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Full-Text Articles in Biochemistry, Biophysics, and Structural Biology

Cell Type–Dependent Mechanisms For Formin-Mediated Assembly Of Filopodia, Lorna E. Young, Ernest G. Heimsath, Henry N. Higgs Oct 2015

Cell Type–Dependent Mechanisms For Formin-Mediated Assembly Of Filopodia, Lorna E. Young, Ernest G. Heimsath, Henry N. Higgs

Open Dartmouth: Faculty Open Access Scholarship

Filopodia are finger-like protrusions from the plasma membrane and are of fundamental importance to cellular physiology, but the mechanisms governing their assembly are still in question. One model, called convergent elongation, proposes that filopodia arise from Arp2/3 complex-nucleated dendritic actin networks, with factors such as formins elongating these filaments into filopodia. We test this model using constitutively active constructs of two formins, FMNL3 and mDia2. Surprisingly, filopodial assembly requirements differ between suspension and adherent cells. In suspension cells, Arp2/3 complex is required for filopodial assembly through either formin. In contrast, a subset of filopodia remains after Arp2/3 ...


Coupling Between Cytoplasmic Concentration Gradients Through Local Control Of Protein Mobility In The Caenorhabditis Elegans Zygote, Youjun Wu, Huaiying Zhang, Erik E. Griffin Jun 2015

Coupling Between Cytoplasmic Concentration Gradients Through Local Control Of Protein Mobility In The Caenorhabditis Elegans Zygote, Youjun Wu, Huaiying Zhang, Erik E. Griffin

Open Dartmouth: Faculty Open Access Scholarship

Cell polarity is characterized by the asymmetric distribution of factors at the cell cortex and in the cytoplasm. Although mechanisms that establish cortical asymmetries have been characterized, less is known about how persistent cytoplasmic asymmetries are generated. During the asymmetric division of the Caenorhabditis elegans zygote, the PAR proteins orchestrate the segregation of the cytoplasmic RNA-binding proteins MEX-5/6 to the anterior cytoplasm and PIE-1, POS-1, and MEX-1 to the posterior cytoplasm. In this study, we find that MEX-5/6 control the segregation of GFP::PIE-1, GFP::POS-1, and GFP::MEX-1 by locally increasing their mobility in the anterior cytoplasm ...


Differential Interactions Of The Formins Inf2, Mdia1, And Mdia2 With Microtubules, Jeremie Gaillard, Bvinay Ramabhadran, Emmanuelle Neumanne, Pinar Gurel, Laurent Blanchoin, Marylin Vantard, Henry N. Higgs Sep 2011

Differential Interactions Of The Formins Inf2, Mdia1, And Mdia2 With Microtubules, Jeremie Gaillard, Bvinay Ramabhadran, Emmanuelle Neumanne, Pinar Gurel, Laurent Blanchoin, Marylin Vantard, Henry N. Higgs

Open Dartmouth: Faculty Open Access Scholarship

A number of cellular processes use both microtubules and actin filaments, but the molecular machinery linking these two cytoskeletal elements remains to be elucidated in detail. Formins are actin-binding proteins that have multiple effects on actin dynamics, and one formin, mDia2, has been shown to bind and stabilize microtubules through its formin homology 2 (FH2) domain. Here we show that three formins, INF2, mDia1, and mDia2, display important differences in their interactions with microtubules and actin. Constructs containing FH1, FH2, and C-terminal domains of all three formins bind microtubules with high affinity (K(d) < 100 nM). However, only mDia2 binds microtubules at 1:1 stoichiometry, with INF2 and mDia1 showing saturating binding at approximately 1:3 (formin dimer:tubulin dimer). INF2-FH1FH2C is a potent microtubule-bundling protein, an effect that results in a large reduction in catastrophe rate. In contrast, neither mDia1 nor mDia2 is a potent microtubule bundler. The C-termini of mDia2 and INF2 have different functions in microtubule interaction, with mDia2's C-terminus required for high-affinity binding and INF2's C-terminus required for bundling. mDia2's C-terminus directly binds microtubules with submicromolar affinity. These formins also differ in their abilities to bind actin and microtubules simultaneously. Microtubules strongly inhibit actin polymerization by mDia2, whereas they moderately inhibit mDia1 and have no effect on INF2. Conversely, actin monomers inhibit microtubule binding/bundling by INF2 but do not affect mDia1 or mDia2. These differences in interactions with microtubules and actin suggest differential function in cellular processes requiring both cytoskeletal elements.


Insulin Stimulates The Phosphorylation Of The Exocyst Protein Sec8 In Adipocytes, Patrick D. Lyons, Grantley R. Peck, Arminja N. Kettenbach, Scott A. Gerber, Liya Roudaia, Gustav E. Lienhard Aug 2009

Insulin Stimulates The Phosphorylation Of The Exocyst Protein Sec8 In Adipocytes, Patrick D. Lyons, Grantley R. Peck, Arminja N. Kettenbach, Scott A. Gerber, Liya Roudaia, Gustav E. Lienhard

Open Dartmouth: Faculty Open Access Scholarship

The signal transduction pathway leading from the insulin receptor to stimulate the fusion of vesicles containing the glucose transporter GLUT4 with the plasma membrane in adipocytes and muscle cells is not completely understood. Current evidence suggests that in addition to the Rab GTPase-activating protein AS160, at least one other substrate of Akt (also called protein kinase B), which is as yet unidentified, is required. Sec8 is a component of the exocyst complex that has been previously implicated in GLUT4 trafficking. In the present study, we report that insulin stimulates the phosphorylation of Sec8 on Ser-32 in 3T3-L1 adipocytes. On the ...


The Yeast Orthologue Of Grasp65 Forms A Complex With A Coiled-Coil Protein That Contributes To Er To Golgi Traffic, Rudy Behnia, Francis A. Barr, John J. Flanagan, Charles Barlowe, Sean Munro Jan 2007

The Yeast Orthologue Of Grasp65 Forms A Complex With A Coiled-Coil Protein That Contributes To Er To Golgi Traffic, Rudy Behnia, Francis A. Barr, John J. Flanagan, Charles Barlowe, Sean Munro

Open Dartmouth: Faculty Open Access Scholarship

The mammalian Golgi protein GRASP65 is required in assays that reconstitute cisternal stacking and vesicle tethering. Attached to membranes by an N-terminal myristoyl group, it recruits the coiled-coil protein GM130. The relevance of this system to budding yeasts has been unclear, as they lack an obvious orthologue of GM130, and their only GRASP65 relative (Grh1) lacks a myristoylation site and has even been suggested to act in a mitotic checkpoint. In this study, we show that Grh1 has an N-terminal amphipathic helix that is N-terminally acetylated and mediates association with the cis-Golgi. We find that Grh1 forms a complex with ...


Pv1 Is A Key Structural Component For The Formation Of The Stomatal And Fenestral Diaphragms, Radu V. Stan, Eugene Tkachenko, Ingrid R. Niesman May 2004

Pv1 Is A Key Structural Component For The Formation Of The Stomatal And Fenestral Diaphragms, Radu V. Stan, Eugene Tkachenko, Ingrid R. Niesman

Open Dartmouth: Faculty Open Access Scholarship

PV1 is an endothelial-specific integral membrane glycoprotein associated with the stomatal diaphragms of caveolae, transendothelial channels, and vesiculo-vacuolar organelles and the diaphragms of endothelial fenestrae. Multiple PV1 homodimers are found within each stomatal and fenestral diaphragm. We investigated the function of PV1 within these diaphragms and their regulation and found that treatment of endothelial cells in culture with phorbol myristate acetate (PMA) led to upregulation of PV1. This correlated with de novo formation of stomatal diaphragms of caveolae and transendothelial channels as well as fenestrae upon PMA treatment. The newly formed diaphragms could be labeled with anti-PV1 antibodies. The upregulation ...


The C. Elegans Heterochronic Gene Lin-46 Affects Developmental Timing At Two Larval Stages And Encodes A Relative Of The Scaffolding Protein Gephyrin, A. S.-R. Pepper, Jill E. Mccane, Kevin Kemper, Dennis Au Yeung, Rosalind C. Lee, Victor Ambros, Eric G. Moss Apr 2004

The C. Elegans Heterochronic Gene Lin-46 Affects Developmental Timing At Two Larval Stages And Encodes A Relative Of The Scaffolding Protein Gephyrin, A. S.-R. Pepper, Jill E. Mccane, Kevin Kemper, Dennis Au Yeung, Rosalind C. Lee, Victor Ambros, Eric G. Moss

Open Dartmouth: Faculty Open Access Scholarship

The succession of developmental events in the C. elegans larva is governed by the heterochronic genes. When mutated, these genes cause either precocious or retarded developmental phenotypes, in which stage-specific patterns of cell division and differentiation are either skipped or reiterated, respectively. We identified a new heterochronic gene, lin-46, from mutations that suppress the precocious phenotypes caused by mutations in the heterochronic genes lin-14 and lin-28. lin-46 mutants on their own display retarded phenotypes in which cell division patterns are reiterated and differentiation is prevented in certain cell lineages. Our analysis indicates that lin-46 acts at a step immediately downstream ...


Meiotic Cohesion Requires Accumulation Of Ord On Chromosomes Before Condensation, Eric M. Balicky, Matthew W. Endres, Cary Lai, Sharon E. Bickel Sep 2002

Meiotic Cohesion Requires Accumulation Of Ord On Chromosomes Before Condensation, Eric M. Balicky, Matthew W. Endres, Cary Lai, Sharon E. Bickel

Open Dartmouth: Faculty Open Access Scholarship

Cohesion between sister chromatids is a prerequisite for accurate chromosome segregation during mitosis and meiosis. To allow chromosome condensation during prophase, the connections that hold sister chromatids together must be maintained but still permit extensive chromatin compaction. In Drosophila, null mutations in the orientation disruptor (ord) gene lead to meiotic nondisjunction in males and females because cohesion is absent by the time that sister kinetochores make stable microtubule attachments. We provide evidence that ORD is concentrated within the extrachromosomal domains of the nuclei ofDrosophila primary spermatocytes during early G2, but accumulates on the meiotic chromosomes by mid to late ...


Sec34p, A Protein Required For Vesicle Tethering To The Yeast Golgi Apparatus, Is In A Complex With Sec35p, Susan M. Vanrheenen, Xiaochun Cao, Stephanie K. Sapperstein, Elbert C. Chiang, Vladimir V. Lupashin, Charles Barlowe, M. Gerard Waters Nov 1999

Sec34p, A Protein Required For Vesicle Tethering To The Yeast Golgi Apparatus, Is In A Complex With Sec35p, Susan M. Vanrheenen, Xiaochun Cao, Stephanie K. Sapperstein, Elbert C. Chiang, Vladimir V. Lupashin, Charles Barlowe, M. Gerard Waters

Open Dartmouth: Faculty Open Access Scholarship

A screen for mutants of Saccharomyces cerevisiae secretory pathway components previously yielded sec34, a mutant that accumulates numerous vesicles and fails to transport proteins from the ER to the Golgi complex at the restrictive temperature (Wuestehube, L.J., R. Duden, A. Eun, S. Hamamoto, P. Korn, R. Ram, and R. Schekman. 1996. Genetics. 142:393–406). We find that SEC34 encodes a novel protein of 93-kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec34-2 is suppressed by the rab GTPase Ypt1p that functions early in the secretory pathway, or by the dominant form of the ER to Golgi complex ...


Docking Of Yeast Vacuoles Is Catalyzed By The Ras-Like Gtpase Ypt7p After Symmetric Priming By Sec18p (Nsf), Andreas Mayer, William Wickner Jan 1997

Docking Of Yeast Vacuoles Is Catalyzed By The Ras-Like Gtpase Ypt7p After Symmetric Priming By Sec18p (Nsf), Andreas Mayer, William Wickner

Open Dartmouth: Faculty Open Access Scholarship

Vacuole inheritance in yeast involves the for- mation of tubular and vesicular “segregation struc- tures” which migrate into the bud and fuse there to es- tablish the daughter cell vacuole. Vacuole fusion has been reconstituted in vitro and may be used as a model for an NSF-dependent reaction of priming, docking, and fusion. We have developed biochemical and micro- scopic assays for the docking step of in vitro vacuole fusion and characterized its requirements. The vacu- oles must be primed for docking by the action of Sec17p ( a -SNAP) and Sec18p (NSF). Priming is neces- sary for both fusion partners ...


A Novel Iron-Regulated Metal Transporter From Plants Identified By Functional Expression In Yeast., David Eide, Margaret Broderius, Jeanette Fett, Mary Lou Guerinot May 1996

A Novel Iron-Regulated Metal Transporter From Plants Identified By Functional Expression In Yeast., David Eide, Margaret Broderius, Jeanette Fett, Mary Lou Guerinot

Open Dartmouth: Faculty Open Access Scholarship

Iron is an essential nutrient for virtually all organisms. The IRT1 (iron-regulated transporter) gene of the plant Arabidopsis thaliana, encoding a probable Fe(II) transporter, was cloned by functional expression in a yeast strain defective for iron uptake. Yeast expressing IRT1 possess a novel Fe(II) uptake activity that is strongly inhibited by Cd. IRT1 is predicted to be an integral membrane protein with a metal-binding domain. Data base comparisons and Southern blot analysis indicated that IRT1 is a member of a gene family in Arabidopsis. Related sequences were also found in the genomes of rice, yeast, nematodes, and humans ...


Differential Regulation Of Collagenase Gene Expression By Retinoic Acid Receptors--Alpha, Beta And Gamma, Luying Pan, Stephen H. Chamberlain, David T. Auble, Constance E. Brinckerhoff Jun 1992

Differential Regulation Of Collagenase Gene Expression By Retinoic Acid Receptors--Alpha, Beta And Gamma, Luying Pan, Stephen H. Chamberlain, David T. Auble, Constance E. Brinckerhoff

Open Dartmouth: Faculty Open Access Scholarship

The mechanisms involved in retinoic acid (RA)-mediated regulation of the collagenase gene in a rabbit synovial fibroblast cell line (HIG82) were investigated. When HIG82 cells are cotransfected with expression vectors containing cDNAs for retinoic acid receptor (RAR) α1, β2, or γ1 and collagenase promoter-driven CAT reporter constructs, only RAR-γ1 represses basal CAT expression upon RA treatment, while RAR-α1, β2, and γ1 all suppress phorbol-induced CAT expression. Thus, transcriptional regulation of collagenase by RA is mediated by RARs in an RAR-type specific manner. Using mutatlonal and deletional analysis, we find that interaction between elements within 182 bp collagenase promoter plays ...