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Title: An improved smaller biotin ligase for BioID proximity labeling.
Creator: Kim, Dae In, Jensen, Samuel C, Noble, Kyle A, Kc, Birendra, Roux, Kenneth H, Motamedchaboki, Khatereh, Roux, Kyle J
Abstract/Description: The BioID method uses a promiscuous biotin ligase to detect protein-protein associations as well as proximate proteins in living cells. Here we report improvements to the BioID method centered on BioID2, a substantially smaller promiscuous biotin ligase. BioID2 enables more-selective targeting of fusion proteins, requires less biotin supplementation, and exhibits enhanced labeling of proximate proteins. Thus BioID2 improves the efficiency of screening for protein-protein associations. We also...
Show moreThe BioID method uses a promiscuous biotin ligase to detect protein-protein associations as well as proximate proteins in living cells. Here we report improvements to the BioID method centered on BioID2, a substantially smaller promiscuous biotin ligase. BioID2 enables more-selective targeting of fusion proteins, requires less biotin supplementation, and exhibits enhanced labeling of proximate proteins. Thus BioID2 improves the efficiency of screening for protein-protein associations. We also demonstrate that the biotinylation range of BioID2 can be considerably modulated using flexible linkers, thus enabling application-specific adjustment of the biotin-labeling radius.
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Date Issued: 2016-04-15
Identifier: FSU_pmch_26912792, 10.1091/mbc.E15-12-0844, PMC4831873, 26912792, 26912792, mbc.E15-12-0844
Format: Citation

Title: Changes in single-molecule integrin dynamics linked to local cellular behavior.
Creator: Jaqaman, Khuloud, Galbraith, James A, Davidson, Michael W, Galbraith, Catherine G
Abstract/Description: Recent advances in light microscopy permit visualization of the behavior of individual molecules within dense macromolecular ensembles in live cells. It is now conceptually possible to relate the dynamic organization of molecular machinery to cellular function. However, inherent heterogeneities, as well as disparities between spatial and temporal scales, pose substantial challenges in deriving such a relationship. New approaches are required to link discrete single-molecule behavior with...
Show moreRecent advances in light microscopy permit visualization of the behavior of individual molecules within dense macromolecular ensembles in live cells. It is now conceptually possible to relate the dynamic organization of molecular machinery to cellular function. However, inherent heterogeneities, as well as disparities between spatial and temporal scales, pose substantial challenges in deriving such a relationship. New approaches are required to link discrete single-molecule behavior with continuous cellular-level processes. Here we combined intercalated molecular and cellular imaging with a computational framework to detect reproducible transient changes in the behavior of individual molecules that are linked to cellular behaviors. Applying our approach to integrin transmembrane receptors revealed a spatial density gradient underlying characteristic molecular density increases and mobility decreases, indicating the subsequent onset of local protrusive activity. Integrin mutants further revealed that these density and mobility transients are separable and depend on different binding domains within the integrin cytoplasmic tail. Our approach provides a generalizable paradigm for dissecting dynamic spatiotemporal molecular behaviors linked to local cellular events.
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Date Issued: 2016-05-15
Identifier: FSU_pmch_27009207, 10.1091/mbc.E16-01-0018, PMC4865314, 27009207, 27009207, mbc.E16-01-0018
Format: Citation

Title: Phosphatase 2A negatively regulates mitotic exit in Saccharomyces cerevisiae.
Creator: Wang, Yanchang, Ng, Tuen-Yung
Abstract/Description: In budding yeast Saccharomyces cerevisiae, Cdc5 kinase is a component of mitotic exit network (MEN), which inactivates cyclin-dependent kinase (CDK) after chromosome segregation. cdc5-1 mutants arrest at telophase at the nonpermissive temperature due to the failure of CDK inactivation. To identify more negative regulators of MEN, we carried out a genetic screen for genes that are toxic to cdc5-1 mutants when overexpressed. Genes that encode the B-regulatory subunit (Cdc55) and the three...
Show moreIn budding yeast Saccharomyces cerevisiae, Cdc5 kinase is a component of mitotic exit network (MEN), which inactivates cyclin-dependent kinase (CDK) after chromosome segregation. cdc5-1 mutants arrest at telophase at the nonpermissive temperature due to the failure of CDK inactivation. To identify more negative regulators of MEN, we carried out a genetic screen for genes that are toxic to cdc5-1 mutants when overexpressed. Genes that encode the B-regulatory subunit (Cdc55) and the three catalytic subunits (Pph21, Pph22, and Pph3) of phosphatase 2A (PP2A) were isolated. In addition to cdc5-1, overexpression of CDC55, PPH21, or PPH22 is also toxic to other temperature-sensitive mutants that display defects in mitotic exit. Consistently, deletion of CDC55 partially suppresses the temperature sensitivity of these mutants. Moreover, in the presence of spindle damage, PP2A mutants display nuclear localized Cdc14, the key player in MEN pathway, indicative of MEN activation. All the evidence suggests the negative role of PP2A in mitotic exit. Finally, our genetic and biochemical data suggest that PP2A regulates the phosphorylation of Tem1, which acts at the very top of MEN pathway.
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Date Issued: 2006-01-01
Identifier: FSU_pmch_16079183, 10.1091/mbc.E04-12-1109, PMC1345648, 16079183, 16079183, E04-12-1109
Format: Citation

Title: Drosophila pericentrin requires interaction with calmodulin for its function at centrosomes and neuronal basal bodies but not at sperm basal bodies.
Creator: Galletta, Brian J, Guillen, Rodrigo X, Fagerstrom, Carey J, Brownlee, Chris W, Lerit, Dorothy A, Megraw, Timothy L, Rogers, Gregory C, Rusan, Nasser M
Abstract/Description: Pericentrin 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...
Show morePericentrin 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.
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Date Issued: 2014-09-15
Identifier: FSU_pmch_25031429, 10.1091/mbc.E13-10-0617, PMC4161505, 25031429, 25031429, mbc.E13-10-0617
Format: Citation

Title: Regulation of the transcription factor YY1 in mitosis through phosphorylation of its DNA-binding domain.
Creator: Rizkallah, Raed, Hurt, Myra M
Abstract/Description: Yin-Yang 1 (YY1) is a ubiquitously expressed zinc finger transcription factor. It regulates a vast array of genes playing critical roles in development, differentiation, and cell cycle. Very little is known about the mechanisms that regulate the functions of YY1. It has long been proposed that YY1 is a phosphoprotein; however, a direct link between phosphorylation and the function of YY1 has never been proven. Investigation of the localization of YY1 during mitosis shows that it is...
Show moreYin-Yang 1 (YY1) is a ubiquitously expressed zinc finger transcription factor. It regulates a vast array of genes playing critical roles in development, differentiation, and cell cycle. Very little is known about the mechanisms that regulate the functions of YY1. It has long been proposed that YY1 is a phosphoprotein; however, a direct link between phosphorylation and the function of YY1 has never been proven. Investigation of the localization of YY1 during mitosis shows that it is distributed to the cytoplasm during prophase and remains excluded from DNA until early telophase. Immunostaining studies show that YY1 is distributed equally between daughter cells and rapidly associates with decondensing chromosomes in telophase, suggesting a role for YY1 in early marking of active and repressed genes. The exclusion of YY1 from DNA in prometaphase HeLa cells correlated with an increase in the phosphorylation of YY1 and loss of DNA-binding activity that can be reversed by dephosphorylation. We have mapped three phosphorylation sites on YY1 during mitosis and show that phosphorylation of two of these sites can abolish the DNA-binding activity of YY1. These results demonstrate a novel mechanism for the inactivation of YY1 through phosphorylation of its DNA-binding domain.
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Date Issued: 2009-11-01
Identifier: FSU_pmch_19793915, 10.1091/mbc.E09-04-0264, PMC2777106, 19793915, 19793915, E09-04-0264
Format: Citation

Title: Slk19 clusters kinetochores and facilitates chromosome bipolar attachment.
Creator: Richmond, Daniel, Rizkallah, Raed, Liang, Fengshan, Hurt, Myra M, Wang, Yanchang
Abstract/Description: In all eukaryotic cells, DNA is packaged into multiple chromosomes that are linked to microtubules through a large protein complex called a kinetochore. Previous data show that the kinetochores are clustered together during most of the cell cycle, but the mechanism and the biological significance of kinetochore clustering are unknown. As a kinetochore protein in budding yeast, the role of Slk19 in the stability of the anaphase spindle has been well studied, but its function in chromosome...
Show moreIn all eukaryotic cells, DNA is packaged into multiple chromosomes that are linked to microtubules through a large protein complex called a kinetochore. Previous data show that the kinetochores are clustered together during most of the cell cycle, but the mechanism and the biological significance of kinetochore clustering are unknown. As a kinetochore protein in budding yeast, the role of Slk19 in the stability of the anaphase spindle has been well studied, but its function in chromosome segregation has remained elusive. Here we show that Slk19 is required for kinetochore clustering when yeast cells are treated with the microtubule-depolymerizing agent nocodazole. We further find that slk19Δ mutant cells exhibit delayed kinetochore capture and chromosome bipolar attachment after the disruption of the kinetochore-microtubule interaction by nocodazole, which is likely attributed to defective kinetochore clustering. In addition, we show that Slk19 interacts with itself, suggesting that the dimerization of Slk19 may mediate the interaction between kinetochores for clustering. Therefore Slk19 likely acts as kinetochore glue that clusters kinetochores to facilitate efficient and faithful chromosome segregation.
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Date Issued: 2013-03-01
Identifier: FSU_pmch_23283988, 10.1091/mbc.E12-07-0552, PMC3583661, 23283988, 23283988, mbc.E12-07-0552
Format: Citation

Title: The Seckel syndrome and centrosomal protein Ninein localizes asymmetrically to stem cell centrosomes but is not required for normal development, behavior, or DNA damage response in Drosophila.
Creator: Zheng, Yiming, Mennella, Vito, Marks, Steven, Wildonger, Jill, Elnagdi, Esraa, Agard, David, Megraw, Timothy L
Abstract/Description: Ninein (Nin) is a centrosomal protein whose gene is mutated in Seckel syndrome (SCKL, MIM 210600), an inherited recessive disease that results in primordial dwarfism, cognitive deficiencies, and increased sensitivity to genotoxic stress. Nin regulates neural stem cell self-renewal, interkinetic nuclear migration, and microtubule assembly in mammals. Nin is evolutionarily conserved, yet its role in cell division and development has not been investigated in a model organism. Here we...
Show moreNinein (Nin) is a centrosomal protein whose gene is mutated in Seckel syndrome (SCKL, MIM 210600), an inherited recessive disease that results in primordial dwarfism, cognitive deficiencies, and increased sensitivity to genotoxic stress. Nin regulates neural stem cell self-renewal, interkinetic nuclear migration, and microtubule assembly in mammals. Nin is evolutionarily conserved, yet its role in cell division and development has not been investigated in a model organism. Here we characterize the single Nin orthologue in Drosophila Drosophila Nin localizes to the periphery of the centrosome but not at centriolar structures as in mammals. However, Nin shares the property of its mammalian orthologue of promoting microtubule assembly. In neural and germline stem cells, Nin localizes asymmetrically to the younger (daughter) centrosome, yet it is not required for the asymmetric division of stem cells. In wing epithelia and muscle, Nin localizes to noncentrosomal microtubule-organizing centers. Surprisingly, loss of nin expression from a nin mutant does not significantly affect embryonic and brain development, fertility, or locomotor performance of mutant flies or their survival upon exposure to DNA-damaging agents. Although it is not essential, our data suggest that Nin plays a supportive role in centrosomal and extracentrosomal microtubule organization and asymmetric stem cell division.
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Date Issued: 2016-06-01
Identifier: FSU_pmch_27053665, 10.1091/mbc.E15-09-0655, PMC4884065, 27053665, 27053665, mbc.E15-09-0655
Format: Citation

Title: Sequential posttranslational modifications regulate PKC degradation.
Creator: Wang, Yan, Wang, Yangbo, Zhang, Huijun, Gao, Yingwei, Huang, Chao, Zhou, Aiwu, Zhou, Yi, Li, Yong
Abstract/Description: Cross-talk among different types of posttranslational modifications (PTMs) has emerged as an important regulatory mechanism for protein function. Here we elucidate a mechanism that controls PKCα stability via a sequential cascade of PTMs. We demonstrate that PKCα dephosphorylation decreases its sumoylation, which in turn promotes its ubiquitination and ultimately enhances its degradation via the ubiquitin-proteasome pathway. These findings provide a molecular explanation for the activation...
Show moreCross-talk among different types of posttranslational modifications (PTMs) has emerged as an important regulatory mechanism for protein function. Here we elucidate a mechanism that controls PKCα stability via a sequential cascade of PTMs. We demonstrate that PKCα dephosphorylation decreases its sumoylation, which in turn promotes its ubiquitination and ultimately enhances its degradation via the ubiquitin-proteasome pathway. These findings provide a molecular explanation for the activation-induced down-regulation of PKC proteins.
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Date Issued: 2016-01-15
Identifier: FSU_pmch_26564794, 10.1091/mbc.E15-09-0624, PMC4713141, 26564794, 26564794, mbc.E15-09-0624
Format: Citation

Title: The molecular function of the yeast polo-like kinase Cdc5 in Cdc14 release during early anaphase.
Creator: Liang, Fengshan, Jin, Fengzhi, Liu, Hong, Wang, Yanchang
Abstract/Description: In the budding yeast Saccharomyces cerevisiae, Cdc14 is sequestered within the nucleolus before anaphase entry through its association with Net1/Cfi1, a nucleolar protein. Protein phosphatase PP2A(Cdc55) dephosphorylates Net1 and keeps it as a hypophosphorylated form before anaphase. Activation of the Cdc fourteen early anaphase release (FEAR) pathway after anaphase entry induces a brief Cdc14 release from the nucleolus. Some of the components in the FEAR pathway, including Esp1, Slk19, and...
Show moreIn the budding yeast Saccharomyces cerevisiae, Cdc14 is sequestered within the nucleolus before anaphase entry through its association with Net1/Cfi1, a nucleolar protein. Protein phosphatase PP2A(Cdc55) dephosphorylates Net1 and keeps it as a hypophosphorylated form before anaphase. Activation of the Cdc fourteen early anaphase release (FEAR) pathway after anaphase entry induces a brief Cdc14 release from the nucleolus. Some of the components in the FEAR pathway, including Esp1, Slk19, and Spo12, inactivate PP2A(Cdc55), allowing the phosphorylation of Net1 by mitotic cyclin-dependent kinase (Cdk) (Clb2-Cdk1). However, the function of another FEAR component, the Polo-like kinase Cdc5, remains elusive. Here, we show evidence indicating that Cdc5 promotes Cdc14 release primarily by stimulating the degradation of Swe1, the inhibitory kinase for mitotic Cdk. First, we found that deletion of SWE1 partially suppresses the FEAR defects in cdc5 mutants. In contrast, high levels of Swe1 impair FEAR activation. We also demonstrated that the accumulation of Swe1 in cdc5 mutants is responsible for the decreased Net1 phosphorylation. Therefore, we conclude that the down-regulation of Swe1 protein levels by Cdc5 promotes FEAR activation by relieving the inhibition on Clb2-Cdk1, the kinase for Net1 protein.
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Date Issued: 2009-08-01
Identifier: FSU_pmch_19570916, 10.1091/mbc.E08-10-1049, PMC2777927, 19570916, 19570916, E08-10-1049
Format: Citation

Title: The function and regulation of budding yeast Swe1 in response to interrupted DNA synthesis.
Creator: Liu, Hong, Wang, Yanchang
Abstract/Description: Periodically regulated cyclin-dependent kinase (Cdk) is required for DNA synthesis and mitosis. Hydroxyurea (HU) inhibits DNA synthesis by depleting dNTPs, the basic unit for DNA synthesis. HU treatment triggers the S-phase checkpoint, which arrests cells at S-phase, inhibits late origin firing and stabilizes replication forks. Using budding yeast as a model system, we found that Swe1, a negative regulator of Cdk, appears at S-phase and accumulates in HU treatment cells. Interestingly, this...
Show morePeriodically regulated cyclin-dependent kinase (Cdk) is required for DNA synthesis and mitosis. Hydroxyurea (HU) inhibits DNA synthesis by depleting dNTPs, the basic unit for DNA synthesis. HU treatment triggers the S-phase checkpoint, which arrests cells at S-phase, inhibits late origin firing and stabilizes replication forks. Using budding yeast as a model system, we found that Swe1, a negative regulator of Cdk, appears at S-phase and accumulates in HU treatment cells. Interestingly, this accumulation is not dependent on S-phase checkpoint. Deltahsl1, Deltahsl7, and cdc5-2 mutants, which have defects in Swe1 degradation, show HU sensitivity because of high Swe1 protein levels. We further demonstrated that their HU sensitivity is not a result of DNA damage accumulation or incomplete DNA synthesis; instead the sensitivity is due to their dramatically delayed recovery from HU-induced S-phase arrest. Strikingly, our in vivo data indicate that Swe1 inhibits the kinase activity of Clb2-Cdk1, but not that of Clb5-Cdk1. Therefore, S-phase accumulated Swe1 prevents Clb2-Cdk1-mediated mitotic activities, but has little effects on Clb5-Cdk1-associated S-phase progression.
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Date Issued: 2006-06-01
Identifier: FSU_pmch_16571676, 10.1091/mbc.E05-11-1093, PMC1474790, 16571676, 16571676, E05-11-1093
Format: Citation

Title: Ubiquilin/Dsk2 promotes inclusion body formation and vacuole (lysosome)-mediated disposal of mutated huntingtin.
Creator: Chuang, Kun-Han, Liang, Fengshan, Higgins, Ryan, Wang, Yanchang
Abstract/Description: Ubiquilin proteins contain a ubiquitin-like domain (UBL) and ubiquitin-associated domain(s) that interact with the proteasome and ubiquitinated substrates, respectively. Previous work established the link between ubiquilin mutations and neurodegenerative diseases, but the function of ubiquilin proteins remains elusive. Here we used a misfolded huntingtin exon I containing a 103-polyglutamine expansion (Htt103QP) as a model substrate for the functional study of ubiquilin proteins. We found...
Show moreUbiquilin proteins contain a ubiquitin-like domain (UBL) and ubiquitin-associated domain(s) that interact with the proteasome and ubiquitinated substrates, respectively. Previous work established the link between ubiquilin mutations and neurodegenerative diseases, but the function of ubiquilin proteins remains elusive. Here we used a misfolded huntingtin exon I containing a 103-polyglutamine expansion (Htt103QP) as a model substrate for the functional study of ubiquilin proteins. We found that yeast ubiquilin mutant (dsk2Δ) is sensitive to Htt103QP overexpression and has a defect in the formation of Htt103QP inclusion bodies. Our evidence further suggests that the UBL domain of Dsk2 is critical for inclusion body formation. Of interest, Dsk2 is dispensable for Htt103QP degradation when Htt103QP is induced for a short time before noticeable inclusion body formation. However, when the inclusion body forms after a long Htt103QP induction, Dsk2 is required for efficient Htt103QP clearance, as well as for autophagy-dependent delivery of Htt103QP into vacuoles (lysosomes). Therefore our data indicate that Dsk2 facilitates vacuole-mediated clearance of misfolded proteins by promoting inclusion body formation. Of importance, the defect of inclusion body formation in dsk2 mutants can be rescued by human ubiquilin 1 or 2, suggesting functional conservation of ubiquilin proteins.
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Date Issued: 2016-07-01
Identifier: FSU_pmch_27170182, 10.1091/mbc.E16-01-0026, PMC4927277, 27170182, 27170182, mbc.E16-01-0026
Format: Citation

Department of Biomedical Sciences (9)	+ -
National High Magnetic Field Laboratory (1)	+ -
Department of Biological Science (1)	+ -

Animals (4)	+ -
Humans (4)	+ -
Saccharomyces cerevisiae Proteins/genetics (4)	+ -
Cell Cycle Proteins/genetics (3)	+ -
Cell Cycle Proteins/metabolism (3)	+ -

Phosphorylation (3)	+ -
Protein-Tyrosine Kinases/metabolism (3)	+ -
Saccharomyces cerevisiae Proteins/metabolism (3)	+ -
Saccharomyces cerevisiae/genetics (3)	+ -
Amino Acid Sequence (2)	+ -
CDC2 Protein Kinase/metabolism (2)	+ -
CHO Cells (2)	+ -
Cricetulus (2)	+ -
DNA/genetics (2)	+ -
Mitosis (2)	+ -
Nocodazole/pharmacology (2)	+ -
Nuclear Proteins/genetics (2)	+ -
Nuclear Proteins/metabolism (2)	+ -
Protein Kinases/genetics (2)	+ -
Protein Kinases/metabolism (2)	+ -
Protein Tyrosine Phosphatases/genetics (2)	+ -
Protein Tyrosine Phosphatases/metabolism (2)	+ -
Protein-Serine-Threonine Kinases (2)	+ -
Protein-Tyrosine Kinases/genetics (2)	+ -
Recombinant Fusion Proteins/genetics (2)	+ -
Recombinant Fusion Proteins/metabolism (2)	+ -
Saccharomyces cerevisiae/cytology (2)	+ -
Anaphase/genetics (1)	+ -
Anaphase/physiology (1)	+ -
Basal Bodies/metabolism (1)	+ -

journal article (11)	+ -
text (11)	+ -

Wang, Yanchang (5)	+ -
Liang, Fengshan (3)	+ -
Hurt, Myra M (2)	+ -
Liu, Hong (2)	+ -
Megraw, Timothy L (2)	+ -

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