G. C., & Bosco, G. (2015). Condensin II Regulates Interphase Chromatin Organization Through the Mrg-Binding Motif of Cap-H2. G3 (Bethesda, Md.), 5(5), 803-17.More infoThe spatial organization of the genome within the eukaryotic nucleus is a dynamic process that plays a central role in cellular processes such as gene expression, DNA replication, and chromosome segregation. Condensins are conserved multi-subunit protein complexes that contribute to chromosome organization by regulating chromosome compaction and homolog pairing. Previous work in our laboratory has shown that the Cap-H2 subunit of condensin II physically and genetically interacts with the Drosophila homolog of human MORF4-related gene on chromosome 15 (MRG15). Like Cap-H2, Mrg15 is required for interphase chromosome compaction and homolog pairing. However, the mechanism by which Mrg15 and Cap-H2 cooperate to maintain interphase chromatin organization remains unclear. Here, we show that Cap-H2 localizes to interband regions on polytene chromosomes and co-localizes with Mrg15 at regions of active transcription across the genome. We show that co-localization of Cap-H2 on polytene chromosomes is partially dependent on Mrg15. We have identified a binding motif within Cap-H2 that is essential for its interaction with Mrg15, and have found that mutation of this motif results in loss of localization of Cap-H2 on polytene chromosomes and results in partial suppression of Cap-H2-mediated compaction and homolog unpairing. Our data are consistent with a model in which Mrg15 acts as a loading factor to facilitate Cap-H2 binding to chromatin and mediate changes in chromatin organization.
- Galletta, B. J., Guillen, R. X., Fagerstrom, C. J., Brownlee, C. W., Lerit, D. A., Megraw, T. L., Rogers, G. C., & Rusan, N. M. (2014). Drosophila pericentrin requires interaction with calmodulin for its function at centrosomes and neuronal basal bodies but not at sperm basal bodies. Molecular biology of the cell, 25(18), 2682-94.More infoPericentrin is a critical centrosomal protein required for organizing pericentriolar material (PCM) in mitosis. Mutations in pericentrin cause the human genetic disorder Majewski/microcephalic osteodysplastic primordial dwarfism type II, making a detailed understanding of its regulation extremely important. Germaine to pericentrin's function in organizing PCM is its ability to localize to the centrosome through the conserved C-terminal PACT domain. Here we use Drosophila pericentrin-like-protein (PLP) to understand how the PACT domain is regulated. We show that the interaction of PLP with calmodulin (CaM) at two highly conserved CaM-binding sites in the PACT domain controls the proper targeting of PLP to the centrosome. Disrupting the PLP-CaM interaction with single point mutations renders PLP inefficient in localizing to centrioles in cultured S2 cells and Drosophila neuroblasts. Although levels of PCM are unaffected, it is highly disorganized. We also demonstrate that basal body formation in the male testes and the production of functional sperm does not rely on the PLP-CaM interaction, whereas production of functional mechanosensory neurons does.
- Nye, J., Buster, D. W., & Rogers, G. C. (2014). The use of cultured Drosophila cells for studying the microtubule cytoskeleton. Methods in molecular biology (Clifton, N.J.), 1136, 81-101.More infoCultured Drosophila cell lines have been developed into a powerful tool for studying a wide variety of cellular processes. Their ability to be easily and cheaply cultured as well as their susceptibility to protein knockdown via double-stranded RNA-mediated interference (RNAi) has made them the model system of choice for many researchers in the fields of cell biology and functional genomics. Here we describe basic techniques for gene knockdown, transgene expression, preparation for fluorescence microscopy, and centrosome enrichment using cultured Drosophila cells with an emphasis on studying the microtubule cytoskeleton.
- Skwarek, L. C., Windler, S. L., de Vreede, G., Rogers, G. C., & Bilder, D. (2014). The F-box protein Slmb restricts the activity of aPKC to polarize epithelial cells. Development (Cambridge, England), 141(15), 2978-83.More infoThe Par-3/Par-6/aPKC complex is the primary determinant of apical polarity in epithelia across animal species, but how the activity of this complex is restricted to allow polarization of the basolateral domain is less well understood. In Drosophila, several multiprotein modules antagonize the Par complex through a variety of means. Here we identify a new mechanism involving regulated protein degradation. Strong mutations in supernumerary limbs (slmb), which encodes the substrate adaptor of an SCF-class E3 ubiquitin ligase, cause dramatic loss of polarity in imaginal discs accompanied by tumorous proliferation defects. Slmb function is required to restrain apical aPKC activity in a manner that is independent of endolysosomal trafficking and parallel to the Scribble module of junctional scaffolding proteins. The involvement of the Slmb E3 ligase in epithelial polarity, specifically limiting Par complex activity to distinguish the basolateral domain, points to parallels with polarization of the C. elegans zygote.
- Brownlee, C. W., & Rogers, G. C. (2013). Show me your license, please: deregulation of centriole duplication mechanisms that promote amplification. Cellular and molecular life sciences : CMLS, 70(6), 1021-34.More infoCentrosomes are organelles involved in generating and organizing the interphase microtubule cytoskeleton, mitotic spindles and cilia. At the centrosome core are a pair of centrioles, structures that act as the duplicating elements of this organelle. Centrioles function to recruit and organize pericentriolar material which nucleates microtubules. While centrioles are relatively simple in construction, the mechanics of centriole biogenesis remain an important yet poorly understood process. More mysterious still are the regulatory mechanisms that oversee centriole assembly. The fidelity of centriole duplication is critical as defects in either the assembly or number of centrioles promote aneuploidy, primary microcephaly, birth defects, ciliopathies and tumorigenesis. In addition, some pathogens employ mechanisms to promote centriole overduplication to the detriment of the host cell. This review summarizes our current understanding of this important topic, highlighting the need for further study if new therapeutics are to be developed to treat diseases arising from defects of centrosome duplication.
- Buster, D. W., Daniel, S. G., Nguyen, H. Q., Windler, S. L., Skwarek, L. C., Peterson, M., Roberts, M., Meserve, J. H., Hartl, T., Klebba, J. E., Bilder, D., Bosco, G., & Rogers, G. C. (2013). SCFSlimb ubiquitin ligase suppresses condensin II-mediated nuclear reorganization by degrading Cap-H2. The Journal of cell biology, 201(1), 49-63.More infoCondensin complexes play vital roles in chromosome condensation during mitosis and meiosis. Condensin II uniquely localizes to chromatin throughout the cell cycle and, in addition to its mitotic duties, modulates chromosome organization and gene expression during interphase. Mitotic condensin activity is regulated by phosphorylation, but mechanisms that regulate condensin II during interphase are unclear. Here, we report that condensin II is inactivated when its subunit Cap-H2 is targeted for degradation by the SCF(Slimb) ubiquitin ligase complex and that disruption of this process dramatically changed interphase chromatin organization. Inhibition of SCF(Slimb) function reorganized interphase chromosomes into dense, compact domains and disrupted homologue pairing in both cultured Drosophila cells and in vivo, but these effects were rescued by condensin II inactivation. Furthermore, Cap-H2 stabilization distorted nuclear envelopes and dispersed Cid/CENP-A on interphase chromosomes. Therefore, SCF(Slimb)-mediated down-regulation of condensin II is required to maintain proper organization and morphology of the interphase nucleus.
- Klebba, J. E., Buster, D. W., Nguyen, A. L., Swatkoski, S., Gucek, M., Rusan, N. M., & Rogers, G. C. (2013). Polo-like kinase 4 autodestructs by generating its Slimb-binding phosphodegron. Current biology : CB, 23(22), 2255-61.More infoPolo-like kinase 4 (Plk4) is a conserved master regulator of centriole assembly. Previously, we found that Drosophila Plk4 protein levels are actively suppressed during interphase. Degradation of interphase Plk4 prevents centriole overduplication and is mediated by the ubiquitin-ligase complex SCF(Slimb/βTrCP). Since Plk4 stability depends on its activity, we studied the consequences of inactivating Plk4 or perturbing its phosphorylation state within its Slimb-recognition motif (SRM). Mass spectrometry of in-vitro-phosphorylated Plk4 and Plk4 purified from cells reveals that it is directly responsible for extensively autophosphorylating and generating its Slimb-binding phosphodegron. Phosphorylatable residues within this regulatory region were systematically mutated to determine their impact on Plk4 protein levels and centriole duplication when expressed in S2 cells. Notably, autophosphorylation of a single residue (Ser293) within the SRM is critical for Slimb binding and ubiquitination. Our data also demonstrate that autophosphorylation of numerous residues flanking S293 collectively contribute to establishing a high-affinity binding site for SCF(Slimb). Taken together, our findings suggest that Plk4 directly generates its own phosphodegron and can do so without the assistance of an additional kinase(s).
- Smith, H. F., Roberts, M. A., Nguyen, H. Q., Peterson, M., Hartl, T. A., Wang, X., Klebba, J. E., Rogers, G. C., & Bosco, G. (2013). Maintenance of interphase chromosome compaction and homolog pairing in Drosophila is regulated by the condensin cap-h2 and its partner Mrg15. Genetics, 195(1), 127-46.More infoDynamic regulation of chromosome structure and organization is critical for fundamental cellular processes such as gene expression and chromosome segregation. Condensins are conserved chromosome-associated proteins that regulate a variety of chromosome dynamics, including axial shortening, lateral compaction, and homolog pairing. However, how the in vivo activities of condensins are regulated and how functional interactors target condensins to chromatin are not well understood. To better understand how Drosophila melanogaster condensin is regulated, we performed a yeast two-hybrid screen and identified the chromo-barrel domain protein Mrg15 to interact with the Cap-H2 condensin subunit. Genetic interactions demonstrate that Mrg15 function is required for Cap-H2-mediated unpairing of polytene chromosomes in ovarian nurse cells and salivary gland cells. In diploid tissues, transvection assays demonstrate that Mrg15 inhibits transvection at Ubx and cooperates with Cap-H2 to antagonize transvection at yellow. In cultured cells, we show that levels of chromatin-bound Cap-H2 protein are partially dependent on Mrg15 and that Cap-H2-mediated homolog unpairing is suppressed by RNA interference depletion of Mrg15. Thus, maintenance of interphase chromosome compaction and homolog pairing status requires both Mrg15 and Cap-H2. We propose a model where the Mrg15 and Cap-H2 protein-protein interaction may serve to recruit Cap-H2 to chromatin and facilitates compaction of interphase chromatin.
- Mennella, V., Keszthelyi, B., McDonald, K. L., Chhun, B., Kan, F., Rogers, G. C., Huang, B., & Agard, D. A. (2012). Subdiffraction-resolution fluorescence microscopy reveals a domain of the centrosome critical for pericentriolar material organization. Nature cell biology, 14(11), 1159-68.More infoAs the main microtubule-organizing centre in animal cells, the centrosome has a fundamental role in cell function. Surrounding the centrioles, the pericentriolar material (PCM) provides a dynamic platform for nucleating microtubules. Although the importance of the PCM is established, its amorphous electron-dense nature has made it refractory to structural investigation. By using SIM and STORM subdiffraction-resolution microscopies to visualize proteins critical for centrosome maturation, we demonstrate that the PCM is organized into two main structural domains: a layer juxtaposed to the centriole wall, and proteins extending farther away from the centriole organized in a matrix. Analysis of Pericentrin-like protein (PLP) reveals that its carboxy terminus is positioned at the centriole wall, it radiates outwards into the matrix and is organized in clusters having quasi-nine-fold symmetry. By RNA-mediated interference (RNAi), we show that PLP fibrils are required for interphase recruitment and proper mitotic assembly of the PCM matrix.
- Roberts, D. M., Pronobis, M. I., Alexandre, K. M., Rogers, G. C., Poulton, J. S., Schneider, D. E., Jung, K., McKay, D. J., & Peifer, M. (2012). Defining components of the ß-catenin destruction complex and exploring its regulation and mechanisms of action during development. PloS one, 7(2), e31284.More infoA subset of signaling pathways play exceptionally important roles in embryonic and post-embryonic development, and mis-regulation of these pathways occurs in most human cancers. One such pathway is the Wnt pathway. The primary mechanism keeping Wnt signaling off in the absence of ligand is regulated proteasomal destruction of the canonical Wnt effector ßcatenin (or its fly homolog Armadillo). A substantial body of evidence indicates that SCF(βTrCP) mediates βcat destruction, however, an essential role for Roc1 has not been demonstrated in this process, as would be predicted. In addition, other E3 ligases have also been proposed to destroy βcat, suggesting that βcat destruction may be regulated differently in different tissues.
- Slevin, L. K., Nye, J., Pinkerton, D. C., Buster, D. W., Rogers, G. C., & Slep, K. C. (2012). The structure of the plk4 cryptic polo box reveals two tandem polo boxes required for centriole duplication. Structure (London, England : 1993), 20(11), 1905-17.More infoCentrioles are key microtubule polarity determinants. Centriole duplication is tightly controlled to prevent cells from developing multipolar spindles, a situation that promotes chromosomal instability. A conserved component in the duplication pathway is Plk4, a polo kinase family member that localizes to centrioles in M/G1. To limit centriole duplication, Plk4 levels are controlled through trans-autophosphorylation that primes ubiquitination. In contrast to Plks 1-3, Plk4 possesses a unique central region called the "cryptic polo box." Here, we present the crystal structure of this region at 2.3 Å resolution. Surprisingly, the structure reveals two tandem homodimerized polo boxes, PB1-PB2, that form a unique winged architecture. The full PB1-PB2 cassette is required for binding the centriolar protein Asterless as well as robust centriole targeting. Thus, with its C-terminal polo box (PB3), Plk4 has a triple polo box architecture that facilitates oligomerization, targeting, and promotes trans-autophosphorylation, limiting centriole duplication to once per cell cycle.
- Brownlee, C. W., Klebba, J. E., Buster, D. W., & Rogers, G. C. (2011). The Protein Phosphatase 2A regulatory subunit Twins stabilizes Plk4 to induce centriole amplification. The Journal of cell biology, 195(2), 231-43.More infoCentriole duplication is a tightly regulated process that must occur only once per cell cycle; otherwise, supernumerary centrioles can induce aneuploidy and tumorigenesis. Plk4 (Polo-like kinase 4) activity initiates centriole duplication and is regulated by ubiquitin-mediated proteolysis. Throughout interphase, Plk4 autophosphorylation triggers its degradation, thus preventing centriole amplification. However, Plk4 activity is required during mitosis for proper centriole duplication, but the mechanism stabilizing mitotic Plk4 is unknown. In this paper, we show that PP2A (Protein Phosphatase 2A(Twins)) counteracts Plk4 autophosphorylation, thus stabilizing Plk4 and promoting centriole duplication. Like Plk4, the protein level of PP2A's regulatory subunit, Twins (Tws), peaks during mitosis and is required for centriole duplication. However, untimely Tws expression stabilizes Plk4 inappropriately, inducing centriole amplification. Paradoxically, expression of tumor-promoting simian virus 40 small tumor antigen (ST), a reported PP2A inhibitor, promotes centrosome amplification by an unknown mechanism. We demonstrate that ST actually mimics Tws function in stabilizing Plk4 and inducing centriole amplification.
- Buster, D. W., Nye, J., Klebba, J. E., & Rogers, G. C. (2010). Preparation of Drosophila S2 cells for light microscopy. Journal of visualized experiments : JoVE.More infoThe ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature and genome sequence database. Cultured Drosophila S2 cells, the product of disassociated 20-24 hour old embryos, possess all these properties. Consequently, S2 cells are extremely well-suited for the analysis of cellular processes, including the discovery of the genes encoding the molecular components of the process or mechanism of interest. The features of S2 cells that are most responsible for their utility are the ease with which they are maintained, their exquisite sensitivity to double-stranded (ds)RNA-mediated interference (RNAi), and their tractability to fluorescence microscopy as either live or fixed cells. S2 cells can be grown in a variety of media, including a number of inexpensive, commercially-available, fully-defined, serum-free media. In addition, they grow optimally and quickly at 21-24 degrees C and can be cultured in a variety of containers. Unlike mammalian cells, S2 cells do not require a regulated atmosphere, but instead do well with normal air and can even be maintained in sealed flasks. Complementing the ease of RNAi in S2 cells is the ability to readily analyze experimentally-induced phenotypes by phase or fluorescence microscopy of fixed or live cells. S2 cells grow in culture as a single monolayer but do not display contact inhibition. Instead, cells tend to grow in colonies in dense cultures. At low density, S2 cultures grown on glass or tissue culture-treated plastic are round and loosely-attached. However, the cytology of S2 cells can be greatly improved by inducing them to flatten extensively by briefly culturing them on a surface coated with the lectin, concanavalin A (ConA). S2 cells can also be stably transfected with fluorescently-tagged markers to label structures or organelles of interest in live or fixed cells. Therefore, the usual scenario for the microscopic analysis of cells is this: first, S2 cells (which can possess transgenes to express tagged markers) are treated by RNAi to eliminate a target protein(s). RNAi treatment time can be adjusted to allow for differences in protein turn-over kinetics and to minimize cell trauma/death if the target protein is important for viability. Next, the treated cells are transferred to a dish containing a coverslip pre-coated with conA to induce cells to spread and tightly adhere to the glass. Finally, cells are imaged with the researcher's choice of microscopy modes. S2 cells are particularly good for studies requiring extended visualization of live cells since these cells stay healthy at room temperature and normal atmosphere.
- Rogers, G. C. (2010). Spindle assembly: more than just microtubules. Current biology : CB, 20(8), R364-6.More infoDo actin dynamics play an active role in mitotic spindle assembly? A new study demonstrates that cortical actin polymerization assists with the earliest phase of spindle pole migration.
- Taylor, S. M., Nevis, K. R., Park, H. L., Rogers, G. C., Rogers, S. L., Cook, J. G., & Bautch, V. L. (2010). Angiogenic factor signaling regulates centrosome duplication in endothelial cells of developing blood vessels. Blood, 116(16), 3108-17.More infoRegulated vascular endothelial growth factor (VEGF) signaling is required for proper angiogenesis, and excess VEGF signaling results in aberrantly formed vessels that do not function properly. Tumor endothelial cells have excess centrosomes and are aneuploid, properties that probably contribute to the morphologic and functional abnormalities of tumor vessels. We hypothesized that endothelial cell centrosome number is regulated by signaling via angiogenic factors, such as VEGF. We found that endothelial cells in developing vessels exposed to elevated VEGF signaling display centrosome overduplication. Signaling from VEGF, through either MEK/ERK or AKT to cyclin E/Cdk2, is amplified in association with centrosome overduplication, and blockade of relevant pathway components rescued the centrosome overduplication defect. Endothelial cells exposed to elevated FGF also had excess centrosomes, suggesting that multiple angiogenic factors regulate centrosome number. Endothelial cells with excess centrosomes survived and formed aberrant spindles at mitosis. Developing vessels exposed to elevated VEGF signaling also exhibited increased aneuploidy of endothelial cells, which is associated with cellular dysfunction. These results provide the first link between VEGF signaling and regulation of the centrosome duplication cycle, and suggest that endothelial cell centrosome overduplication contributes to aberrant angiogenesis in developing vessel networks exposed to excess angiogenic factors.
- Rath, U., Rogers, G. C., Tan, D., Gomez-Ferreria, M. A., Buster, D. W., Sosa, H. J., & Sharp, D. J. (2009). The Drosophila kinesin-13, KLP59D, impacts Pacman- and Flux-based chromosome movement. Molecular biology of the cell, 20(22), 4696-705.More infoChromosome movements are linked to the active depolymerization of spindle microtubule (MT) ends. Here we identify the kinesin-13 family member, KLP59D, as a novel and uniquely important regulator of spindle MT dynamics and chromosome motility in Drosophila somatic cells. During prometaphase and metaphase, depletion of KLP59D, which targets to centrosomes and outer kinetochores, suppresses the depolymerization of spindle pole-associated MT minus ends, thereby inhibiting poleward tubulin Flux. Subsequently, during anaphase, loss of KLP59D strongly attenuates chromatid-to-pole motion by suppressing the depolymerization of both minus and plus ends of kinetochore-associated MTs. The mechanism of KLP59D's impact on spindle MT plus and minus ends appears to differ. Our data support a model in which KLP59D directly depolymerizes kinetochore-associated plus ends during anaphase, but influences minus ends indirectly by localizing the pole-associated MT depolymerase KLP10A. Finally, electron microscopy indicates that, unlike the other Drosophila kinesin-13s, KLP59D is largely incapable of oligomerizing into MT-associated rings in vitro, suggesting that such structures are not a requisite feature of kinetochore-based MT disassembly and chromosome movements.
- Rogers, G. C., Rusan, N. M., Roberts, D. M., Peifer, M., & Rogers, S. L. (2009). The SCF Slimb ubiquitin ligase regulates Plk4/Sak levels to block centriole reduplication. The Journal of cell biology, 184(2), 225-39.More infoRestricting centriole duplication to once per cell cycle is critical for chromosome segregation and genomic stability, but the mechanisms underlying this block to reduplication are unclear. Genetic analyses have suggested an involvement for Skp/Cullin/F box (SCF)-class ubiquitin ligases in this process. In this study, we describe a mechanism to prevent centriole reduplication in Drosophila melanogaster whereby the SCF E3 ubiquitin ligase in complex with the F-box protein Slimb mediates proteolytic degradation of the centrosomal regulatory kinase Plk4. We identified SCF(Slimb) as a regulator of centriole duplication via an RNA interference (RNAi) screen of Cullin-based ubiquitin ligases. We found that Plk4 binds to Slimb and is an SCF(Slimb) target. Both Slimb and Plk4 localize to centrioles, with Plk4 levels highest at mitosis and absent during S phase. Using a Plk4 Slimb-binding mutant and Slimb RNAi, we show that Slimb regulates Plk4 localization to centrioles during interphase, thus regulating centriole number and ensuring the block to centriole reduplication.
- Rusan, N. M., & Rogers, G. C. (2009). Centrosome function: sometimes less is more. Traffic (Copenhagen, Denmark), 10(5), 472-81.More infoTight regulation of centrosome duplication is critical to ensure that centrosome number doubles once and only once per cell cycle. Superimposed onto this centrosome duplication cycle is a functional centrosome cycle in which they alternate between phases of quiescence and robust microtubule (MT) nucleation and MT-anchoring activities. In vertebrate cycling cells, interphase centrioles accumulate less pericentriolar material (PCM), reducing their MT nucleation capacity. In mitosis, centrosomes mature, accumulating more PCM to increase their nucleation and anchoring capacities to form robust MT asters. Interestingly, functional cycles of centrosomes can be altered to suit the cell's needs. Some interphase centrosomes function as a microtubule-organizing center by increasing their ability to anchor MTs to form centrosomal radial arrays. Other interphase centrosomes maintain their MT nucleation capacity but reduce/eliminate their MT-anchoring capacity. Recent work demonstrates that Drosophila cells take this to the extreme, whereby centrioles lose all detectable PCM during interphase, offering an explanation as to how centrosome-deficient flies develop to adulthood. Drosophila stem cells further modify the functional cycle by differentially regulating their two centrioles - a situation that seems important for stem cell asymmetric divisions, as misregulation of centrosome duplication in stem/progenitor cells can promote tumor formation. Here, we review recent findings that describe variations in the functional cycle of centrosomes.