A signature-based method for indexing cell cycle phase distribution from microarray profiles.
Mizuno H, Nakanishi Y, Ishii N, Sarai A, Kitada K. BMC Genomics. 2009, 10:137.
Background
The cell cycle machinery interprets oncogenic signals and reflects the biology of cancers. To date,... more
Background
The cell cycle machinery interprets oncogenic signals and reflects the biology of cancers. To date, various methods for cell cycle phase estimation such as mitotic index, S phase fraction, and immunohistochemistry have provided valuable information on cancers (e.g. proliferation rate). However, those methods rely on one or few measurements and the scope of the information is limited. There is a need for more systematic cell cycle analysis methods.
Results
We developed a signature-based method for indexing cell cycle phase distribution from microarray profiles under consideration of cycling and non-cycling cells. A cell cycle signature masterset, composed of genes which express preferentially in cycling cells and in a cell cycle-regulated manner, was created to index the proportion of cycling cells in the sample. Cell cycle signature subsets, composed of genes whose expressions peak at specific stages of the cell cycle, were also created to index the proportion of cells in the corresponding stages. The method was validated using cell cycle datasets and quiescence-induced cell datasets. Analyses of a mouse tumor model dataset and human breast cancer datasets revealed variations in the proportion of cycling cells. When the influence of non-cycling cells was taken into account, "buried" cell cycle phase distributions were depicted that were oncogenic-event specific in the mouse tumor model dataset and were associated with patients' prognosis in the human breast cancer datasets.
Conclusion
The signature-based cell cycle analysis method presented in this report, would potentially be of value for cancer characterization and diagnostics.
Dynamic Mitochondrial Networks in Cancer
Published on the Scientific American Blog
Research projects evolve in a fortuitous manner, often guided by a convergence of novel observations, intuition,... more
Research projects evolve in a fortuitous manner, often guided by a convergence of novel observations, intuition, helpful colleagues and unique personal circumstances. It is precisely this constellation that prompted two cardiologists to study the mitochondrial networks in lung cancer cells.
In 2008, my colleague and friend Stephen Archer, a Professor of Medicine at the University of Chicago, asked me whether I would be interested in studying the role of mitochondrial networks in lung cancer cells. My first response was the question “Do mitochondria really form networks?”, because at that time the expression “mitochondria” evoked images of scattered oval-like organelles, a textbook image of electron microscopy.......
Inhibition of mitochondrial fission prevents cell cycle progression in lung cancer
Published in the FASEB Journal
Mitochondria exist in dynamic networks that undergo fusion and fission. Mitochondrial fusion and fission are mediated... more Mitochondria exist in dynamic networks that undergo fusion and fission. Mitochondrial fusion and fission are mediated by several GTPases in the outer mitochondrial membrane, notably mitofusin-2 (Mfn-2), which promotes fusion, and dynamin-related protein (Drp-1), which promotes fission. We report that human lung cancer cell lines exhibit an imbalance of Drp-1/Mfn-2 expression, which promotes a state of mitochondrial fission. Lung tumor tissue samples from patients demonstrated a similar increase in Drp-1 and decrease in Mfn-2 when compared to adjacent healthy lung. Complementary approaches to restore mitochondrial network formation in lung cancer cells by overexpression of Mfn-2, Drp-1 inhibition, or Drp-1 knockdown resulted in a marked reduction of cancer cell proliferation and an increase in spontaneous apoptosis. The number of cancer cells in S phase decreased from 32.4 ± 0.6 to 6.4 ± 0.3% with Drp-1 inhibition (P<0.001). In a xenotransplantation model, Mfn-2 gene therapy or Drp-1 inhibition could regress tumor growth. The tumor volume decreased from 205.6 ± 59 to 70.6 ± 15 mm(3) (P<0.05) with Mfn-2 overexpression and from 186.0 ± 19 to 87.0 ± 6 mm(3) (P<0.01) with therapeutic Drp-1 inhibition. Impaired fusion and enhanced fission contribute fundamentally to the proliferation/apoptosis imbalance in cancer and constitute promising novel therapeutic targets.
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Rab35 GTPase and OCRL phosphatase remodel lipids and F-actin for successful cytokinesis.
Dambournet D, Machicoane M, Chesneau L, Sachse M, Rocancourt M, El Marjou A, Formstecher E, Salomon R, Goud B, Echard A.
Nat Cell Biol. 2011 Jun 26;13(8):981-8.
Abscission is the least understood step of cytokinesis. It consists of the final cut of the intercellular bridge... more Abscission is the least understood step of cytokinesis. It consists of the final cut of the intercellular bridge connecting the sister cells at the end of mitosis, and is thought to involve membrane trafficking as well as lipid and cytoskeleton remodelling. We previously identified the Rab35 GTPase as a regulator of a fast recycling endocytic pathway that is essential for post-furrowing cytokinesis stages. Here, we report that the phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)) 5-phosphatase OCRL, which is mutated in Lowe syndrome patients, is an effector of the Rab35 GTPase in cytokinesis abscission. GTP-bound (active) Rab35 directly interacts with OCRL and controls its localization at the intercellular bridge. Depletion of Rab35 or OCRL inhibits cytokinesis abscission and is associated with local abnormal PtdIns(4,5)P(2) and F-actin accumulation in the intercellular bridge. These division defects are also found in cell lines derived from Lowe patients and can be corrected by the addition of low doses of F-actin depolymerization drugs. Our data demonstrate that PtdIns(4,5)P(2) hydrolysis is important for normal cytokinesis abscission to locally remodel the F-actin cytoskeleton in the intercellular bridge. They also reveal an unexpected role for the phosphatase OCRL in cell division and shed new light on the pleiotropic phenotypes associated with Lowe disease.
The RabGAP Proteins Gyp5p and Gyl1p Recruit the BAR Domain Protein Rvs167p for Polarized Exocytosis.
Prigent M, Boy-Marcotte E, Chesneau L, Gibson K, Dupré-Crochet S, Tisserand H, Verbavatz JM, Cuif MH.
Traffic. 2011 Aug;12(8):1084-97.
The Rab GTPase-activating proteins (GAP) Gyp5p and Gyl1p are involved in the control of polarized exocytosis at the... more The Rab GTPase-activating proteins (GAP) Gyp5p and Gyl1p are involved in the control of polarized exocytosis at the small-bud stage in Saccharomyces cerevisiae. Both Gyp5p and Gyl1p interact with the N-Bin1/Amphiphysin/Rvs167 (BAR) domain protein Rvs167p, but the biological function of this interaction is unclear. We show here that Gyp5p and Gyl1p recruit Rvs167p to the small-bud tip, where it plays a role in polarized exocytosis. In gyp5Δgyl1Δ cells, Rvs167p is not correctly localized to the small-bud tip. Both P473L mutation in the SH3 domain of Rvs167p and deletion of the proline-rich regions of Gyp5p and Gyl1p disrupt the interaction of Rvs167p with Gyp5p and Gyl1p and impair the localization of Rvs167p to the tips of small buds. We provide evidence for the accumulation of secretory vesicles in small buds of rvs167Δ cells and for defective Bgl2p secretion in rvs167Δ cultures enriched in small-budded cells at 13°C, implicating Rvs167p in polarized exocytosis. Moreover, both the accumulation of secretory vesicles in Rvs167p P473L cells cultured at 13°C and secretion defects in cells producing Gyp5p and Gyl1p without proline-rich regions strongly suggest that the function of Rvs167p in exocytosis depends on its ability to interact with Gyp5p and Gyl1p.
Gyp5p and Gyl1p are involved in the control of polarized exocytosis in budding yeast.
Chesneau L, Dupré S, Burdina A, Roger J, Le Panse S, Jacquet M, Cuif MH. J Cell Sci. 2004 Sep 15;117(Pt 20):4757-67.
We report here elements for functional characterization of two members of the Saccharomyces cerevisiae Ypt/Rab GTPase... more We report here elements for functional characterization of two members of the Saccharomyces cerevisiae Ypt/Rab GTPase activating proteins family (GAP): Gyp5p, a potent GAP in vitro for Ypt1p and Sec4p, and the protein Ymr192wp/APP2 that we propose to rename Gyl1p (GYp like protein). Immunofluorescence experiments showed that Gyp5p and Gyl1p partly colocalize at the bud emergence site, at the bud tip and at the bud neck during cytokinesis. Subcellular fractionation and co-immunoprecipitation experiments showed that Gyp5p and Gyl1p co-fractionate with post-Golgi vesicles and plasma membrane, and belong to the same protein complexes in both localizations. We found by co-immunoprecipitation experiments that a fraction of Gyp5p interacts with Sec4p, a small GTPase involved in exocytosis, and that a fraction of Gyl1p associates at the plasma membrane with the Gyp5p/Sec4p complexes. We showed also that GYP5 genetically interacts with SEC2, which encodes the Sec4p exchange factor. Examination of the gyp5Deltagyl1Delta mutants grown at 13 degrees C revealed a slight growth defect, a secretion defect and an accumulation of secretory vesicles in the small-budded cells. These data suggest that Gyp5p and Gyl1p are involved in control of polarized exocytosis.
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Caractérisation fonctionnelle de deux protéines à domaine Ypt/Rab GAP, Gyp5p et Gyl1p chez Saccharomyces cerevisiae
Thèse de Laurent Chesneau
Thèse présentée pour obtenir le grade de
DOCTEUR EN SCIENCES DE L’UNIVERSITE PARIS-SUD 11
Spécialité Biologie Moléculaire de la Cellule
Thèse soutenue le 30 Janvier 2007 devant la Commission d’Examen composée de
M. Rosine HAGUENAUER-TSAPIS (Présidente)
M. Bruno GOUD (Rapporteur)
M. Robert ARKOWITZ (Rapporteur)
M. Barbara WINSOR (Examinatrice)
M. Michel JACQUET (Examinateur)
M. Marie-Hélène CUIF (Directrice de Thèse)
Les GTPases de la famille Ypt/Rab sont impliquées dans la régulation du trafic vésiculaire, et plus particulièrement... more
Les GTPases de la famille Ypt/Rab sont impliquées dans la régulation du trafic vésiculaire, et plus particulièrement dans les étapes de transport, d’arrimage et de fusion des vésicules. La régulation de leur activité GTPase est importante pour la régulation de leurs fonctions. L’activité GTPase des protéines Ypt/Rab est stimulée par des protéines activatrices de GTPase. Chez la levure Saccharomyces cerevisiae, il existe 10 protéines portant un domaine catalytique des protéines activatrices des GTPases Ypt/Rab. Gyp5p et Gyl1p sont deux protéines paralogues qui font partie de cette famille de protéines. In vitro, Gyp5p stimule l’activité GTPase de Ypt1p, qui est impliquée dans le transport entre le réticulum endoplasmique et l’appareil de Golgi, et de Sec4p, impliquée dans l’exocytose. Gyl1p, qui possède un domaine catalytique partiellement dégénéré, n’a pas d’activité stimulatrice de GTPase connue.
L’objectif de mon travail de thèse était de rechercher les fonctions biologiques de Gyp5p et de Gyl1p.
Nous avons montré que Gyp5p et Gyl1p sont situées aux sites de croissance polarisée, où elles sont co-localisées avec Sec4p. Gyp5p est présente dans un complexe contenant Sec4p dans une fraction enrichie en vésicules post-golgiennes, et Gyp5p et Gyl1p sont présentes dans un complexe contenant Sec4p dans une fraction enrichie en membrane plasmique. SEC2, qui code pour le facteur d’échange de Sec4p, et GYP5 présentent un effet génétique antagoniste par rapport à la croissance cellulaire. Les cellules dépourvues de Gyp5p et Gyl1p présentent des défauts de sécrétion, et accumulent des vésicules uniquement chez les jeunes bourgeons à 13°C. Ces résultats montrent que Gyp5p et Gyl1p sont impliquées dans l’exocytose polarisée.
Gyp5p et Gyl1p présentent une localisation dynamique au cours du cycle cellulaire. Gyp5p et Gyl1p sont co-localisées aux étapes de croissance polarisée, mais sont dissociées au cours de la croissance isotropique. Nous avons montré que Gyp5p est délocalisée en l’absence de Gyl1p, et Gyl1p est délocalisée en l’absence de Gyp5p, ce qui montre que la localisation de Gyp5p et Gyl1p est interdépendante. Nous avons montré que Gyp5p et Gyl1p sont capables d’interagir directement in vitro. Nous avons observé que les sous-unités du polarisome Bni1p, Bud6p et Spa2p sont nécessaires à la localisation de Gyp5p et Gyl1p aux sites de croissance polarisée. Le polarisome est un complexe
dont une des fonctions est de réguler la formation de câbles d’actine au sommet du bourgeon par la formine Bni1p. Des défauts de localisation de Gyp5p et Gyl1p ont également été observés en l’absence de l’autre formine Bnr1p. Ces résultats suggèrent que la localisation de Gyp5p et Gyl1p serait dépendante des câbles d’actine.
Nous avons également montré qu’une partie de Gyp5p et de Gyl1p est présente dans un complexe avec Rvs167p. Rvs167p est une protéine connue pour être impliquée dans l’endocytose. Une partie de Rvs167p est co-localisée avec Gyp5p et Gyl1p aux sites de croissance polarisée. Mais en l’absence de Gyp5p et de Gyl1p, Rvs167p n’est plus présente au sommet des jeunes bourgeons et au cou du bourgeon lors de la cytocinèse. Gyp5p et Gyl1p sont donc nécessaire à la localisation de Rvs167p aux sites de croissance polarisée.
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Interdependence of the Ypt/RabGAP Gyp5p and Gyl1p for recruitment to the sites of polarized growth.
Chesneau L, Prigent M, Boy-Marcotte E, Daraspe J, Fortier G, Jacquet M, Verbavatz JM, Cuif MH. Traffic. 2008 Apr;9(4):608-22.
Gyp5p and Gyl1p are two members of the Ypt/Rab guanosine triphosphatases-activating proteins involved in the control... more Gyp5p and Gyl1p are two members of the Ypt/Rab guanosine triphosphatases-activating proteins involved in the control of polarized exocytosis in Saccharomyces cerevisiae. We had previously shown that Gyp5p and Gyl1p colocalize at the sites of polarized growth and belong to the same complex in subcellular fractions enriched in plasma membrane or secretory vesicles. Here, we investigate the interaction between Gyp5p and Gyl1p as well as the mechanism of their localization to the sites of polarized growth. We show that purified recombinant Gyp5p and Gyl1p interact directly in vitro. In vivo, both Gyp5p and Gyl1p are mutually required to concentrate at the sites of polarized growth. Moreover, the localization of Gyp5p and Gyl1p to the sites of polarized growth requires the formins Bni1p and Bnr1p and depends on actin cables. We show that, in a sec6-4 mutant, blocking secretion leads to coaccumulation of Gyp5p and Gyl1p, together with Sec4p. Electron microscopy experiments demonstrate that Gyp5p is associated with secretory vesicles. Altogether, our results indicate that both Gyp5p and Gyl1p access the sites of polarized growth by transport on secretory vesicles. Two polarisome components, Spa2p and Bud6p, are involved in maintaining Gyp5p and Gyl1p colocalized at the sites of polarized growth.
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Seen by: APC15 drives the turnover of MCC-CDC20 to make the spindle assembly checkpoint responsive to kinetochore attachment.
by Mark Collins
Jörg Mansfeld, Philippe Collin, Mark O. Collins, Jyoti S. Choudhary & Jonathon Pines. Nature Cell Biolgy. 2011 Sep 18. doi: 10.1038/ncb2347
Faithful chromosome segregation during mitosis depends on the spindle assembly checkpoint (SAC), which monitors... more Faithful chromosome segregation during mitosis depends on the spindle assembly checkpoint (SAC), which monitors kinetochore attachment to the mitotic spindle. Unattached kinetochores generate mitotic checkpoint proteins complexes (MCCs) that bind and inhibit the anaphase-promoting complex, or cyclosome (APC/C). How the SAC proficiently inhibits the APC/C but still allows its rapid activation when the last kinetochore attaches to the spindle is important for the understanding of how cells maintain genomic stability. We show that the APC/C subunit APC15 is required for the turnover of the APC/C co-activator CDC20 and release of MCCs during SAC signalling but not for APC/C activity per se. In the absence of APC15, MCCs and ubiquitylated CDC20 remain 'locked' onto the APC/C, which prevents the ubiquitylation and degradation of cyclin B1 when the SAC is satisfied. We conclude that APC15 mediates the constant turnover of CDC20 and MCCs on the APC/C to allow the SAC to respond to the attachment state of kinetochores.
Robustness of Boolean dynamics under knockouts
Co-authored with Nils Bertschinger, Johannes Rauh, Eckehard Olbrich, and Konstantin Klemm
The response to a knockout of a node is a characteristic feature of a networked dynamical system. Knockout resilience... more The response to a knockout of a node is a characteristic feature of a networked dynamical system. Knockout resilience in the dynamics of the remaining nodes is a sign of robustness. Here we study the effect of knockouts for binary state sequences and their implementations in terms of Boolean threshold networks. Besides random sequences with biologically plausible constraints, we analyze the cell cycle sequence of the species Saccharomyces cerevisiae and the Boolean networks implementing it. Comparing with an appropriate null model we do not find evidence that the yeast wildtype network is optimized for high knockout resilience. Our notion of knockout resilience weakly correlates with the size of the basin of attraction, which has also been considered a measure of robustness.
Regulatory networks and connected components of the neutral space - A look at functional islands
Co-authored with Konstantin Klemm
The functioning of a living cell is largely determined by the structure of its regulatory network, comprising... more The functioning of a living cell is largely determined by the structure of its regulatory network, comprising non-linear interactions between regulatory genes. An important factor for the stability and evolvability of such regulatory systems is neutrality – typically a large number of alternative network structures give rise to the necessary dynamics. Here we study the discretized regulatory dynamics of the yeast cell cycle [Li et al., PNAS, 2004] and the set of networks capable of reproducing it, which we call functional. Among these, the empirical yeast wildtype network is close to optimal with respect to sparse wiring. Under point mutations, which establish or delete single interactions, the neutral space of functional networks is fragmented into ≈ 4.7 × 108 components. One of the smaller ones contains the wildtype network. On average, functional networks reachable from the wildtype by mutations are sparser, have higher noise resilience and fewer fixed point attractors as compared with networks outside of this wildtype component.
Simple Rules for Complex Processes: New Lessons from the Budding Yeast Cell Cycle
by Umut Eser
A preview about our paper.
Two papers in this issue of Molecular Cell, Doncic et al., 2011 and Eser et al., 2011, present some satisfyingly... more Two papers in this issue of Molecular Cell, Doncic et al., 2011 and Eser et al., 2011, present some satisfyingly simple ideas for the organization of the complex network that controls cell cycle progression and cell fate specification in budding yeast.
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Seen by: Quantitative Proteomics Reveals the Basis for the Biochemical Specificity of the Cell-Cycle Machinery
by Mark Collins
Felicia Walton Pagliuca*, Mark O. Collins*, Agata Lichawska, Philip Zegerman, Jyoti S. Choudhary, Jonathon Pines. Molecular Cell, Volume 43, Issue 3, 406-417, 5 August 2011
Cyclin-dependent kinases comprise the conserved machinery that drives progress through the cell cycle, but how they do... more Cyclin-dependent kinases comprise the conserved machinery that drives progress through the cell cycle, but how they do this in mammalian cells is still unclear. To identify the mechanisms by which cyclin-cdks control the cell cycle, we performed a time-resolved analysis of the in vivo interactors of cyclins E1, A2, and B1 by quantitative mass spectrometry. This global analysis of context-dependent protein interactions reveals the temporal dynamics of cyclin function in which networks of cyclin-cdk interactions vary according to the type of cyclin and cell-cycle stage. Our results explain the temporal specificity of the cell-cycle machinery, thereby providing a biochemical mechanism for the genetic requirement for multiple cyclins in vivo and reveal how the actions of specific cyclins are coordinated to control the cell cycle. Furthermore, we identify key substrates (Wee1 and c15orf42/Sld3) that reveal how cyclin A is able to promote both DNA replication and mitosis.
HDAC4 promotes growth of colon cancer cells via repression of p21.
by Dr. Gary Kao and Dr. Jay Dorsey
Wilson AJ, Byun DS, Nasser S, Murray LB, Ayyanar K, Arango D, Figueroa M, Melnick A, Kao GD, Augenlicht LH, Mariadason JM.
SourceDepartment of Oncology, Albert Einstein Cancer Center, Montefiore Medical Center, Bronx, NY 10467, USA.
The class II Histone deacetylase (HDAC), HDAC4, is expressed in a tissue-specific manner, and it represses... more
The class II Histone deacetylase (HDAC), HDAC4, is expressed in a tissue-specific manner, and it represses differentiation of specific cell types. We demonstrate here that HDAC4 is expressed in the proliferative zone in small intestine and colon and that its expression is down-regulated during intestinal differentiation in vivo and in vitro. Subcellular localization studies demonstrated HDAC4 expression was predominantly nuclear in proliferating HCT116 cells and relocalized to the cytoplasm after cell cycle arrest. Down-regulating HDAC4 expression by small interfering RNA (siRNA) in HCT116 cells induced growth inhibition and apoptosis in vitro, reduced xenograft tumor growth, and increased p21 transcription. Conversely, overexpression of HDAC4 repressed p21 promoter activity. p21 was likely a direct target of HDAC4, because HDAC4 down-regulation increased p21 mRNA when protein synthesis was inhibited by cycloheximide. The importance of p21 repression in HDAC4-mediated growth promotion was demonstrated by the failure of HDAC4 down-regulation to induce growth arrest in HCT116 p21-null cells. HDAC4 down-regulation failed to induce p21 when Sp1 was functionally inhibited by mithramycin or siRNA-mediated down-regulation. HDAC4 expression overlapped with that of Sp1, and a physical interaction was demonstrated by coimmunoprecipitation. Chromatin immunoprecipitation (ChIP) and sequential ChIP analyses demonstrated Sp1-dependent binding of HDAC4 to the proximal p21 promoter, likely directed through the HDAC4-HDAC3-N-CoR/SMRT corepressor complex. Consistent with increased transcription, HDAC4 or SMRT down-regulation resulted in increased histone H3 acetylation at the proximal p21 promoter locus. These studies identify HDAC4 as a novel regulator of colon cell proliferation through repression of p21.