Apg9p/Cvt7p is an integral membrane protein required for transport vesicle formation in the Cvt and autophagy pathways.
AuthorsNoda T,Kim J,Huang WP,Baba M,Tokunaga C,Ohsumi Y,Klionsky DJ
JournalThe Journal of cell biology
PubMed ID10662773
In nutrient-rich, vegetative conditions, the yeast Saccharomyces cerevisiae transports a resident protease, aminopeptidase I (API), to the vacuole by the cytoplasm to vacuole targeting (Cvt) pathway, thus contributing to the degradative capacity of this organelle. When cells subsequently encounter starvation conditions, the machinery that recruited precursor API (prAPI) also sequesters ... More
Glutamate induces the rapid formation of spine head protrusions in hippocampal slice cultures.
AuthorsRichards DA,Mateos JM,Hugel S,de Paola V,Caroni P,Gähwiler BH,McKinney RA
JournalProceedings of the National Academy of Sciences of the United States of America
PubMed ID15831587
Synaptic plasticity at neuronal connections has been well characterized functionally by using electrophysiological approaches, but the structural basis for this phenomenon remains controversial. We have studied the dynamic interactions between presynaptic and postsynaptic structures labeled with FM 4-64 and a membrane-targeted GFP, respectively, in hippocampal slices. Under conditions of reduced ... More
Fab1p is essential for PtdIns(3)P 5-kinase activity and the maintenance of vacuolar size and membrane homeostasis.
The Saccharomyces cerevisiae FAB1 gene encodes a 257-kD protein that contains a cysteine-rich RING-FYVE domain at its NH2-terminus and a kinase domain at its COOH terminus. Based on its sequence, Fab1p was initially proposed to function as a phosphatidylinositol 4-phosphate (PtdIns(4)P) 5-kinase (). Additional sequence analysis of the Fab1p kinase ... More
Slow spontaneous secretion from single large dense-core vesicles monitored in neuroendocrine cells.
AuthorsStenovec M, Kreft M, Poberaj I, Betz WJ, Zorec R
JournalFASEB J
PubMed ID15180959
Hormones are released from cells by passing through an exocytotic pore that forms after vesicle and plasma membrane fusion. In stimulated exocytosis vesicle content is discharged swiftly. Although rapid vesicle discharge has also been proposed to mediate basal secretion, this has not been studied directly. We investigated basal hormone release ... More
Traffic-independent function of the Sar1p/COPII machinery in proteasomal sorting of the cystic fibrosis transmembrane conductance regulator.
AuthorsFu L, Sztul E
JournalJ Cell Biol
PubMed ID12538638
'Newly synthesized proteins that do not fold correctly in the ER are targeted for ER-associated protein degradation (ERAD) through distinct sorting mechanisms; soluble ERAD substrates require ER-Golgi transport and retrieval for degradation, whereas transmembrane ERAD substrates are retained in the ER. Retained transmembrane proteins are often sequestered into specialized ER ... More
Novel genes involved in endosomal traffic in yeast revealed by suppression of a targeting-defective plasma membrane ATPase mutant.
AuthorsLuo W, Chang A
JournalJ Cell Biol
PubMed ID9265642
'A novel genetic selection was used to identify genes regulating traffic in the yeast endosomal system. We took advantage of a temperature-sensitive mutant in PMA1, encoding the plasma membrane ATPase, in which newly synthesized Pma1 is mislocalized to the vacuole via the endosome. Diversion of mutant Pma1 from vacuolar delivery ... More
Molecular dynamics of a presynaptic active zone protein studied in Munc13-1-enhanced yellow fluorescent protein knock-in mutant mice.
AuthorsKalla S, Stern M, Basu J, Varoqueaux F, Reim K, Rosenmund C, Ziv NE, Brose N
JournalJ Neurosci
PubMed ID17167095
'GFP (green fluorescent protein) fusion proteins have revolutionized research on protein dynamics at synapses. However, corresponding analyses usually involve protein expression methods that override endogenous regulatory mechanisms, and therefore cause overexpression and temporal or spatial misexpression of exogenous fusion proteins, which may seriously compromise the physiological validity of such experiments. ... More
Osmotic stress-induced increase of phosphatidylinositol 3,5-bisphosphate requires Vac14p, an activator of the lipid kinase Fab1p.
AuthorsBonangelino CJ, Nau JJ, Duex JE, Brinkman M, Wurmser AE, Gary JD, Emr SD, Weisman LS
JournalJ Cell Biol
PubMed ID11889142
'Phosphatidylinositol 3,5-bisphosphate (PtdIns[3,5]P(2)) was first identified as a non-abundant phospholipid whose levels increase in response to osmotic stress. In yeast, Fab1p catalyzes formation of PtdIns(3,5)P(2) via phosphorylation of PtdIns(3)P. We have identified Vac14p, a novel vacuolar protein that regulates PtdIns(3,5)P(2) synthesis by modulating Fab1p activity in both the absence and ... More
A selective transport route from Golgi to late endosomes that requires the yeast GGA proteins.
AuthorsBlack MW, Pelham HR
JournalJ Cell Biol
PubMed ID11062260
'Pep12p is a yeast syntaxin located primarily in late endosomes. Using mutagenesis of a green fluorescent protein chimera we have identified a sorting signal FSDSPEF, which is required for transport of Pep12p from the exocytic pathway to late endosomes, from which it can, when overexpressed, reach the vacuole. When this ... More
Rapid and reversible chemical inactivation of synaptic transmission in genetically targeted neurons.
AuthorsKarpova AY, Tervo DG, Gray NW, Svoboda K
JournalNeuron
PubMed ID16337911
'Inducible and reversible silencing of selected neurons in vivo is critical to understanding the structure and dynamics of brain circuits. We have developed Molecules for Inactivation of Synaptic Transmission (MISTs) that can be genetically targeted to allow the reversible inactivation of neurotransmitter release. MISTs consist of modified presynaptic proteins that ... More
The yeast endosomal t-SNARE, Pep12p, functions in the absence of its transmembrane domain.
AuthorsGerrard SR, Mecklem AB, Stevens TH
JournalTraffic
PubMed ID11208059
'Delivery of proteins to the vacuole of the yeast Saccharomyces cerevisiae requires the function of two distinct SNARE complexes. Pep12p and Vam3p are both t-SNAREs of the syntaxin family that are components of these SNARE complexes. We have used a genetic approach to address the role of Pep12p in vacuolar ... More
An in vivo membrane fusion assay implicates SpoIIIE in the final stages of engulfment during Bacillus subtilis sporulation.
AuthorsSharp MD, Pogliano K
JournalProc Natl Acad Sci U S A
PubMed ID10588743
'Shortly after the synthesis of the two cells required for sporulation in Bacillus subtilis, the membranes of the larger mother cell begin to migrate around and engulf the smaller forespore cell. At the completion of this process the leading edges of the migrating membrane meet and fuse, releasing the forespore ... More
A family of proteins with gamma-adaptin and VHS domains that facilitate trafficking between the trans-Golgi network and the vacuole/lysosome.
AuthorsHirst J, Lui WW, Bright NA, Totty N, Seaman MN, Robinson MS
JournalJ Cell Biol
PubMed ID10747088
'We have cloned and characterized members of a novel family of proteins, the GGAs. These proteins contain an NH(2)-terminal VHS domain, one or two coiled-coil domains, and a COOH-terminal domain homologous to the COOH-terminal "ear" domain of gamma-adaptin. However, unlike gamma-adaptin, the GGAs are not associated with clathrin-coated vesicles or ... More
GFP-tagged mutant prion protein forms intra-axonal aggregates in transgenic mice.
AuthorsMedrano AZ, Barmada SJ, Biasini E, Harris DA,
JournalNeurobiol Dis
PubMed ID18514536
'A nine-octapeptide insertional mutation in the prion protein (PrP) causes a fatal neurodegenerative disorder in both humans and transgenic mice. To determine the precise cellular localization of this mutant PrP (designated PG14), we have generated transgenic mice expressing PG14-EGFP, a fluorescent fusion protein that can be directly visualized in vivo. ... More
Two distinct regions in a yeast myosin-V tail domain are required for the movement of different cargoes.
AuthorsCatlett NL, Duex JE, Tang F, Weisman LS
JournalJ Cell Biol
PubMed ID10931864
'The Saccharomyces cerevisiae myosin-V, Myo2p, is essential for polarized growth, most likely through transport of secretory vesicles to the developing bud. Myo2p is also required for vacuole movement, a process not essential for growth. The globular region of the myosin-V COOH-terminal tail domain is proposed to bind cargo. Through random ... More
A vital stain for studying membrane dynamics in bacteria: a novel mechanism controlling septation during Bacillus subtilis sporulation.
AuthorsPogliano J, Osborne N, Sharp MD, Abanes-De Mello A, Perez A, Sun YL, Pogliano K
JournalMol Microbiol
PubMed ID10096082
'At the onset of sporulation in Bacillus subtilis, two potential division sites are assembled at each pole, one of which will be used to synthesize the asymmetrically positioned sporulation septum. Using the vital stain FM 4-64 to label the plasma membrane of living cells, we examined the fate of these ... More
Geminating pollen has tubular vacuoles, displays highly dynamic vacuole biogenesis, and requires VACUOLESS1 for proper function.
AuthorsHicks GR, Rojo E, Hong S, Carter DG, Raikhel NV
JournalPlant Physiol
PubMed ID14988481
'Vacuoles perform multiple functions in plants, and VCL1 (VACUOLESS1) is essential for biogenesis with loss of expression in the vcl1 mutant leading to lethality. Vacuole biogenesis plays a prominent role in gametophytes, yet is poorly understood. Given the importance of VCL1, we asked if it contributes to vacuole biogenesis during ... More
Clusters of GABAA receptors on cultured hippocampal cells correlate only partially with functional synapses.
AuthorsKannenberg K, Sieghart W, Reuter H
JournalEur J Neurosci
PubMed ID10103120
'We describe a method to label gamma-aminobutyric acid (GABA)A receptors on the surface of living hippocampal neurons in primary culture, and we compare the distribution of receptors with that of active synapses. To visualize GABAA receptors, the affinity-purified antibody beta3(1-13), recognizing the extracellular N-termini of the GABAA receptor beta2- and ... More
GIT1 functions in a motile, multi-molecular signaling complex that regulates protrusive activity and cell migration.
AuthorsManabe R, Kovalenko M, Webb DJ, Horwitz AR
JournalJ Cell Sci
PubMed ID11896197
'GIT1 is a multidomain protein that is thought to function as an integrator of signaling pathways controlling vesicle trafficking, adhesion and cytoskeletal organization. It regulates ARF GTPases and has binding domains for paxillin and PIX, which is a PAK-binding protein and an exchange factor for Rac. We show that GIT1 ... More
Long-term staining of live Merkel cells with FM dyes.
AuthorsFukuda J, Ishimine H, Masaki Y
JournalCell Tissue Res
PubMed ID12658440
'Live Merkel cells in the skin and hair follicles are known to incorporate a fluorescence dye, quinacrine, which has been utilized to identify and dissect the cells for experiments. Quinacrine fluorescence of the cells is, however, quickly lost and quinacrine-stained Merkel cells soon become difficult to identify in tissue culture. ... More
ATP-dependent interaction of the cytosolic domains of the inwardly rectifying K+ channel Kir6.2 revealed by fluorescence resonance energy transfer.
AuthorsTsuboi T, Lippiat JD, Ashcroft FM, Rutter GA
JournalProc Natl Acad Sci U S A
PubMed ID14681552
'ATP-sensitive K(+) (K(ATP)) channels play important roles in the regulation of membrane excitability in many cell types. ATP inhibits channel activity by binding to a specific site formed by the N and C termini of the pore-forming subunit, Kir6.2, but the structural changes associated with this interaction remain unclear. Here, ... More
New insights into the bioenergetics of mitochondrial disorders using intracellular ATP reporters.
AuthorsGajewski CD, Yang L, Schon EA, Manfredi G
JournalMol Biol Cell
PubMed ID12972552
'Mutations in mitochondrial DNA (mtDNA) cause impairment of ATP synthesis. It was hypothesized that high-energy compounds, such as ATP, are compartmentalized within cells and that different cell functions are sustained by different pools of ATP, some deriving from mitochondrial oxidative phosphorylation (OXPHOS) and others from glycolysis. Therefore, an OXPHOS dysfunction ... More
Real-time three-dimensional imaging of lipid signal transduction: apical membrane insertion of epithelial Na(+) channels.
'In the distal tubule, Na(+) resorption is mediated by epithelial Na(+) channels (ENaC). Hormones such as aldosterone, vasopressin, and insulin modulate ENaC membrane targeting, assembly, and/or kinetic activity, thereby regulating salt and water homeostasis. Insulin binds to a receptor on the basal membrane to initiate a signal transduction cascade that ... More
Positive and negative regulation of a SNARE protein by control of intracellular localization.
AuthorsNakanishi H, de los Santos P, Neiman AM
JournalMol Biol Cell
PubMed ID14742704
'In Saccharomyces cerevisiae, the developmentally regulated Soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) protein Spo20p mediates the fusion of vesicles with the prospore membrane, which is required for the formation of spores. Spo20p is subject to both positive and negative regulation by separate sequences in its aminoterminal domain. We ... More
Dynamic organization of vacuolar and microtubule structures during cell cycle progression in synchronized tobacco BY-2 cells.
AuthorsKutsuna N, Hasezawa S
JournalPlant Cell Physiol
PubMed ID12354913
'In higher plant cells, vacuoles show considerable diversity in their shapes and functions. The roles of vacuoles in the storage, osmoregulation, digestion and secretory pathway are well established; however, their functions in cell morphogenesis and cell division are still unclear. To observe the dynamic changes of vacuoles in living plant ... More
An endosome-to-plasma membrane pathway involved in trafficking of a mutant plasma membrane ATPase in yeast.
AuthorsLuo W, Chang A
JournalMol Biol Cell
PubMed ID10679016
'The plasma membrane ATPase, encoded by PMA1, is delivered to the cell surface via the secretory pathway. Previously, we characterized a temperature-sensitive pma1 mutant in which newly synthesized Pma1-7 is not delivered to the plasma membrane but is mislocalized instead to the vacuole at 37 degrees C. Several vps mutants, ... More
Neural cell adhesion molecule promotes accumulation of TGN organelles at sites of neuron-to-neuron contacts.
AuthorsSytnyk V, Leshchyns'ka I, Delling M, Dityateva G, Dityatev A, Schachner M
JournalJ Cell Biol
PubMed ID12438412
'Transformation of a contact between axon and dendrite into a synapse is accompanied by accumulation of the synaptic machinery at this site, being delivered in intracellular organelles mainly of TGN origin. Here, we report that in cultured hippocampal neurons, TGN organelles are linked via spectrin to clusters of the neural ... More
A heterodimer of thioredoxin and I(B)2 cooperates with Sec18p (NSF) to promote yeast vacuole inheritance.
AuthorsXu Z, Mayer A, Muller E, Wickner W
JournalJ Cell Biol
PubMed ID9015301
'Early in S phase, the vacuole (lysosome) of Saccharomyces cerevisiae projects a stream of vesicles and membranous tubules into the bud where they fuse and establish the daughter vacuole. This inheritance reaction can be studied in vitro with isolated vacuoles. Rapid and efficient homotypic fusion between salt-washed vacuoles requires the ... More
Regulation of dendritic spine motility in cultured hippocampal neurons.
AuthorsKorkotian E, Segal M
JournalJ Neurosci
PubMed ID11487635
'Regulation of dendritic spine motility was studied in dissociated cultures of the rat and mouse hippocampus, using green fluorescent protein-labeled neurons or neurons loaded with the calcium-sensitive dye Oregon Green-1. Cells were time-lapse-photographed on a confocal laser-scanning microscope at high resolution to detect movements as well as spontaneous fluctuations of ... More
Endocytosis and vacuolar morphology in Saccharomyces cerevisiae are altered in response to ethanol stress or heat shock.
AuthorsMeaden PG, Arneborg N, Guldfeldt LU, Siegumfeldt H, Jakobsen M
JournalYeast
PubMed ID10487923
'The vital lipophilic dye N-(3-triethylammoniumpropyl)-4-[6-(4-(diethylamino)phenyl]hexatrie nyl ) pyridinium dibromide (FM 4-64) was used to study the effect of ethanol stress and heat shock on endocytosis in the yeast Saccharomyces cerevisiae. Yeast cells stained with FM 4-64 were placed in a culture chamber and the internalization of the dye was monitored ... More
Identification of a novel family of nonclassic yeast phosphatidylinositol transfer proteins whose function modulates phospholipase D activity and Sec14p-independent cell growth.
AuthorsLi X, Routt SM, Xie Z, Cui X, Fang M, Kearns MA, Bard M, Kirsch DR, Bankaitis VA
JournalMol Biol Cell
PubMed ID10848624
'Yeast phosphatidylinositol transfer protein (Sec14p) is essential for Golgi function and cell viability. We now report a characterization of five yeast SFH (Sec Fourteen Homologue) proteins that share 24-65% primary sequence identity with Sec14p. We show that Sfh1p, which shares 64% primary sequence identity with Sec14p, is nonfunctional as a ... More
Cell fusion during yeast mating requires high levels of a-factor mating pheromone.
AuthorsBrizzio V, Gammie AE, Nijbroek G, Michaelis S, Rose MD
JournalJ Cell Biol
PubMed ID8991086
'During conjugation, two yeast cells fuse to form a single zygote. Cell fusion requires extensive remodeling of the cell wall, both to form a seal between the two cells and to remove the intervening material. The two plasma membranes then fuse to produce a continuous cytoplasm. We report the characterization ... More
Structure, function, and motility of vacuoles in filamentous fungi
AuthorsCole L, Orlovich DA, Ashford AE
JournalFungal Genet Biol
PubMed ID9742195
'Current information on the structure and function of motile tubular vacuoles in Pisolithus tinctorius and other fungi is reviewed. The use of fluorochromes to label the vacuole lumen is evaluated and observations on the structure and motility of vacuoles in P. tinctorius are differentiated from possible artifacts. The styryl dyes ... More
Chemical genetic analysis of Apg1 reveals a non-kinase role in the induction of autophagy.
AuthorsAbeliovich H, Zhang C, Dunn WA, Shokat KM, Klionsky DJ
JournalMol Biol Cell
PubMed ID12589048
'Macroautophagy is a catabolic membrane trafficking phenomenon that is observed in all eukaryotic cells in response to various stimuli, such as nitrogen starvation and challenge with specific hormones. In the yeast Saccharomyces cerevisiae, the induction of autophagy involves a direct signal transduction mechanism that affects membrane dynamics. In this system, ... More
Modification of a ubiquitin-like protein Paz2 conducted micropexophagy through formation of a novel membrane structure.
AuthorsMukaiyama H, Baba M, Osumi M, Aoyagi S, Kato N, Ohsumi Y, Sakai Y
JournalMol Biol Cell
PubMed ID13679515
'Microautophagy is a versatile process in which vacuolar or lysosomal membranes directly sequester cytosolic targets for degradation. Recent genetic evidence suggested that microautophagy uses molecular machineries essential for macroautophagy, but the details of this process are still unknown. In this study, a ubiquitin-like protein Paz2 essential for micropexophagy in the ... More
MinCD-dependent regulation of the polarity of SpoIIIE assembly and DNA transfer.
AuthorsSharp MD, Pogliano K
JournalEMBO J
PubMed ID12426398
'During Bacillus subtilis sporulation, the SpoIIIE DNA translocase moves a trapped chromosome across the sporulation septum into the forespore. The direction of DNA translocation is controlled by the specific assembly of SpoIIIE in the mother cell and subsequent export of DNA into the forespore. We present evidence that the MinCD ... More
Role of endocytosis in the transfection of L929 fibroblasts by polyethylenimine/DNA complexes.
AuthorsRémy-Kristensen A, Clamme JP, Vuilleumier C, Kuhry JG, Mély Y
JournalBiochim Biophys Acta
PubMed ID11513802
'Polyethylenimine (PEI) is one of the most efficient nonviral vectors for gene therapy. The aim of this study was to investigate the role of endocytosis in the transfection of synchronized L929 fibroblasts by PEI/DNA complexes. This was performed by confocal microscopy and flow cytometry, using the endocytosis marker FM4-64 and ... More
Bro1 is an endosome-associated protein that functions in the MVB pathway in Saccharomyces cerevisiae.
AuthorsOdorizzi G, Katzmann DJ, Babst M, Audhya A, Emr SD
JournalJ Cell Sci
PubMed ID12668726
'Multivesicular bodies are late endosomal compartments containing lumenal vesicles that are formed by inward budding of the limiting endosomal membrane. In the yeast Saccharomyces cerevisiae, integral membrane proteins are sorted into the lumenal vesicles of multivesicular bodies, and this process requires the class E subset of VPS genes. We show ... More
Varying division planes of secondary constrictions in spheroidal Escherichia coli cells.
AuthorsZaritsky A, Woldringh CL, Fishov I, Vischer NO, Einav M
JournalMicrobiology
PubMed ID10376816
'Planes of successive divisions in Escherichia coli have been proposed to be either parallel or perpendicular to each other, restricted to one or two dimensions. To test the hypothesis that divisions can occur in planes alternating in three dimensions, a method was developed to generate cells with secondary constrictions during ... More
Confocal laser scanning microscopy of hamster cerebellum using FM4-64 as intracellular staining.
AuthorsCastejón O, Sims P
JournalScanning
PubMed ID10070780
'The FM4-64, a member of the family of fluorescent dyes, has been applied to the cerebellar cortex to evaluate its properties as an intracellular stain and intracortical tracer. Slabs of hamster cerebellum, 1-2 mm thick, were incubated in 10, 30, and 100 microns solutions of FM4-64 in sodium phosphate buffer ... More
Loss of halophytism by interference with SOS1 expression.
AuthorsOh DH, Leidi E, Zhang Q, Hwang SM, Li Y, Quintero FJ, Jiang X, D'Urzo MP, Lee SY, Zhao Y, Bahk JD, Bressan RA, Yun DJ, Pardo JM, Bohnert HJ,
JournalPlant Physiol
PubMed ID19571313
'The contribution of SOS1 (for Salt Overly Sensitive 1), encoding a sodium/proton antiporter, to plant salinity tolerance was analyzed in wild-type and RNA interference (RNAi) lines of the halophytic Arabidopsis (Arabidopsis thaliana)-relative Thellungiella salsuginea. Under all conditions, SOS1 mRNA abundance was higher in Thellungiella than in Arabidopsis. Ectopic expression of ... More
A protein containing a serine-rich domain with vesicle fusing properties mediates cell cycle-dependent cytosolic pH regulation.
AuthorsBrazill DT, Caprette DR, Myler HA, Hatton RD, Ammann RR, Lindsey DF, Brock DA, Gomer RH
JournalJ Biol Chem
PubMed ID10747962
'Initial differentiation in Dictyostelium involves both asymmetric cell division and a cell cycle-dependent mechanism. We previously identified a gene, rtoA, which when disrupted randomizes the cell cycle-dependent mechanism without affecting either the underlying cell cycle or asymmetric differentiation. We find that in wild-type cells, RtoA levels vary during the cell ... More
Proton pumps populate the contractile vacuoles of Dictyostelium amoebae.
AuthorsHeuser J, Zhu Q, Clarke M
JournalJ Cell Biol
PubMed ID8509452
'Amoebae of the eukaryotic microorganism Dictyostelium discoideum were found to contain an interconnected array of tubules and cisternae whose membranes were studded with 15-nm-diameter "pegs." Comparison of the ultrastructure and freeze-fracture behavior of these pegs with similar structures found in other cells and tissues indicated that they were the head ... More
Interdependent assembly of specific regulatory lipids and membrane fusion proteins into the vertex ring domain of docked vacuoles.
AuthorsFratti RA, Jun Y, Merz AJ, Margolis N, Wickner W
JournalJ Cell Biol
PubMed ID15611334
'Membrane microdomains are assembled by lipid partitioning (e.g., rafts) or by protein-protein interactions (e.g., coated vesicles). During docking, yeast vacuoles assemble "vertex" ring-shaped microdomains around the periphery of their apposed membranes. Vertices are selectively enriched in the Rab GTPase Ypt7p, the homotypic fusion and vacuole protein sorting complex (HOPS)-VpsC Rab ... More
Assembly of new individual excitatory synapses: time course and temporal order of synaptic molecule recruitment.
AuthorsFriedman HV, Bresler T, Garner CC, Ziv NE
JournalNeuron
PubMed ID10939331
'Time-lapse microscopy, retrospective immunohistochemistry, and cultured hippocampal neurons were used to determine the time frame of individual glutamatergic synapse assembly and the temporal order in which specific molecules accumulate at new synaptic junctions. New presynaptic boutons capable of activity-evoked vesicle recycling were observed to form within 30 min of initial ... More
Septal localization of forespore membrane proteins during engulfment in Bacillus subtilis.
AuthorsRubio A, Pogliano K
JournalEMBO J
PubMed ID15044948
'In Bacillus subtilis, many membrane proteins localize to the sporulation septum, where they play key roles in spore morphogenesis and cell-specific gene expression, but the mechanism for septal targeting is not well understood. SpoIIQ, a forespore-expressed protein, is involved in engulfment and forespore-specific gene expression. We find that SpoIIQ dynamically ... More
Clathrin-mediated endocytosis is essential in Trypanosoma brucei.
AuthorsAllen CL, Goulding D, Field MC
JournalEMBO J
PubMed ID14517238
'In Trypanosoma brucei, the plasma membrane is dominated by glycosylphosphatidylinositol (GPI)-anchored proteins. Endocytic activity correlates with expression levels of the clathrin heavy chain TbCLH, and additional evidence suggests that rapid endocytosis may play a role in evasion of the immune response. TbCLH is present on both endocytic vesicles and post-Golgi ... More
Yeast exocytic v-SNAREs confer endocytosis.
AuthorsGurunathan S, Chapman-Shimshoni D, Trajkovic S, Gerst JE
JournalMol Biol Cell
PubMed ID11029060
'In yeast, homologues of the synaptobrevin/VAMP family of v-SNAREs (Snc1 and Snc2) confer the docking and fusion of secretory vesicles at the cell surface. As no v-SNARE has been shown to confer endocytosis, we examined whether yeast lacking the SNC genes, or possessing a temperature-sensitive allele of SNC1 (SNC1(ala43)), are ... More
A novel RING finger protein complex essential for a late step in protein transport to the yeast vacuole.
AuthorsRieder SE, Emr SD
JournalMol Biol Cell
PubMed ID9362071
'Protein transport to the lysosome-like vacuole in yeast is mediated by multiple pathways, including the biosynthetic routes for vacuolar hydrolases, the endocytic pathway, and autophagy. Among the more than 40 genes required for vacuolar protein sorting (VPS) in Saccharomyces cerevisiae, mutations in the four class C VPS genes result in ... More
N-Glycosylation at two sites critically alters thiazide binding and activity of the rat thiazide-sensitive Na(+):Cl(-) cotransporter.
AuthorsHoover RS, Poch E, Monroy A, Vázquez N, Nishio T, Gamba G, Hebert SC
JournalJ Am Soc Nephrol
PubMed ID12538726
'The rat thiazide-sensitive Na-Cl cotransporter (rNCC) is expressed in the renal distal convoluted tubule and is the site of action of an important class of antihypertensive agents, the thiazide diuretics. The amino acid sequence contains two potential N-linked glycosylation consensus sites, N404 and N424. Either enzymatic deglycosylation or tunicamycin reduced ... More
Quantitative and dynamic assessment of the contribution of the ER to phagosome formation.
AuthorsTouret N, Paroutis P, Terebiznik M, Harrison RE, Trombetta S, Pypaert M, Chow A, Jiang A, Shaw J, Yip C, Moore HP, van der Wel N, Houben D, Peters PJ, de Chastellier C, Mellman I, Grinstein S,
JournalCell
PubMed ID16213220
'Phagosomes were traditionally thought to originate from an invagination and scission of the plasma membrane to form a distinct intracellular vacuole. An alternative model implicating the endoplasmic reticulum (ER) as a major component of nascent and maturing phagosomes was recently proposed (Gagnon et al., 2002). To reconcile these seemingly disparate ... More
The Saccharomyces cerevisiae homologue of human Wiskott-Aldrich syndrome protein Las17p interacts with the Arp2/3 complex.
AuthorsMadania A, Dumoulin P, Grava S, Kitamoto H, Schärer-Brodbeck C, Soulard A, Moreau V, Winsor B
JournalMol Biol Cell
PubMed ID10512884
'Yeast Las17 protein is homologous to the Wiskott-Aldrich Syndrome protein, which is implicated in severe immunodeficiency. Las17p/Bee1p has been shown to be important for actin patch assembly and actin polymerization. Here we show that Las17p interacts with the Arp2/3 complex. LAS17 is an allele-specific multicopy suppressor of ARP2 and ARP3 ... More
Cytoplasmic and nuclear delivery of a TAT-derived peptide and a beta-peptide after endocytic uptake into HeLa cells.
AuthorsPotocky TB, Menon AK, Gellman SH
JournalJ Biol Chem
PubMed ID14517218
'Several short, highly cationic peptides are able to enter the cytoplasm and nucleus of cells from the extracellular medium. The mechanism of entry is unknown. A number of fluorescence-based studies suggested that these molecules cross the plasma membrane by an energy-independent process, directly gaining access to the cytoplasm. Recent reports ... More
Cooperative binding of the cytoplasm to vacuole targeting pathway proteins, Cvt13 and Cvt20, to phosphatidylinositol 3-phosphate at the pre-autophagosomal structure is required for selective autophagy.
AuthorsNice DC, Sato TK, Stromhaug PE, Emr SD, Klionsky DJ
JournalJ Biol Chem
PubMed ID12048214
'Autophagy is a catabolic membrane-trafficking mechanism involved in cell maintenance and development. Most components of autophagy also function in the cytoplasm to vacuole targeting (Cvt) pathway, a constitutive biosynthetic pathway required for the transport of aminopeptidase I (Ape1). The protein components of autophagy and the Cvt pathway include a putative ... More
Expression of the vesicular acetylcholine transporter, proteins involved in exocytosis, and functional calcium signaling in varicosities and soma of a murine septal cell line.
'The expression and localization of the vesicular acetylcholine transporter in a septal cell line, SN56, were investigated. Immunoprecipitation and immunoblot analysis of postnuclear supernatants indicated that this cell line expresses reasonable amounts of the transporter. Immunofluorescence and confocal microscopy experiments showed that the vesicular transporter is present in varicosities and ... More
TEDS site phosphorylation of the yeast myosins I is required for ligand-induced but not for constitutive endocytosis of the G protein-coupled receptor Ste2p.
'The yeast myosins I Myo3p and Myo5p have well established functions in the polarization of the actin cytoskeleton and in the endocytic uptake of the G protein-coupled receptor Ste2p. A number of results suggest that phosphorylation of the conserved TEDS serine of the myosin I motor head by the Cdc42p ... More
Bacterial DNA segregation by dynamic SopA polymers.
AuthorsLim GE, Derman AI, Pogliano J
JournalProc Natl Acad Sci U S A
PubMed ID16306264
'Many bacterial plasmids and chromosomes rely on ParA ATPases for proper positioning within the cell and for efficient segregation to daughter cells. Here we demonstrate that the F-plasmid-partitioning protein SopA polymerizes into filaments in an ATP-dependent manner in vitro, and that the filaments elongate at a rate that is similar ... More
AtIREG2 encodes a tonoplast transport protein involved in iron-dependent nickel detoxification in Arabidopsis thaliana roots.
AuthorsSchaaf G, Honsbein A, Meda AR, Kirchner S, Wipf D, von Wirén N
JournalJ Biol Chem
PubMed ID16790430
'Iron acquisition in Arabidopsis depends mainly on AtIRT1, a Fe2+ transporter in the plasma membrane of root cells. However, substrate specificity of AtIRT1 is low, leading to an excess accumulation of other transition metals in iron-deficient plants. In the present study we describe AtIREG2 as a nickel transporter at the ... More
Regulated degradation of an endoplasmic reticulum membrane protein in a tubular lysosome in Leishmania mexicana.
'The cell surface of the human parasite Leishmania mexicana is coated with glycosylphosphatidylinositol (GPI)-anchored macromolecules and free GPI glycolipids. We have investigated the intracellular trafficking of green fluorescent protein- and hemagglutinin-tagged forms of dolichol-phosphate-mannose synthase (DPMS), a key enzyme in GPI biosynthesis in L. mexicana promastigotes. These functionally active chimeras ... More
ARF is required for maintenance of yeast Golgi and endosome structure and function.
AuthorsGaynor EC, Chen CY, Emr SD, Graham TR
JournalMol Biol Cell
PubMed ID9487133
'ADP ribosylation factor (ARF) is thought to play a critical role in recruiting coatomer (COPI) to Golgi membranes to drive transport vesicle budding. Yeast strains harboring mutant COPI proteins exhibit defects in retrograde Golgi to endoplasmic reticulum protein transport and striking cargo-selective defects in anterograde endoplasmic reticulum to Golgi protein ... More
The clathrin adaptor complex 1 directly binds to a sorting signal in Ste13p to reduce the rate of its trafficking to the late endosome of yeast.
AuthorsFoote C, Nothwehr SF
JournalJ Cell Biol
PubMed ID16702232
'Yeast trans-Golgi network (TGN) membrane proteins maintain steady-state localization by constantly cycling to and from endosomes. In this study, we examined the trafficking itinerary and molecular requirements for delivery of a model TGN protein A(F-->A)-alkaline phosphatase (ALP) to the prevacuolar/endosomal compartment (PVC). A(F-->A)-ALP was found to reach the PVC via ... More
Differential vesicular targeting and time course of synaptic secretion of the mammalian neurotrophins.
AuthorsBrigadski T, Hartmann M, Lessmann V
JournalJ Neurosci
PubMed ID16107647
'Neurotrophins are a family of secreted neuronal survival and plasticity factors comprising NGF, BDNF, neurotrophin-3 (NT-3), and NT-4. Whereas synaptic secretion of BDNF has been described, the routes of intracellular targeting and secretion of NGF, NT-3, and NT-4 in neurons are poorly understood. To allow for a direct comparison of ... More
Cell-penetrating peptides. A reevaluation of the mechanism of cellular uptake.
AuthorsRichard JP, Melikov K, Vives E, Ramos C, Verbeure B, Gait MJ, Chernomordik LV, Lebleu B
JournalJ Biol Chem
PubMed ID12411431
'Cellular uptake of a family of cationic cell-penetrating peptides (examples include Tat peptides and penetratin) have been ascribed in the literature to a mechanism that does not involve endocytosis. In this work we reevaluate the mechanisms of cellular uptake of Tat 48-60 and (Arg)(9). We demonstrate here that cell fixation, ... More
Clathrin is required for the function of the mitotic spindle.
AuthorsRoyle SJ, Bright NA, Lagnado L
JournalNature
PubMed ID15858577
'Clathrin has an established function in the generation of vesicles that transfer membrane and proteins around the cell. The formation of clathrin-coated vesicles occurs continuously in non-dividing cells, but is shut down during mitosis, when clathrin concentrates at the spindle apparatus. Here, we show that clathrin stabilizes fibres of the ... More
Membrane transport in Caenorhabditis elegans: an essential role for VPS34 at the nuclear membrane.
AuthorsRoggo L, Bernard V, Kovacs AL, Rose AM, Savoy F, Zetka M, Wymann MP, Müller F
JournalEMBO J
PubMed ID11927551
'Here we present a detailed genetic analysis of let-512/vps34 that encodes the Caenorhabditis elegans homologue of the yeast phosphatidylinositol 3-kinase Vps34p. LET-512/VPS34 has essential functions and is ubiquitously expressed in all tissues and developmental stages. It accumulates at a perinuclear region, and mutations in let-512/vps34 result in an expansion of ... More
Early stages of the secretory pathway, but not endosomes, are required for Cvt vesicle and autophagosome assembly in Saccharomyces cerevisiae.
AuthorsReggiori F, Wang CW, Nair U, Shintani T, Abeliovich H, Klionsky DJ
JournalMol Biol Cell
PubMed ID15004240
'The Cvt pathway is a biosynthetic transport route for a distinct subset of resident yeast vacuolar hydrolases, whereas macroautophagy is a nonspecific degradative mechanism that allows cell survival during starvation. Yet, these two vacuolar trafficking pathways share a number of identical molecular components and are morphologically very similar. For example, ... More
Cell cycle synchronization of Escherichia coli using the stringent response, with fluorescence labeling assays for DNA content and replication.
AuthorsFerullo DJ, Cooper DL, Moore HR, Lovett ST,
JournalMethods
PubMed ID19245839
'We describe a method for synchronization of the cell cycle in the bacterium Escherichia coli. Treatment of asynchronous cultures with the amino acid analog, dl-serine hydroxamate, induces the stringent response, with concomitant arrest of DNA replication at initiation. Following release of the stringent response, cells initiate DNA replication in synchrony, ... More
Assembly of presynaptic active zones from cytoplasmic transport packets.
AuthorsAhmari SE, Buchanan J, Smith SJ
JournalNat Neurosci
PubMed ID10769383
'Little is known about presynaptic assembly during central nervous system synaptogenesis. Here we used time-lapse fluorescence imaging, immunocytochemistry and electron microscopy to study hippocampal neuronal cultures transfected with a fusion construct of the presynaptic vesicle protein VAMP and green fluorescent protein. Our results suggest that major cytoplasmic and membrane-associated protein ... More
Constitutive sharing of recycling synaptic vesicles between presynaptic boutons.
AuthorsDarcy KJ, Staras K, Collinson LM, Goda Y
JournalNat Neurosci
PubMed ID16462738
'The synaptic vesicle cycle is vital for sustained neurotransmitter release. It has been assumed that functional synaptic vesicles are replenished autonomously at individual presynaptic terminals. Here we tested this assumption by using FM dyes in combination with fluorescence recovery after photobleaching and correlative light and electron microscopy in cultured rat ... More
Continuous and transient vesicle cycling at a ribbon synapse.
AuthorsRouze NC, Schwartz EA
JournalJ Neurosci
PubMed ID9786969
'Optical methods were used to study the Ca2+ dependence of vesicle cycling in bipolar cells isolated from goldfish retinas. Uniformly raising the Ca2+ concentration to between 0.8 and 20 microM produced a continuous vesicle cycle of balanced exocytosis and endocytosis with a maximum rate equivalent to the turnover of the ... More
GPI-anchored influenza hemagglutinin induces hemifusion to both red blood cell and planar bilayer membranes.
AuthorsMelikyan GB, White JM, Cohen FS
JournalJ Cell Biol
PubMed ID7593189
'Under fusogenic conditions, fluorescent dye redistributed from the outer monolayer leaflet of red blood cells (RBCs) to cells expressing glycophosphatidylinositol-anchored influenza virus hemagglutinin (GPI-HA) without transfer of aqueous dye. This suggests that hemifusion, but not full fusion, occurred (Kemble, G. W., T. Danieli, and J. M. White. 1994. Cell. 76:383-391). ... More
New components of a system for phosphate accumulation and polyphosphate metabolism in Saccharomyces cerevisiae revealed by genomic expression analysis.
AuthorsOgawa N, DeRisi J, Brown PO
JournalMol Biol Cell
PubMed ID11102525
'The PHO regulatory pathway is involved in the acquisition of phosphate (P(i)) in the yeast Saccharomyces cerevisiae. When extracellular P(i) concentrations are low, several genes are transcriptionally induced by this pathway, which includes the Pho4 transcriptional activator, the Pho80-Pho85 cyclin-CDK pair, and the Pho81 CDK inhibitor. In an attempt to ... More
Hierarchy of protein assembly at the vertex ring domain for yeast vacuole docking and fusion.
AuthorsWang L, Merz AJ, Collins KM, Wickner W
JournalJ Cell Biol
PubMed ID12566429
'Vacuole tethering, docking, and fusion proteins assemble into a "vertex ring" around the apposed membranes of tethered vacuoles before catalyzing fusion. Inhibitors of the fusion reaction selectively interrupt protein assembly into the vertex ring, establishing a causal assembly hierarchy: (a) The Rab GTPase Ypt7p mediates vacuole tethering and forms the ... More
FM dyes label sterol-rich plasma membrane domains and are internalized independently of the cytoskeleton in characean internodal cells.
AuthorsKlima A, Foissner I,
JournalPlant Cell Physiol
PubMed ID18757863
'We applied the endocytic markers FM1-43, FM4-64 and filipin to internodal cells of the green alga Chara corallina. Both FM dyes stained stable, long-living plasma membrane patches with a diameter of up to 1 microm. After 5 min, FM dyes labeled cortical, trembling structures up to 500 nm in size. ... More
The mechanism of action of the Pseudomonas aeruginosa-encoded type III cytotoxin, ExoU.
AuthorsSato H, Frank DW, Hillard CJ, Feix JB, Pankhaniya RR, Moriyama K, Finck-Barbançon V, Buchaklian A, Lei M, Long RM, Wiener-Kronish J, Sawa T
JournalEMBO J
PubMed ID12805211
'Pseudomonas aeruginosa delivers the toxin ExoU to eukaryotic cells via a type III secretion system. Intoxication with ExoU is associated with lung injury, bacterial dissemination and sepsis in animal model and human infections. To search for ExoU targets in a genetically tractable system, we used controlled expression of the toxin ... More
Identification and characterization of Saccharomyces cerevisiae mutants defective in fluid-phase endocytosis.
AuthorsWiederkehr A, Meier KD, Riezman H
JournalYeast
PubMed ID11378903
'A mutant library generated by the European Functional Analysis Network (EUROFAN) was screened for strains defective in fluid-phase endocytosis. Accumulation of Lucifer yellow in the vacuole was used as a marker for efficient endocytosis. Fourteen mutants, including ede1Delta, rcy1Delta, sys1Delta and tlg2Delta, previously described to be involved in membrane trafficking, ... More
Distinct roles for the yeast phosphatidylinositol 4-kinases, Stt4p and Pik1p, in secretion, cell growth, and organelle membrane dynamics.
AuthorsAudhya A, Foti M, Emr SD
JournalMol Biol Cell
PubMed ID10930462
'The yeast Saccharomyces cerevisiae possesses two genes that encode phosphatidylinositol (PtdIns) 4-kinases, STT4 and PIK1. Both gene products phosphorylate PtdIns at the D-4 position of the inositol ring to generate PtdIns(4)P, which plays an essential role in yeast viability because deletion of either STT4 or PIK1 is lethal. Furthermore, although ... More
Polarized hyphal growth in Candida albicans requires the Wiskott-Aldrich Syndrome protein homolog Wal1p.
AuthorsWalther A, Wendland J
JournalEukaryot Cell
PubMed ID15075276
'The yeast-to-hypha transition is a key feature in the cell biology of the dimorphic human fungal pathogen Candida albicans. Reorganization of the actin cytoskeleton is required for this dimorphic switch in Candida. We show that C. albicans WAL1 mutants with both copies of the Wiskott-Aldrich syndrome protein (WASP) homolog deleted ... More
Visualizing postendocytic traffic of synaptic vesicles at hippocampal synapses.
AuthorsLi Z, Murthy VN
JournalNeuron
PubMed ID11545718
'We have investigated mechanisms in postendocytic processing of synaptic vesicles at hippocampal synapses, using synaptobrevin/vesicle-associated membrane protein (VAMP) tagged with variants of the green fluorescent protein. Following exocytosis, VAMP is retrieved at synaptic and adjoining axonal regions. Retrieved VAMP-containing vesicles return to synaptic vesicle clusters at a rate slower than ... More
Distinct mechanisms for aerenchyma formation in leaf sheaths of rice genotypes displaying a quiescence or escape strategy for flooding tolerance.
AuthorsParlanti S, Kudahettige NP, Lombardi L, Mensuali-Sodi A, Alpi A, Perata P, Pucciariello C,
JournalAnn Bot
PubMed ID21489969
'Rice is one of the few crops able to withstand periods of partial or even complete submergence. One of the adaptive traits of rice is the constitutive presence and further development of aerenchyma which enables oxygen to be transported to submerged organs. The development of lysigenous aerenchyma is promoted by ... More
Characterization and imaging of A6 epithelial cell clones expressing fluorescently labeled ENaC subunits.
AuthorsBlazer-Yost BL, Butterworth M, Hartman AD, Parker GE, Faletti CJ, Els WJ, Rhodes SJ
JournalAm J Physiol Cell Physiol
PubMed ID11443062
'A6 model renal epithelial cells were stably transfected with enhanced green fluorescent protein (EGFP)-tagged alpha- or beta-subunits of the epithelial Na(+) channel (ENaC). Transfected RNA and proteins were both expressed in low abundance, similar to the endogenous levels of ENaC in native cells. In living cells, laser scanning confocal microscopy ... More
The dynamic control of kiss-and-run and vesicular reuse probed with single nanoparticles.
AuthorsZhang Q, Li Y, Tsien RW,
JournalScience
PubMed ID19213879
'Vesicular secretion of neurotransmitter is essential for neuronal communication. Kiss-and-run is a mode of membrane fusion and retrieval without the full collapse of the vesicle into the plasma membrane and de novo regeneration. The importance of kiss-and-run during efficient neurotransmission has remained in doubt. We developed an approach for loading ... More
Functional analysis of conserved structural elements in yeast syntaxin Vam3p.
AuthorsWang Y, Dulubova I, Rizo J, Südhof TC
JournalJ Biol Chem
PubMed ID11349128
'Vam3p, a syntaxin-like SNARE protein involved in yeast vacuole fusion, is composed of a three-helical N-terminal domain, a canonical SNARE motif, and a C-terminal transmembrane region (TMR). Surprisingly, we find that the N-terminal domain of Vam3p is not essential for fusion, although analogous domains in other syntaxins are indispensible for ... More
Analysis of three separate probes suggests the absence of endocytosis in Neurospora crassa hyphae.
AuthorsTorralba S, Heath IB
JournalFungal Genet Biol
PubMed ID12431457
'Reports of the existence of endocytosis in filamentous fungi have been conflicting and inconclusive. For this reason, we have tested three independent markers in Neurospora crassa: the electron opaque marker lanthanum (La) and the fluorescent probes Lucifer yellow (LY) and FM4-64. Both La and LY were endocytosed by Saccharomyces cerevisiae ... More
Imaging membrane potential in dendritic spines.
AuthorsNuriya M, Jiang J, Nemet B, Eisenthal KB, Yuste R
JournalProc Natl Acad Sci U S A
PubMed ID16407122
'Dendritic spines mediate most excitatory inputs in the brain. Although it is clear that spines compartmentalize calcium, it is still unknown what role, if any, they play in integrating synaptic inputs. To investigate the electrical function of spines directly, we used second harmonic generation (SHG) imaging of membrane potential in ... More
PSD-95 involvement in maturation of excitatory synapses.
AuthorsEl-Husseini AE, Schnell E, Chetkovich DM, Nicoll RA, Bredt DS
JournalScience
PubMed ID11082065
'PSD-95 is a neuronal PDZ protein that associates with receptors and cytoskeletal elements at synapses, but whose function is uncertain. We found that overexpression of PSD-95 in hippocampal neurons can drive maturation of glutamatergic synapses. PSD-95 expression enhanced postsynaptic clustering and activity of glutamate receptors. Postsynaptic expression of PSD-95 also ... More
The C2 domain of the Rsp5 ubiquitin ligase binds membrane phosphoinositides and directs ubiquitination of endosomal cargo.
AuthorsDunn R, Klos DA, Adler AS, Hicke L
JournalJ Cell Biol
PubMed ID15078904
'Ubiquitin ligases of the Nedd4 family regulate membrane protein trafficking by modifying both cargo proteins and the transport machinery with ubiquitin. Here, we investigate the role of the yeast Nedd4 homologue, Rsp5, in protein sorting into vesicles that bud into the multivesicular endosome (MVE) en route to the vacuole. A ... More
Carbon nanotubes as intracellular protein transporters: generality and biological functionality.
AuthorsKam NW, Dai H
JournalJ Am Chem Soc
PubMed ID15839702
'Various proteins adsorb spontaneously on the sidewalls of acid-oxidized single-walled carbon nanotubes. This simple nonspecific binding scheme can be used to afford noncovalent protein-nanotube conjugates. The proteins are found to be readily transported inside various mammalian cells with nanotubes acting as the transporter via the endocytosis pathway. Once released from ... More
Phosphate starvation in fungi induces the replacement of phosphatidylcholine with the phosphorus-free betaine lipid diacylglyceryl-n,n,n-trimethylhomoserine.
'Diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) is a phosphorus-free betaine-lipid analog of phosphatidylcholine (PtdCho) synthesized by many soil bacteria, algae, and nonvascular plants. Synthesis of DGTS and other phosphorus-free lipids in bacteria occurs in response to phosphorus (P) deprivation and results in the replacement of phospholipids by nonphosphorous lipids. The genes encoding DGTS biosynthetic ... More
Simultaneous monitoring of three key neuronal functions in primary neuronal cultures.
AuthorsEvans GJ, Cousin MA
JournalJ Neurosci Methods
PubMed ID17049620
'The coupling of Ca(2+) influx to synaptic vesicle (SV) recycling in nerve terminals is essential for neurotransmitter release and thus neuronal communication. Both of these parameters have been monitored using fluorescent reporter dyes such as fura-2 and FM1-43 in single central nerve terminals. However, their simultaneous monitoring has been hampered ... More
The yeast actin-related protein Arp2p is required for the internalization step of endocytosis.
AuthorsMoreau V, Galan JM, Devilliers G, Haguenauer-Tsapis R, Winsor B
JournalMol Biol Cell
PubMed ID9243513
'The Saccharomyces cerevisiae actin-related protein Arp2p is an essential component of the actin cytoskeleton. We have tested its potential role in the endocytic and exocytic pathways by using a temperature-sensitive allele, arp2-1. The fate of the plasma membrane transporter uracil permease was followed to determine whether Arp2p plays a role ... More
Characterization of yeast V-ATPase mutants lacking Vph1p or Stv1p and the effect on endocytosis.
AuthorsPerzov N, Padler-Karavani V, Nelson H, Nelson N
JournalJ Exp Biol
PubMed ID11948198
'Subunit a of V-ATPase in the yeast Saccharomyces cerevisiae, in contrast to its other subunits, is encoded by two genes VPH1 and STV1. While disruption of any other gene encoding the V-ATPase subunits results in growth arrest at pH 7.5, null mutants of Vph1p or Stv1p can grow at this ... More
Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging.
AuthorsCentonze VE, White JG
JournalBiophys J
PubMed ID9746543
'Multiphoton excitation fluorescence imaging generates an optical section of sample by restricting fluorophore excitation to the plane of focus. High photon densities, achieved only in the focal volume of the objective, are sufficient to excite the fluorescent probe molecules by density-dependent, multiphoton excitation processes. We present comparisons of confocal with ... More
Cytoarchitectonic arrangement and intracortical circuits of hamster cerebellum. A study by means of confocal scanning laser microscopy.
AuthorsCastejón O, Sims P
JournalBiocell
PubMed ID10904543
'The FM4-64, a member of the family of fluorescent dyes, has been applied to the cerebellar cortex to study its cytoarchitectonic arrangement and intracortical course of mossy and climbing fibers. Slabs of hamster cerebellum, 1-2 mm thick, were incubated in 10, 30 and 100 microM solutions of FM4-64 in 0.1 ... More
Actin has a molecular scaffolding, not propulsive, role in presynaptic function.
AuthorsSankaranarayanan S, Atluri PP, Ryan TA
JournalNat Neurosci
PubMed ID12536209
'We used actin tagged with enhanced green fluorescent protein (EGFP-actin) to characterize the distribution and dynamics of actin in living presynaptic terminals in rat CNS neurons. Actin was preferentially concentrated around--and appeared to surround--the presynaptic vesicle cluster. In resting terminals, approximately 30% of actin was found to be in a ... More
Ubiquitin interactions of NZF zinc fingers.
AuthorsAlam SL, Sun J, Payne M, Welch BD, Blake BK, Davis DR, Meyer HH, Emr SD, Sundquist WI
JournalEMBO J
PubMed ID15029239
'Ubiquitin (Ub) functions in many different biological pathways, where it typically interacts with proteins that contain modular Ub recognition domains. One such recognition domain is the Npl4 zinc finger (NZF), a compact zinc-binding module found in many proteins that function in Ub-dependent processes. We now report the solution structure of ... More
The kinetics of synaptic vesicle pool depletion at CNS synaptic terminals.
AuthorsFernández-Alfonso T, Ryan TA
JournalNeuron
PubMed ID15046726
'During sustained action potential (AP) firing at nerve terminals, the rates of endocytosis compared to exocytosis determine how quickly the available synaptic vesicle pool is depleted, in turn influencing presynaptic efficacy. Mechanisms, including rapid kiss-and-run endocytosis as well as local, preferential recycling of docked vesicles, have been proposed as a ... More
Staurosporine blocks evoked release of FM1-43 but not acetylcholine from frog motor nerve terminals.
AuthorsHenkel AW, Betz WJ
JournalJ Neurosci
PubMed ID8613758
'The protein kinase inhibitor staurosporine inhibited, and often abolished, activity-dependent destaining of frog motor nerve terminals that had been preloaded with the fluorescent dye FM1-43. Staurosporine did not, however, block synaptic transmission; staurosporine treated muscles twitched in response to nerve stimulation, and the amplitudes of evoked end plate potentials were ... More
Endosome fusion in living cells overexpressing GFP-rab5.
AuthorsRoberts RL, Barbieri MA, Pryse KM, Chua M, Morisaki JH, Stahl PD
JournalJ Cell Sci
PubMed ID10523503
'CHO and BHK cells which overexpress either wild-type rab5 or rab5:Q79L, a constitutively active rab5 mutant, develop enlarged cytoplasmic vesicles that exhibit many characteristics of early endosomes including immunoreactivity for rab5 and transferrin receptor. Time-lapse video microscopy shows the enlarged endosomes arise primarily by fusion of smaller vesicles. These fusion ... More
Cell biology of the human thiamine transporter-1 (hTHTR1). Intracellular trafficking and membrane targeting mechanisms.
AuthorsSubramanian VS, Marchant JS, Parker I, Said HM
JournalJ Biol Chem
PubMed ID12454006
'The human thiamine transporter hTHTR1 is involved in the cellular accumulation of thiamine (vitamin B1) in many tissues. Thiamine deficiency disorders, such as thiamine-responsive megaloblastic anemia (TRMA), which is associated with specific mutations within hTHTR1, likely impairs the functionality and/or intracellular targeting of hTHTR1. Unfortunately, nothing is known about the ... More