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Biomedical subjects

R B Kelly

Publications and source records attributed to R B Kelly.

At least 19 recordsLinked to original sources
PubMed2001

The neuronal form of adaptor protein-3 is required for synaptic vesicle formation from endosomes.

Heterotetrameric adaptor complexes vesiculate donor membranes. One of the adaptor protein complexes, AP-3, is present in two forms; one form is expressed in all tissues of the body, whereas the other is restricted to brain. Mice lacking both the ubiquitous and neuronal forms of AP-3 exhibit neurological disorders that are not observed in mice that are mutant only in the ubiquitous form. To begin to understand the role of neuronal AP-3 in neurological disease, we investigated its function in in vitro assays as well as its localization in neural tissue. In the presence of GTPgammaS both ubiquitous and neuronal forms of AP-3 can bind to purified synaptic vesicles. However, only the neuronal form of AP-3 can produce synaptic vesicles from endosomes in vitro. We also identified that the expression of neuronal AP-3 is limited to varicosities of neuronal-like processes and is expressed in most axons of the brain. Although the AP-2/clathrin pathway is the major route of vesicle production and the relatively minor neuronal AP-3 pathway is not necessary for viability, the absence of the latter could lead to the neurological abnormalities seen in mice lacking the expression of AP-3 in brain. In this study we have identified the first brain-specific function for a neuronal adaptor complex.

Adaptor Proteins, Vesicular Transport↗
PubMed2001

The AP2 binding site of synaptotagmin 1 is not an internalization signal but a regulator of endocytosis.

One characteristic linking members of the synaptotagmin family to endocytosis is their ability to bind the heterotetrameric AP2 complex via their C2B domain. By using CD4/synaptotagmin 1 chimeras, we found that the internalization signal of synaptotagmin 1 lies at the extreme COOH-terminus of the protein and can function in the absence of the C2B domain that contains the AP2 binding site. However, although not essential for internalization, the C2B domain of synaptotagmin 1 appeared to control the recognition of the internalization motif. By mutagenesis, two sites have been identified that modify regulation by the C2B domain in the neuroendocrine PC12 cell line. Mutation of a dilysine motif in the beta sandwich core of the domain eliminates endocytosis. This site is known to be a site of protein-protein interaction. Mutations in the calcium binding region, or in its close proximity, also affect internalization in PC12 cells. In fibroblasts, the C2B domain inhibits the COOH-terminal internalization signal, resulting in an absence of internalization in those cells. Thus, internalization of synaptotagmin 1 is controlled by the presence of a latent internalization signal in the COOH-terminal region and a regulatory region in the C2B domain. We propose that internalization of synaptotagmin 1 is regulated in this way to allow it to couple the processes of endocytosis and calcium-mediated exocytosis in cells of the neuroendocrine lineage.

Adaptor Protein Complex alpha Subunits↗
PubMed2001

Endocytotic mechanisms in synapses.

Nerve terminals are highly enriched in proteins needed for endocytosis. Although constitutive and ligand-stimulated endocytosis take place in nerve terminals, the primary type is compensatory endocytosis--the process by which a cell retrieves the additional membrane added to cell surface by a regulated secretory event. This process has been extensively characterized using electrophysiological techniques. Except for an unusual form of coupled exo- and endocytosis called kiss-and-run release, compensatory endocytosis appears to use basically the same clathrin-mediated mechanisms as the constitutive and ligand stimulated type. The remarkable speed and selectivity of compensatory endocytosis may be achieved by concentrating the machinery at specialized sites in the nerve terminal adjacent to exocytosis sites and by the use of neuronal isoforms of the proteins that mediate endocytosis.

Animals↗
PubMed2001

Rab4 regulates formation of synaptic-like microvesicles from early endosomes in PC12 cells.

Early endosomes in PC12 cells are an important site for the formation of synaptic-like microvesicles and constitutive recycling vesicles. By immunogold electron microscopy, the small GTPase rab4 was localized to early endosomes and numerous small vesicles in the cell periphery and Golgi area of PC12 cells. Overexpression of GTPase-deficient Q67Lrab4 increased the number of early endosome-associated and cytoplasmic vesicles, whereas expression of GDP-bound S22Nrab4 significantly increased the length of early endosomal tubules. In parallel, Q67Lrab4 induced a shift in rab4, VAMP2, and TfR label from early endosomes to peripheral vesicles, whereas S22Nrab4 increased early endosome labeling of all three proteins. These observations were corroborated by early endosome budding assays. Together, our data document a thus far unrecognized role for rab4 in the formation of synaptic-like microvesicles and add to our understanding of the formation of constitutive recycling vesicles from early endosomes.

Animals↗
PubMed2000

Syndapin isoforms participate in receptor-mediated endocytosis and actin organization.

Syndapin I (SdpI) interacts with proteins involved in endocytosis and actin dynamics and was therefore proposed to be a molecular link between the machineries for synaptic vesicle recycling and cytoskeletal organization. We here report the identification and characterization of SdpII, a ubiquitously expressed isoform of the brain-specific SdpI. Certain splice variants of rat SdpII in other species were named FAP52 and PACSIN 2. SdpII binds dynamin I, synaptojanin, synapsin I, and the neural Wiskott-Aldrich syndrome protein (N-WASP), a stimulator of Arp2/3 induced actin filament nucleation. In neuroendocrine cells, SdpII colocalizes with dynamin, consistent with a role for syndapin in dynamin-mediated endocytic processes. The src homology 3 (SH3) domain of SdpI and -II inhibited receptor-mediated internalization of transferrin, demonstrating syndapin involvement in endocytosis in vivo. Overexpression of full-length syndapins, but not the NH(2)-terminal part or the SH3 domains alone, had a strong effect on cortical actin organization and induced filopodia. This syndapin overexpression phenotype appears to be mediated by the Arp2/3 complex at the cell periphery because it was completely suppressed by coexpression of a cytosolic COOH-terminal fragment of N-WASP. Consistent with a role in actin dynamics, syndapins localized to sites of high actin turnover, such as filopodia tips and lamellipodia. Our results strongly suggest that syndapins link endocytosis and actin dynamics.

Actins↗
PubMed2000

Direct observation of rates of preventive service delivery in community family practice.

BACKGROUND: Data on preventive service delivery in primary care practice have been limited by indirect methods of measurement. This study describes directly observed rates of preventive service delivery during outpatient visits to community family physicians. METHODS: In a multimethod cross-sectional study, research nurses directly observed consecutive patient visits in the offices of 138 family physicians in Northeast Ohio. Patient eligibility for services recommended by the U.S. Preventive Services Task Force was determined from medical record review. Service delivery was assessed by direct observation of outpatient visits. Rates of delivery of specific preventive services were computed. Global summary measures were calculated for health habit counseling, screening, and immunization services. RESULTS: Among 4,049 visits by established patients with available medical records, wide variation was observed among rates of different preventive services delivered during well-care visits. During illness visits, rates were uniformly low for all preventive services. Counseling services were delivered at only slightly lower rates during illness visits compared to well visits. Patients were up to date on 55% of screening, 24% of immunization, and 9% of health habit counseling services. CONCLUSION: Rates of preventive service delivery are low. Illness visits are important opportunities to deliver preventive services, particularly health habit counseling, to patients. Preventive service delivery summary scores are useful in providing a patient population perspective on the delivery of preventive services and in focusing attention on delivery of a comprehensive portfolio of services.

Adult↗
PubMed2000

Selective inhibition of adaptor complex-mediated vesiculation.

A short while ago, we could only inhibit post-Golgi membrane traffic with crude, unselective tools, such as low temperature or high extracellular sucrose. Molecular dissection of vesiculation steps has revealed unexpected complexity in the coating machinery that has initiated a search for more specific inhibitors. We have learned that membrane vesiculation is driven by a tightly regulated multicomponent, membrane-associated protein machine held together by carefully specified interaction domains. An experimental advantage of such complex interacting machinery is that it is very susceptible to disruption by dominant negative inhibitors or by overexpression. As a result, we now have much more specific inhibitors of post-Golgi membrane traffic. Some, such as dynamin K44A, may be general inhibitors, whereas others can distinguish classes of endocytotic events (10), binding events that require clathrin from those that do not (42), or specific steps of endocytosis (62). Ligand-mediated uptake of EGF and numerous, but not all, GPCRs can be inhibited by overexpression of an ARF GTPase-activating protein that has no effect on transferrin uptake (67). We can look forward to increasingly powerful and selective inhibitors that should help us to navigate successfully the complex routes of post-Golgi membrane traffic.

Adaptor Protein Complex alpha Subunits↗
PubMed2000

The AP-3 complex required for endosomal synaptic vesicle biogenesis is associated with a casein kinase Ialpha-like isoform.

The formation of small vesicles is mediated by cytoplasmic coats the assembly of which is regulated by the activity of GTPases, kinases, and phosphatases. A heterotetrameric AP-3 adaptor complex has been implicated in the formation of synaptic vesicles from PC12 endosomes (). When the small GTPase ARF1 is prevented from hydrolyzing GTP, we can reconstitute AP-3 recruitment to synaptic vesicle membranes in an assembly reaction that requires temperatures above 15 degrees C and the presence of ATP suggesting that an enzymatic step is involved in the coat assembly. We have now found an enzymatic reaction, the phosphorylation of the AP-3 adaptor complex, that is linked with synaptic vesicle coating. Phosphorylation occurs in the beta3 subunit of the complex by a kinase similar to casein kinase 1alpha. The kinase copurifies with neuronal-specific AP-3. In vitro, purified casein kinase I selectively phosphorylates the beta3A and beta3B subunit at its hinge domain. Inhibiting the kinase hinders the recruitment of AP-3 to synaptic vesicles. The same inhibitors that prevent coat assembly in vitro also inhibit the formation of synaptic vesicles in PC12 cells. The data suggest, therefore, that the mechanism of AP-3-mediated vesiculation from neuroendocrine endosomes requires the phosphorylation of the adaptor complex at a step during or after AP-3 recruitment to membranes.

Adaptor Protein Complex alpha Subunits↗
PubMed1999

The endocytic machinery in nerve terminals surrounds sites of exocytosis.

In most models of endocytosis, the endocytic machinery is recruited from the cytoplasm by cytoplasmic tails of the plasma membrane proteins that are to be internalized. This does not appear to be true at synapses where the endocytic machinery required for synaptic vesicle recycling is localized to membrane-associated 'hot spots' [1] [2]. In Drosophila neuromuscular junctions, the multi-domain protein Dap160 is also localized to hot spots [3] and has some characteristics expected of an anchoring protein. Anchoring the endocytic machinery to the plasma membrane might help contribute to the remarkable speed of synaptic vesicle recycling [4]. Here, we report that the endocytic machinery surrounds sites that are believed to be sites of exocytosis. We propose that the radial distribution of the synaptic vesicle recycling machinery already present on the plasma membrane in unstimulated nerve terminals is a fundamental unit of pre-synaptic organization and allows the nerve terminal to extract maximum recycling efficiency out of conventional endocytic machinery.

Animals↗
PubMed1999

SH3-domain-containing proteins function at distinct steps in clathrin-coated vesicle formation.

Several SH3-domain-containing proteins have been implicated in endocytosis by virtue of their interactions with dynamin; however, their functions remain undefined. Here we report the efficient reconstitution of ATP-, GTP-, cytosol- and dynamin-dependent formation of clathrin-coated vesicles in permeabilized 3T3-L1 cells. The SH3 domains of intersectin, endophilin I, syndapin I and amphiphysin II inhibit coated-vesicle formation in vitro through interactions with membrane-associated proteins. Most of the SH3 domains tested selectively inhibit late events involving membrane fission, but the SH3A domain of intersectin uniquely inhibits intermediate events leading to the formation of constricted coated pits. These results suggest that interactions between SH3 domains and their partners function sequentially in endocytic coated-vesicle formation.

3T3 Cells↗
PubMed1999

Synaptic vesicles form by budding from tubular extensions of sorting endosomes in PC12 cells.

The putative role of sorting early endosomes (EEs) in synaptic-like microvesicle (SLMV) formation in the neuroendocrine PC12 cell line was investigated by quantitative immunoelectron microscopy. By BSA-gold internalization kinetics, four distinct endosomal subcompartments were distinguished: primary endocytic vesicles, EEs, late endosomes, and lysosomes. As in other cells, EEs consisted of vacuolar and tubulovesicular subdomains. The SLMV marker proteins synaptophysin and vesicle-associated membrane protein 2 (VAMP-2) localized to both the EE vacuoles and associated tubulovesicles. Quantitative analysis showed that the transferrin receptor and SLMV proteins colocalized to a significantly higher degree in primary endocytic vesicles then in EE-associated tubulovesicles. By incubating PC12 cells expressing T antigen-tagged VAMP (VAMP-TAg) with antibodies against the luminal TAg, the recycling pathway of SLMV proteins was directly visualized. At 15 degrees C, internalized VAMP-TAg accumulated in the vacuolar domain of EEs. Upon rewarming to 37 degrees C, the labeling shifted to the tubular part of EEs and to newly formed SLMVs. Our data delineate a pathway in which SLMV proteins together with transferrin receptor are delivered to EEs, where they are sorted into SLMVs and recycling vesicles, respectively.

Animals↗
PubMed1999

Syndapin I, a synaptic dynamin-binding protein that associates with the neural Wiskott-Aldrich syndrome protein.

The GTPase dynamin has been clearly implicated in clathrin-mediated endocytosis of synaptic vesicle membranes at the presynaptic nerve terminal. Here we describe a novel 52-kDa protein in rat brain that binds the proline-rich C terminus of dynamin. Syndapin I (synaptic, dynamin-associated protein I) is highly enriched in brain where it exists in a high molecular weight complex. Syndapin I can be involved in multiple protein-protein interactions via a src homology 3 (SH3) domain at the C terminus and two predicted coiled-coil stretches. Coprecipitation studies and blot overlay analyses revealed that syndapin I binds the brain-specific proteins dynamin I, synaptojanin, and synapsin I via an SH3 domain-specific interaction. Coimmunoprecipitation of dynamin I with antibodies recognizing syndapin I and colocalization of syndapin I with dynamin I at vesicular structures in primary neurons indicate that syndapin I associates with dynamin I in vivo and may play a role in synaptic vesicle endocytosis. Furthermore, syndapin I associates with the neural Wiskott-Aldrich syndrome protein, an actin-depolymerizing protein that regulates cytoskeletal rearrangement. These characteristics of syndapin I suggest a molecular link between cytoskeletal dynamics and synaptic vesicle recycling in the nerve terminal.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Beta 2-adrenergic receptor endocytic pathway is controlled by a saturable mechanism distinct from that of transferrin receptor.

Agonist-dependent internalization is an important phase of beta 2-adrenergic receptor (beta 2AR) regulation. Recent reports have indicated that early steps of beta 2AR endocytosis may involve mechanisms different from those which regulate the internalization of constitutively recycling receptors, such as transferrin receptor (TfR). In the present study, we addressed this issue by comparing, in the same cells, the endocytic pathway of beta 2AR with that of the TfR. Upon incubation at 15 degrees C, activated beta 2ARs accumulated in peripheral endosomes of HEK-293 cells while they were targeted to perinuclear organelles at 37 degrees C. The temperature block was not specific to beta 2ARs, since both peripheral and perinuclear beta 2AR-containing endosomes comigrated on sucrose gradients with those containing transferrin receptors and were loaded with horseradish peroxidase-coupled transferrin. Endocytosis of beta 2ARs was saturable in HEK-293 cells and did not increase upon overexpression of beta-arrestin 1. TfR endocytosis was unaffected by the simultaneous internalization of overexpressed beta 2AR, indicating that the limiting components which regulate endocytosis of these two receptors are different. In conclusion, ligand activated beta 2AR and constitutively recycling receptors, such as TfR, enter the endocytic pathway via distinct saturable mechanisms but converge in the same endosomal compartments. Our results also indicate that a still unidentified component(s) controls beta 2AR endocytosis.

3,3'-Diaminobenzidine↗
PubMed1999

Deconstructing membrane traffic.

Intracellular movement of proteins and lipids between organelles is usually described in terms of cargo, carriers, traffic and docking, familiar terms that imply parallels to human activities. Over the past century, scientists have been criticized for constructing hypotheses that reflect too much of their current political and cultural values. In this article, concepts of membrane traffic are re-examined to see whether they reflect the cell's view of the world or our own.

Biological Transport↗
PubMed1998

Neuroendocrine synaptic vesicles are formed in vitro by both clathrin-dependent and clathrin-independent pathways.

In the neuroendocrine cell line, PC12, synaptic vesicles can be generated from endosomes by a sorting and vesiculation process that requires the heterotetrameric adaptor protein AP3 and a small molecular weight GTPase of the ADP ribosylation factor (ARF) family. We have now discovered a second pathway that sorts the synaptic vesicle-associated membrane protein (VAMP) into similarly sized vesicles. For this pathway the plasma membrane is the precursor rather than endosomes. Both pathways require cytosol and ATP and are inhibited by GTPgammaS. The second pathway, however, uses AP2 instead of AP3 and is brefeldin A insensitive. The AP2-dependent pathway is inhibited by depletion of clathrin or by inhibitors of clathrin binding, whereas the AP3 pathway is not. The VAMP-containing, plasma membrane-derived vesicles can be readily separated on sucrose gradients from transferrin (Tf)-containing vesicles generated by incubating Tf-labeled plasma membrane preparations at 37 degreesC. Dynamin- interacting proteins are required for the AP2-mediated vesiculation from the plasma membrane, but not from endosomes. Thus, VAMP is sorted into small vesicles by AP3 and ARF1 at endosomes and by AP2 and clathrin at the plasma membrane.

ADP-Ribosylation Factor 1↗