Biomedical subjects

R B Kelly

Publications and source records attributed to R B Kelly.

At least 37 records · Page 2Linked to original sources

PubMed1998

Vesiculation and sorting from PC12-derived endosomes in vitro.

Formation of small vesicles resembling synaptic vesicles can be reconstituted in vitro by incubating labeled homogenates of PC12 cells with ATP and two cytoplasmic proteins, AP3 and ARF1 [Faúndez, V., Horng, J.-T. & Kelly, R. B. (1998) Cell 93, 423-432]. To determine whether AP3 was mediating budding from plasma membranes or endosomes the organelle that generated the synaptic vesicles was characterized. The budding activity was enriched in organelles that labeled at 15 degrees C, but not at 4 degrees C, that excluded a marker of plasma membranes and that contained internalized transferrin, indicating that the precursor was an endosome. Vesicles formed from the endosomal precursor in vitro excluded transferrin. We conclude that ARF-mediated vesiculation into synaptic vesicle-sized organelles uses an endosomal precursor and occurs simultaneously in vitro with sorting of synaptic vesicle proteins from other membrane protein constituents of the endosome.

ADP-Ribosylation Factor 1↗

PubMed1998

Dap160, a neural-specific Eps15 homology and multiple SH3 domain-containing protein that interacts with Drosophila dynamin.

The discovery of overlapping hot spots of dynamin (Estes, P. S., Roos, J., van der Bliek, A., Kelly, R. B., Krishnan, K. S., and Ramaswami, M. (1996) J. Neurosci. 16, 5443-5456) and the heterotetrameric adaptor 2 complex (Gonzalez-Gaitan, M., and Jäckle, H. (1997) Cell 88, 767-776) in Drosophila nerve terminals led to the concept of zones of active endocytosis close to sites of active exocytosis. The proline-rich domain of Drosophila dynamin was used to identify and purify a third component of the endocytosis zones. Dap160 (dynamin-associated protein 160 kDa) is a membrane-associated, dynamin-binding protein of 160 kDa that has four putative src homology 3 domains and an Eps15 homology domain, motifs frequently found in proteins associated with endocytosis. The binding capacities of the four putative src homology 3 domains were examined individually and in combination and shown to bind known proteins that contained proline-rich domains. Each binding site, however, was different in its preference for binding partners. We suggest that Dap160 is a scaffolding protein that helps anchor proteins required for endocytosis at sites where they are needed in the Drosophila nerve terminal.

Adaptor Protein Complex alpha Subunits↗

PubMed1998

A function for the AP3 coat complex in synaptic vesicle formation from endosomes.

Synaptic vesicles can be coated in vitro in a reaction that is ARF-, ATP-, and temperature-dependent and requires synaptic vesicle membrane proteins. The coat is largely made up of the heterotetrameric complex, adaptor protein 3, recently implicated in Golgi-to-vacuole traffic in yeast. Depletion of AP3 from brain cytosol inhibits small vesicle formation from PC12 endosomes in vitro. Budding from washed membranes can be reconstituted with purified AP3 and recombinant ARF1. We conclude that AP3 coating is involved in at least one pathway of small vesicle formation from endosomes.

ADP-Ribosylation Factor 1↗

PubMed1998

GLUT4 and transferrin receptor are differentially sorted along the endocytic pathway in CHO cells.

The trafficking of GLUT4, a facilitative glucose transporter, is examined in transfected CHO cells. In previous work, we expressed GLUT4 in neuroendocrine cells and fibroblasts and found that it was targeted to a population of small vesicles slightly larger than synaptic vesicles (Herman, G.A, F. Bonzelius, A.M. Cieutat, and R.B. Kelly. 1994. Proc. Natl. Acad. Sci. USA. 91: 12750-12754.). In this study, we demonstrate that at 37 degrees C, GLUT4-containing small vesicles (GSVs) are detected after cell surface radiolabeling of GLUT4 whereas uptake of radioiodinated human transferrin does not show appreciable accumulation within these small vesicles. Immunofluorescence microscopy experiments show that at 37 degrees C, cell surface-labeled GLUT4 as well as transferrin is internalized into peripheral and perinuclear structures. At 15 degrees C, endocytosis of GLUT4 continues to occur at a slowed rate, but whereas fluorescently labeled GLUT4 is seen to accumulate within large peripheral endosomes, no perinuclear structures are labeled, and no radiolabeled GSVs are detectable. Shifting cells to 37 degrees C after accumulating labeled GLUT4 at 15 degrees C results in the reappearance of GLUT4 in perinuclear structures and GSV reformation. Cytosol acidification or treatment with hypertonic media containing sucrose prevents the exit of GLUT4 from peripheral endosomes as well as GSV formation, suggesting that coat proteins may be involved in the endocytic trafficking of GLUT4. In contrast, at 15 degrees C, transferrin continues to traffic to perinuclear structures and overall labels structures similar in distribution to those observed at 37 degrees C. Furthermore, treatment with hypertonic media has no apparent effect on transferrin trafficking from peripheral endosomes. Double-labeling experiments after the internalization of both transferrin and surface-labeled GLUT4 show that GLUT4 accumulates within peripheral compartments that exclude the transferrin receptor (TfR) at both 15 degrees and 37 degrees C. Thus, GLUT4 is sorted differently from the transferrin receptor as evidenced by the targeting of each protein to distinct early endosomal compartments and by the formation of GSVs. These results suggest that the sorting of GLUT4 from TfR may occur primarily at the level of the plasma membrane into distinct endosomes and that the organization of the endocytic system in CHO cells more closely resembles that of neuroendocrine cells than previously appreciated.

Animals↗

PubMed1998

Reconstitution of synaptic vesicle biogenesis from PC12 cell membranes.

Neuroendocrine PC12 cells contain small microvesicles that closely resemble synaptic vesicles in their physical and chemical properties. Two defining characteristics of synaptic vesicles are their homogeneous size and their unique protein composition. Since synaptic vesicles arise by endocytosis from the plasma membrane, nerve terminals and PC12 cells must contain the molecular machinery to sort synaptic vesicles from other membrane proteins and pinch off vesicles of the correct diameter from a precursor compartment. A cell-free reconstitution system was developed that generates vesicles from PC12 membrane precursors in the presence of ATP and brain cytosol and is temperature dependent. At 15 degrees C, surface-labeled synaptic vesicle proteins accumulate in a donor compartment, while labeled synaptic vesicles cannot be detected. The block of synaptic vesicle formation at 15 degrees C enables the use of the monoclonal antibody, KT3, a specific marker for the epitope-tagged synaptic vesicle protein, VAMP-TAg, to label precursors in the synaptic vesicle biogenesis pathway. From membranes labeled in vivo at 15 degrees C, vesicles generated in vitro at 37 degreesC had the sedimentation characteristics of neuroendocrine synaptic vesicles on glycerol velocity gradients, and excluded the transferrin receptor. Therefore, vesiculation and sorting can be studied in this cell-free system.

Adenosine Triphosphate↗

PubMed1998

A v-SNARE participates in synaptic vesicle formation mediated by the AP3 adaptor complex.

Reconstitution of synaptic vesicle formation in vitro has revealed a pathway of synaptic vesicle biogenesis from endosomes that requires the heterotetrameric adaptor complex AP3. Because synaptic vesicles have a distinct protein composition, the AP3 complex should selectively recognize some or all of the synaptic vesicle proteins. Here we show that one element of this recognition process is the v-SNARE, VAMP-2, because tetanus toxin, which cleaves VAMP-2, inhibited the formation of synaptic vesicles and their coating with AP3 in vitro. Mutant tetanus toxin and botulinum toxins, which cleave t-SNAREs, did not inhibit synaptic vesicle production. AP3-containing complexes isolated from coated vesicles could be immunoprecipitated by a VAMP-2 antibody. These data imply that AP3 recognizes a component of the fusion machinery, which may prevent the production of inert synaptic vesicles.

Adaptor Protein Complex 3↗

PubMed1998

Critical success factors for promotion and tenure in family medicine departments.

PURPOSE: (1) To summarize the judgments of family medicine department leaders regarding the elements leading to success in promotion and/or tenure, and (2) to compare the views of department leaders with those of family medicine faculty who have been successfully promoted. METHOD: Two surveys were conducted. The first was of 296 associate professor members of the Society of Teachers of Family Medicine in November 1993. The second, conducted in the summer of 1994, was of all 115 U.S. members of the Association of Departments of Family Medicine; surveys were addressed to chairs, directors, or promotion and tenure committee chairs. Both survey instruments requested data regarding each respondent's department, impressions about the promotion and tenure processes at the respondent's institution, and general impressions regarding the characteristics of successful candidates. Comparisons of the responses to the two questionnaires were made using two-tailed t-tests; responses to open-ended questions were analyzed qualitatively by two independent investigators. RESULTS: In all, 75% of the department leaders and 67% of the associate professors returned completed questionnaires. The two groups had similar views about the importance of certain academic activities to success at promotion and tenure. The primary difference between the groups was in their estimates of weekly time available for research and writing activities: the leaders reported that successful candidates spent a mean of 25% of their work-weeks on research and writing activities; the associate professors, on the other hand, reported spending a mean of 15% of their workweeks on these activities. The department leaders described six basic groups of critical success factors. The associate professors emphasized lack of time as a major obstacle to success. CONCLUSION: The findings emphasize the critical importance of protected time for scholarly activities (such as research and writing) if generalists are to be promoted or tenured.

Career Mobility↗

PubMed1998

Illuminating the 'black box'. A description of 4454 patient visits to 138 family physicians.

BACKGROUND: The content and context of family practice outpatient visits have never been fully described, leaving many aspects of family practice in a "black box," unseen by policymakers and understood only in isolation. This article describes community family practices, physicians, patients, and outpatient visits. METHODS: Practicing family physicians in northeast Ohio were invited to participate in a multimethod study of the content of primary care practice. Research nurses directly observed consecutive patient visits, and collected additional data using medical record reviews, patient and physician questionnaires, billing data, practice environment checklists, and ethnographic fieldnotes. RESULTS: Visits by 4454 patients seeing 138 physicians in 84 practices were observed. Outpatient visits to family physicians encompassed a wide variety of patients, problems, and levels of complexity. The average patient paid 4.3 visits to the practice within the past year. The mean visit duration was 10 minutes. Fifty-eight percent of visits were for acute illness, 24% for chronic illness, and 12% for well care. The most common uses of time were history-taking, planning treatment, physical examination, health education, feedback, family information, chatting, structuring the interaction, and patient questions. CONCLUSIONS: Family practice and patient visits are complex, with competing demands and opportunities to address a wide range of problems of individuals and families over time and at various stages of health and illness. Multimethod research in practice settings can identify ways to enhance the competing opportunities of family practice to improve the health of their patients.

Adult↗

PubMed1997

ADP ribosylation factor 1 is required for synaptic vesicle budding in PC12 cells.

Carrier vesicle generation from donor membranes typically progresses through a GTP-dependent recruitment of coats to membranes. Here we explore the role of ADP ribosylation factor (ARF) 1, one of the GTP-binding proteins that recruit coats, in the production of neuroendocrine synaptic vesicles (SVs) from PC12 cell membranes. Brefeldin A (BFA) strongly and reversibly inhibited SV formation in vivo in three different PC12 cell lines expressing vesicle-associated membrane protein-T Antigen derivatives. Other membrane traffic events remained unaffected by the drug, and the BFA effects were not mimicked by drugs known to interfere with formation of other classes of vesicles. The involvement of ARF proteins in the budding of SVs was addressed in a cell-free reconstitution system (Desnos, C., L. Clift-O'Grady, and R.B. Kelly. 1995. J. Cell Biol. 130:1041-1049). A peptide spanning the effector domain of human ARF1 (2-17) and recombinant ARF1 mutated in its GTPase activity, both inhibited the formation of SVs of the correct size. During in vitro incubation in the presence of the mutant ARFs, the labeled precursor membranes acquired different densities, suggesting that the two ARF mutations block at different biosynthetic steps. Cell-free SV formation in the presence of a high molecular weight, ARF-depleted fraction from brain cytosol was significantly enhanced by the addition of recombinant myristoylated native ARF1. Thus, the generation of SVs from PC12 cell membranes requires ARF and uses its GTPase activity, probably to regulate coating phenomena.

ADP-Ribosylation Factor 1↗

PubMed1997

Effect of mutations in vesicle-associated membrane protein (VAMP) on the assembly of multimeric protein complexes.

The assembly of multimeric protein complexes that include vesicle-associated membrane protein 2 (VAMP-2) and the plasma membrane proteins syntaxin 1A and synaptosome-associated protein of 25 kDa (SNAP-25) are thought to reflect the biochemical correlates of synaptic vesicle targeting, priming, or fusion. Using a variety of protein-protein interaction assays and a series of deletion and point mutations, we have investigated the domains of VAMP-2 required for the formation of binary complexes with either syntaxin 1A or SNAP-25 and ternary complexes with both syntaxin 1A and SNAP-25. Deletions within the central conserved domain of VAMP-2 eliminated binding to either syntaxin 1A or both syntaxin 1A and SNAP-25. Although all of the deletion mutants were able to form ternary complexes, only some of these complexes were resistant to denaturation in sodium dodecyl sulfate. These results demonstrate that cooperative interactions result in the formation of at least two biochemically distinct classes of ternary complex. Two point mutations previously shown to have effects on the intracellular trafficking of VAMP-2 (M46A, reduced endocytosis and sorting to synaptic vesicles; N49A, enhanced sorting to synaptic vesicles) lie within a domain required for both syntaxin 1A and SNAP-25 binding. Syntaxin 1A and SNAP-25 binding was reduced by the M46A mutation and enhanced by the N49A mutation, suggesting that a correlation exists between the membrane-trafficking phenotype of the two VAMP-2 point mutants and their competence to form complexes with either syntaxin 1A or SNAP-25.

Animals↗

PubMed1997

ATP-dependent formation of free synaptic vesicles from PC12 membranes in vitro.

Synaptic vesicles are released from membranes during incubation at 37 degrees C in the presence of ATP (adenosine triphosphate). The donor membranes are a rapidly sedimenting fraction derived from the neuroendocrine cell line PC12 (pheochromocytoma 12). These starting membranes contain the synaptic vesicle proteins, synaptophysin and SV2, and the endosomal markers transferrin receptor and cation-independent MPR (mannose 6-phosphate receptor). Incubating the membranes in vitro increased the amount of organelles that migrate as synaptic vesicles in velocity sedimentation gradients. The synaptic vesicle fractions that contain both synaptophysin and SV2 do not contain endosomal markers. A synaptic vesicle increase in vitro is time-, cytosol-, ATP- and temperature-dependent and is inhibited by NEM (N-ethylmaleimide), BFA (brefeldin A) and aluminum fluoride, but not GTP gamma S (guanosine-5'O-C3-thiotriphosphate). The production of synaptic vesicles under these conditions is unlike the de novo generation of vesicles from endosomes (1). Incubation in vitro under the conditions described here may allow the final stages of synaptic vesicle formation, uncoating or undocking, to occur but not the initiation of formation de novo.

Adenosine Triphosphate↗

PubMed1996

Mutational analysis of VAMP domains implicated in Ca2+-induced insulin exocytosis.

Vesicle-associated membrane protein-2 (VAMP-2) and cellubrevin are associated with the membrane of insulin-containing secretory granules and of gamma-aminobutyric acid (GABA)-containing synaptic-like vesicles of pancreatic beta-cells. We found that a point mutation in VAMP-2 preventing targeting to synaptic vesicles also impairs the localization on insulin-containing secretory granules, suggesting a similar requirement for vesicular targeting. Tetanus toxin (TeTx) treatment of permeabilized HIT-T15 cells leads to the proteolytic cleavage of VAMP-2 and cellubrevin and causes the inhibition of Ca2+-triggered insulin exocytosis. Transient transfection of HIT-T15 cells with VAMP-1, VAMP-2 or cellubrevin made resistant to the proteolytic action of TeTx by amino acid replacements in the cleavage site restored Ca2+-stimulated secretion. Wild-type VAMP-2, wild-type cellubrevin or a mutant of VAMP-2 resistant to TeTx but not targeted to secretory granules were unable to rescue Ca2+-evoked insulin release. The transmembrane domain and the N-terminal region of VAMP-2 were not essential for the recovery of stimulated exocytosis, but deletions preventing the binding to SNAP-25 and/or to syntaxin I rendered the protein inactive in the reconstitution assay. Mutations of putative phosphorylation sites or of negatively charged amino acids in the SNARE motif recognized by clostridial toxins had no effect on the ability of VAMP-2 to mediate Ca2+-triggered secretion. We conclude that: (i) both VAMP-2 and cellubrevin can participate in the exocytosis of insulin; (ii) the interaction of VAMP-2 with syntaxin and SNAP-25 is required for docking and/or fusion of secretory granules with the plasma membrane; and (iii) the phosphorylation of VAMP-2 is not essential for Ca2+-stimulated insulin exocytosis.

Botulinum Toxins↗

PubMed1996

Traffic of dynamin within individual Drosophila synaptic boutons relative to compartment-specific markers.

Presynaptic terminals contain several specialized compartments, which have been described by electron microscopy. We show in an identified Drosophila neuromuscular synapse that several of these compartments-synaptic vesicle clusters, presynaptic plasma membrane, presynaptic cytosol, and axonal cytoskeleton-labeled by specific reagents may be resolved from one another by laser scanning confocal microscopy. Using a panel of compartment-specific markers and Drosophila shibire(ts1) mutants to trap an intermediate stage in synaptic vesicle recycling, we have examined the localization and redistribution of dynamin within single synaptic varicosities at the larval neuromuscular junction. Our results suggest that dynamin is not a freely diffusible molecule in resting nerve terminals; rather, it appears localized to synaptic sites by association with yet uncharacterized presynaptic components. In shi(ts1) nerve terminals depleted of synaptic vesicles, dynamin is quantitatively redistributed to the plasma membrane. It is not, however, distributed uniformly over presynaptic plasmalemma; instead, fluorescence images show "hot spots" of dynamin on the plasma membrane of vesicle-depleted nerve terminals. We suggest that these dynamin-rich domains may mark the active zones for synaptic vesicle endocytosis first described at the frog neuromuscular junction.

Animals↗

PubMed1996

Rehearsing the ABCs. Protein translocation.

Recent evidence that vacuolar enzymes in yeast can be delivered directly from the cytosol, rather than via the secretory pathway, alerts us to the increasing evidence for 'non-classical' forms of protein translocation that may involve ABC transporters.

ATP-Binding Cassette Transporters↗

PubMed1996

Association of Drosophila cysteine string proteins with membranes.

Cysteine string proteins are putative synaptic vesicle proteins that lack a transmembrane domain. Our analysis shows that Drosophila cysteine string proteins are extensively modified by hydroxylamine-sensitive fatty acylation. This modification could be responsible for association of csp's with membranes. Extensive deacylation of Dcsp's by a 20 h incubation in 1 M hydroxylamine, pH 7.0, or methanolic KOH produces a protein of 6-7 kDa lower mass than untreated Dcsp's. Surprisingly, the hydroxylamine treatment does not cause release of Dcsp's from membranes. On the other hand, alkaline stripping of membranes isolated from Drosophila brain by 0.1 M sodium carbonate, pH 11.5, causes a significant release of Dcsp's from membranes into the cytosol. These results indicate that fatty acylation may not form the main anchor of Dcsp's in membranes. Taking advantage of the endocytotic block in the Drosophila mutant shibire ts1, we analyzed the acylation states of Dcsp's in two stages during synaptic vesicle recycling and found no evidence for an acylation/deacylation cycle of Dcsp's in the brain nerve terminals.

Acylation↗

PubMed1996

Endocytosis of VAMP is facilitated by a synaptic vesicle targeting signal.

After synaptic vesicles fuse with the plasma membrane and release their contents, vesicle membrane proteins recycle by endocytosis and are targeted to newly formed synaptic vesicles. The membrane traffic of an epitope-tagged form of VAMP-2 (VAMP-TAg) was observed in transfected cells to identify sequence requirements for recycling of a synaptic vesicle membrane protein. In the neuroendocrine PC12 cell line VAMP-TAg is found not only in synaptic vesicles, but also in endosomes and on the plasma membrane. Endocytosis of VAMP-TAg is a rapid and saturable process. At high expression levels VAMP-TAg accumulates at the cell surface. Rapid endocytosis of VAMP-TAg also occurs in transfected CHO cells and is therefore independent of other synaptic proteins. The majority of the measured endocytosis is not directly into synaptic vesicles since mutations in VAMP-TAg that enhance synaptic vesicle targeting did not affect endocytosis. Nonetheless, mutations that inhibited synaptic vesicle targeting, in particular replacement of methionine-46 by alanine, inhibited endocytosis by 85% in PC12 cells and by 35% in CHO cells. These results demonstrate that the synaptic vesicle targeting signal is also used for endocytosis and can be recognized in cells lacking synaptic vesicles.

Amino Acid Sequence↗

PubMed1996

A new pathway for protein export in Saccharomyces cerevisiae.

Several physiologically important proteins lack a classical secretory signal sequence, yet they are secreted from cells. To investigate the secretion mechanism of such proteins, a representative mammalian protein that is exported by a nonclassical mechanism, galectin-1, has been expressed in yeast. Galectin-1 is exported across the yeast plasma membrane, and this export does not require the classical secretory pathway nor the yeast multidrug resistance-like protein Ste6p, the transporter for the peptide a factor. A screen for components of the export machinery has identified genes that are involved in nonclassical export. These findings demonstrate a new pathway for protein export that is distinct from the classical secretory pathway in yeast.

ATP-Binding Cassette Transporters↗

PubMed1996

Academic achievement of successful candidates for tenure and promotion to associate professor.

BACKGROUND AND OBJECTIVES: For faculty to achieve promotion and tenure, a clear understanding is needed of the measures by which performance will be judged. This study describes the specific academic achievements of successful candidates for tenure and promotion to associate professor in family medicine. METHODS: A survey questionnaire was mailed to all members listed in the Society of Teachers of Family Medicine database as holding the rank of associate professor. The questionnaire sought information about items as they were at the time the individual was being considered for promotion to associate professor with tenure. The items included scholarly accomplishments, time for research, areas of scholarly expertise, and departmental policy regarding promotion and tenure. RESULTS: Of the 296 eligible faculty, 197 (67%) returned a completed survey. The mean number of publications of these successfully promoted individuals was 10. The respondents averaged 78% of their publications in peer-reviewed journals and were first author on 64% of their peer-reviewed articles. The mean number of peer-reviewed publications per year was 1.5. Research grant support as principal or coinvestigator was reported by 44%, and respondents averaged 9 hours a week on scholarly activities. Scholarly activity was found to vary significantly by type of school, degree, and tenure track status. CONCLUSIONS: Information reported here can be of value for prospective promotion and tenure candidates, their advisors, and members of review committees.

Achievement↗