[Show abstract][Hide abstract] ABSTRACT: Hsp90 is one of the most abundant and conserved proteins in the cell. Reduced levels or activity of Hsp90 causes defects in many cellular processes and also reveals genetic and non-genetic variation within a population. Despite information about Hsp90 protein-protein interactions, a global view of the Hsp90 regulated proteome in yeast is unavailable. To investigate the degree of dependency of individual yeast proteins on Hsp90, we used the SILAC method coupled with mass spectrometry (MS) to quantify around 4,000 proteins in low-Hsp90 cells. We observed that 904 proteins changed in their abundance by more than 1.5 fold. When compared with the transcriptome of the same population of cells, two-thirds of the mis-regulated proteins were observed to be affected post-transcriptionally, of which the majority were down-regulated. Further analyses indicated that the down-regulated proteins are highly conserved and assume central roles in cellular networks with a high number of protein interacting partners, suggesting that Hsp90 buffers genetic and non-genetic variation through regulating protein network hubs. The down-regulated proteins were enriched for essential proteins previously not known to be Hsp90-dependent. Finally, we observed that down-regulation of transcription factors and mating pathway components by attenuating Hsp90 function led to decreased target gene expression and pheromone response respectively, providing a direct link between observed proteome regulation and cellular phenotypes.
Genome Biology and Evolution 10/2014; 6(10):2851-2865. · 4.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this study, we examine the effect of chemokine (C-C motif) ligand 5 (CCL5)/Regulated on Activation Normal T cell Expressed and Secreted (RANTES), a pro-inflammatory cytokine on osteogenic differentiation of human mesenchymal stem cells (hMSCs). We found CCL5 expression was increased during osteogenic differentiation of hMSCs and CCL5 expression is dependent on the presence of dexamethasone. Knocking down endogenous CCL5 expression blocked osteogenesis, as revealed by decreasing alkaline phosphatase (ALP) activity and a reduction in the expression levels of ALP, bone sialoprotein (BSP), and osteopontin (OPN). Of note, the overexpression of CCL5 was sufficient to increase ALP expression and activity. Moreover, the down-regulation of chemokine (C-C motif) receptor 1 (CCR1), one of the CCL5 receptors, significantly decreased the osteogenesis of hMSCs. Interestingly, the down-regulation of CCR1, but not CCL5, was sufficient to affect the cell numbers during the process of osteogenesis. Our findings reveal that both CCL5 and CCR1 are required for osteogenesis of human MSCs, CCL5 is sufficient for the osteogenesis, and provide a novel link between dexamethasone and CCL5 in human osteogenesis.
[Show abstract][Hide abstract] ABSTRACT: Human embryonic stem cells (hESCs) are functionally unique for their self-renewal ability and pluripotency, but the molecular mechanisms giving rise to these properties are not fully understood. hESCs can differentiate into embryoid bodies (EBs) containing ectoderm, mesoderm, and endoderm. In the miR-200 family, miR-200c was especially enriched in undifferentiated hESCs and significantly downregulated in EBs. The knockdown of the miR-200c in hESCs downregulated Nanog expression, upregulated GATA binding protein 4 (GATA4) expression, and induced hESC apoptosis. The knockdown of GATA4 rescued hESC apoptosis induced by downregulation of miR-200c. miR-200c directly targeted the 3'-untranslated region of GATA4. Interestingly, the downregulation of GATA4 significantly inhibited EB formation in hESCs. Overexpression of miR-200c inhibited EB formation and repressed the expression of ectoderm, endoderm, and mesoderm markers, which could partially be rescued by ectopic expression of GATA4. Fibroblast growth factor (FGF) and activin A/nodal can sustain hESC renewal in the absence of feeder layer. Inhibition of transforming growth factor-β (TGF-β)/activin A/nodal signaling by SB431542 treatment downregulated the expression of miR-200c. Overexpression of miR-200c partially rescued the expression of Nanog/phospho-Smad2 that was downregulated by SB431542 treatment. Our observations have uncovered novel functions of miR-200c and GATA4 in regulating hESC renewal and differentiation.
Stem Cell Research 12/2013; 12(2):338-353. · 4.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Due to the development of drug resistance, the outcome for the majority of patients with acute myeloid leukemia (acute myelogenous leukemia; AML) remains poor. To prevent drug resistance and increase the therapeutic efficacy of treating AML, the development of new combinatory drug therapies is necessary. Sonic hedgehog (Shh) is expressed in AML biopsies and is essential for the drug resistance of cancer stem cells of AML. AML patients are frequently infected by bacteria and exposed to lipopolysaccharide (LPS). LPS itself, its derivatives, and its downstream effectors, such as tumor necrosis factor-α (TNF-α) and interferons (IFNs), have been shown to provoke anti-tumor effects. The application of a Shh inhibitor against AML cells in the presence of LPS/TNF-α/IFNs has not been investigated. We found that the Shh inhibitor cyclopamine in combination with LPS treatment synergistically induced massive cell apoptosis in THP-1 and U937 cells. The cytotoxic effects of this combined drug treatment were confirmed in 5 additional AML cell lines, in primary AML cells, and in an AML mouse model. Replacing cyclopamine with another Shh inhibitor, Sant-1, had the same effect. LPS could be substituted by TNF-α or IFNs to induce AML cell death in combination with cyclopamine. Our results suggest a potential strategy for the development of new therapies employing Shh antagonists in the presence of LPS/TNF-α/IFNs for the treatment of AML patients.
Investigational New Drugs 12/2012; · 3.50 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the budding yeast Saccharomyces cerevisiae, cell cycle progression and cytokinesis at mitotic exit are proposed to be linked by CDC14 phosphatase antagonizing the function of mitotic B-type cyclin (CLBs). We have isolated a temperature-sensitive mutant, cdc14(A280V), with a mutation in the conserved phosphatase domain. Prolonged arrest in the cdc14(A280V) mutant partially uncoupled cell cycle progression from the completion of cytokinesis as measured by bud re-emergence, in the form of elongated apical projections, and DNA re-replication. In contrast to previous mitotic exit mutants, cdc14(A280V) mutants displayed a strong bias for the first apical projection to form in the mother cell body. Using cdc14(A280V) mutant phenotypes, the functions of the B-type cyclins at mitotic exit were investigated. The preference in mother-daughter apical projection formation was observed to be independent of any individual CLB function. However, cdc14(A280V)clb1Δ cells displayed a pronounced increase in apical projections, while cdc14(A280V)clb3Δ cells were observed to form round cellular chains. While cdc14(A280V) cells arrested at mitotic exit, both cdc14(A280V)clb1Δ and cdc14(A280V)clb3Δ cells completed cytokinesis, but failed cell separation. cdc14(A280V)clb2Δ cells displayed a defect in actin ring assembly. These observations differentiate the functions of CLB1, CLB2, and CLB3 at mitotic exit, and are consistent with the hypothesis that CLB activities are antagonized by the CDC14 phosphatase in order to couple cell cycle progression with cytokinesis at mitotic exit.
Fungal Genetics and Biology 07/2011; 48(10):966-78. · 3.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chromosome segregation depends on the spindle checkpoint, which delays anaphase until all chromosomes have bound microtubules and have been placed under tension. The Mad1-Mad2 complex is an essential component of the checkpoint. We studied the consequences of removing one copy of MAD2 in diploid cells of the budding yeast, Saccharomyces cerevisiae. Compared to MAD2/MAD2 cells, MAD2/mad2Δ heterozygotes show increased chromosome loss and have different responses to two insults that activate the spindle checkpoint: MAD2/mad2Δ cells respond normally to antimicrotubule drugs but cannot respond to chromosomes that lack tension between sister chromatids. In MAD2/mad2Δ cells with normal sister chromatid cohesion, removing one copy of MAD1 restores the checkpoint and returns chromosome loss to wild-type levels. We conclude that cells need the normal Mad2:Mad1 ratio to respond to chromosomes that are not under tension.
Molecular biology of the cell 05/2011; 22(14):2448-57. · 5.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The serum-free medium from Japanese encephalitis virus (JEV) infected Baby Hamster Kidney-21 (BHK-21) cell cultures was analyzed by liquid chromatography tandem mass spectrometry (LC-MS) to identify host proteins that were secreted upon viral infection. Five proteins were identified, including the molecular chaperones Hsp90, GRP78, and Hsp70. The functional role of GRP78 in the JEV life cycle was then investigated. Co-migration of GRP78 with JEV particles in sucrose density gradients was observed and co-localization of viral E protein with GRP78 was detected by immunofluorescence analysis in vivo. Knockdown of GRP78 expression by siRNA did not effect viral RNA replication, but did impair mature viral production. Mature viruses that do not co-fractionate with GPR78 displayed a significant decrease in viral infectivity. Our results support the hypothesis that JEV co-opts host cell GPR78 for use in viral maturation and in subsequent cellular infections.
[Show abstract][Hide abstract] ABSTRACT: The serum-free medium from Japanese encephalitis virus (JEV) infected Baby Hamster Kidney-21 (BHK-21) cell cultures was analyzed
by liquid chromatography tandem mass spectrometry (LC-MS) to identify host proteins that were secreted upon viral infection.
Five proteins were identified, including the molecular chaperones Hsp90, GRP78, and Hsp70. The functional role of GRP78 in
the JEV life cycle was then investigated. Co-migration of GRP78 with JEV particles in sucrose density gradients was observed
and co-localization of viral E protein with GRP78 was detected by immunofluorescence analysis in vivo. Knockdown of GRP78 expression by siRNA did not effect viral RNA replication, but did impair mature viral production. Mature
viruses that do not co-fractionate with GPR78 displayed a significant decrease in viral infectivity. Our results support the
hypothesis that JEV co-opts host cell GPR78 for use in viral maturation and in subsequent cellular infections.
[Show abstract][Hide abstract] ABSTRACT: Cell cycle transitions are controlled, in part, by ubiquitin-dependent proteolysis. In mitosis, the metaphase to anaphase transition is governed by an E3 ubiquitin ligase called the cyclosome or Anaphase-Promoting Complex (APC), and a WD40-repeat protein co-factor called Cdc20. In vitro Cdc20-dependent APC (APC(Cdc20)) assays have been useful in the identification and validation of target substrates, and in the study of APC enzymology and regulation. Many aspects of the regulation of cell cycle progression have been discovered in the budding yeast Saccharomyces cerevisiae, and proteins purified from this model organism have been employed in a wide variety of in vitro assays. Here we outline a quantitative in vitro mitotic APC(Cdc20) assay that makes use of a highly active form of the APC that is purified from budding yeast cells arrested in mitosis.
[Show abstract][Hide abstract] ABSTRACT: In the budding yeast Saccharomyces cerevisiae the mitotic spindle must be positioned along the mother-bud axis to activate the mitotic exit network (MEN) in anaphase. To examine MEN proteins during mitotic exit, we imaged the MEN activators Tem1p and Cdc15p and the MEN regulator Bub2p in vivo. Quantitative live cell fluorescence microscopy demonstrated the spindle pole body that segregated into the daughter cell (dSPB) signaled mitotic exit upon penetration into the bud. Activation of mitotic exit was associated with an increased abundance of Tem1p-GFP and the localization of Cdc15p-GFP on the dSPB. In contrast, Bub2p-GFP fluorescence intensity decreased in mid-to-late anaphase on the dSPB. Therefore, MEN protein localization fluctuates to switch from Bub2p inhibition of mitotic exit to Cdc15p activation of mitotic exit. The mechanism that elevates Tem1p-GFP abundance in anaphase is specific to dSPB penetration into the bud and Dhc1p and Lte1p promote Tem1p-GFP localization. Finally, fluorescence recovery after photobleaching (FRAP) measurements revealed Tem1p-GFP is dynamic at the dSPB in late anaphase. These data suggest spindle pole penetration into the bud activates mitotic exit, resulting in Tem1p and Cdc15p persistence at the dSPB to initiate the MEN signal cascade.
Molecular Biology of the Cell 05/2004; 15(4):1519-32. · 4.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The midzone is the domain of the mitotic spindle that maintains spindle bipolarity during anaphase and generates forces required for spindle elongation (anaphase B). Although there is a clear role for microtubule (MT) motor proteins at the spindle midzone, less is known about how microtubule-associated proteins (MAPs) contribute to midzone organization and function. Here, we report that budding yeast Ase1p is a member of a conserved family of midzone-specific MAPs. By size exclusion chromatography and velocity sedimentation, both Ase1p in extracts and purified Ase1p behaved as a homodimer. Ase1p bound and bundled MTs in vitro. By live cell microscopy, loss of Ase1p resulted in a specific defect: premature spindle disassembly in mid-anaphase. Furthermore, when overexpressed, Ase1p was sufficient to trigger spindle elongation in S phase-arrested cells. FRAP revealed that Ase1p has both a very slow rate of turnover within the midzone and limited lateral diffusion along spindle MTs. We propose that Ase1p functions as an MT cross-bridge that imparts matrix-like characteristics to the midzone. MT-dependent networks of spindle midzone MAPs may be one molecular basis for the postulated spindle matrix.
The Journal of Cell Biology 03/2003; 160(4):517-28. · 9.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Accurate distribution of the chromosomes in dividing cells requires coupling of cellular polarity cues with both the orientation of the mitotic spindle and cell cycle progression. Work in budding yeast has demonstrated that cytoplasmic dynein and the kinesin Kip3p define redundant pathways that ensure proper spindle orientation. Furthermore, it has been shown that the Kip3p pathway components Kar9p and Bim1p (Yeb1p) form a complex that provides a molecular link between cortical polarity cues and spindle microtubules. Recently, other studies indicated that the cortical localization of Kar9p depends upon actin cables and Myo2p, a type V myosin. In addition, a BUB2-dependent cell cycle checkpoint has been described that inhibits the mitotic exit network and cytokinesis until proper centrosome position is achieved. Combined, these studies provide molecular insight into how cells link cellular polarity, spindle position and cell cycle progression.
[Show abstract][Hide abstract] ABSTRACT: Correct positioning of the mitotic spindle is critical for cell division and development. Spindle positioning involves a search-and-capture mechanism whereby dynamic microtubules find and then interact with specific sites on the submembrane cortex. Genetic, biochemical, and imaging experiments suggest a mechanism for cortical-microtubule capture. Bim1p, located at microtubule distal ends, bound Kar9p, a protein associated with the daughter cell cortex. Bim1p is the yeast ortholog of human EB1, a binding partner for the adenomatous polyposis coli tumor suppressor. EB1 family proteins may have a general role in linking the microtubule cytoskeleton to cortical polarity determinants.
[Show abstract][Hide abstract] ABSTRACT: Human EB1 is a highly conserved protein that binds to the carboxyl terminus of the human adenomatous polyposis coli (APC) tumor suppressor protein , a domain of APC that is commonly deleted in colorectal neoplasia . EB1 belongs to a family of microtubule-associated proteins that includes Schizosaccharomyces pombe Mal3  and Saccharomyces cerevisiae Bim1p . Bim1p appears to regulate the timing of cytokinesis as demonstrated by a genetic interaction with Act5, a component of the yeast dynactin complex . Whereas the predominant function of the dynactin complex in yeast appears to be in positioning the mitotic spindle , in animal cells, dynactin has been shown to function in diverse processes, including organelle transport, formation of the mitotic spindle, and perhaps cytokinesis    . Here, we demonstrate that human EB1 can be coprecipitated with p150(Glued), a member of the dynactin protein complex. EB1 was also found associated with the intermediate chain of cytoplasmic dynein (CDIC) and with dynamitin (p50), another component of the dynactin complex, but not with dynein heavy chain, in a complex that sedimented at approximately 5S in a sucrose density gradient. The association of EB1 with members of the dynactin complex was independent of APC and was preserved in the absence of an intact microtubule cytoskeleton. The molecular interaction of EB1 with members of the dynactin complex and with CDIC may be important for microtubule-based processes.
Current Biology 05/1999; 9(8):425-8. · 9.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The evolutionarily conserved protein EB1 originally was identified by its physical association with the carboxyl-terminal
portion of the adenomatous polyposis coli (APC) tumor suppressor protein, an APC domain commonly mutated in familial and sporadic
forms of colorectal neoplasia. The subcellular localization of EB1 in epithelial cells was studied by using immunofluorescence
and biochemical techniques. EB1 colocalized both to cytoplasmic microtubules in interphase cells and to spindle microtubules
during mitosis, with pronounced centrosome staining. The cytoskeletal array detected by anti-EB1 antibody was abolished by
incubation of the cells with nocodazole, an agent that disrupts microtubules; upon drug removal, EB1 localized to the microtubule-organizing
center. Immunofluorescence analysis of SW480, a colon cancer cell line that expresses only carboxyl-terminal-deleted APC unable
to interact with EB1, demonstrated that EB1 remained localized to the microtubule cytoskeleton, suggesting that this pattern
of subcellular distribution is not mediated by its interaction with APC. In vitro cosedimentation with taxol-stabilized microtubules demonstrated that a significant fraction of EB1 associated with microtubules.
Recent studies of the yeast EB1 homologues Mal3 and Bim1p have demonstrated that both proteins localize to microtubules and
are important in vivo for microtubule function. Our results demonstrate that EB1 is a novel component of the microtubule cytoskeleton in mammalian
cells. Associating with the mitotic apparatus, EB1 may play a physiologic role connecting APC to cellular division, coordinating
the control of normal growth and differentiation processes in the colonic epithelium.
Proceedings of the National Academy of Sciences 08/1998; 95(18):10596-10601. · 9.81 Impact Factor