Pyridinium Dibromide Research Articles

Endocytosis Is Crucial for Cell Polarity and Apical Membrane Recycling in the Filamentous Fungus Aspergillus oryzae

Establishing the occurrence of endocytosis in filamentous fungi was elusive in the past mainly due to the lack of reliable indicators of endocytosis. Recently, however, it was shown that the fluorescent dye N-(3-triethylammoniumpropyl)-4-(p-diethyl-aminophenyl-hexatrienyl)pyridinium dibromide (FM4-64) and the plasma membrane protein AoUapC (Aspergillus oryzae UapC) fused to enhanced green fluorescent protein (EGFP) were internalized from the plasma membrane by endocytosis. Although the occurrence of endocytosis was clearly demonstrated, its physiological importance in filamentous fungi still remains largely unaddressed. We generated a strain in which A. oryzae end4 (Aoend4), the A. oryzae homolog of Saccharomyces cerevisiae END4/SLA2, was expressed from the Aoend4 locus under the control of a regulatable thiA promoter. The growth of this strain was severely impaired, and its hyphal morphology was altered in the Aoend4-repressed condition. Moreover, in the Aoend4-repressed condition, neither FM4-64 nor AoUapC-EGFP was internalized, indicating defective endocytosis. Furthermore, the localization of a secretory soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) was abnormal in the Aoend4-repressed condition. Aberrant accumulation of cell wall components was also observed by calcofluor white staining and transmission electron microscopy analysis, and several genes that encode cell wall-building enzymes were upregulated, indicating that the regulation of cell wall synthesis is abnormal in the Aoend4-repressed condition, whereas Aopil1 disruptants do not display the phenotype exhibited in the Aoend4-repressed condition. Our results strongly suggest that endocytosis is crucial for the hyphal tip growth in filamentous fungi.

Synapsin IIa Controls the Reserve Pool of Glutamatergic Synaptic Vesicles

Synapsins regulate synaptic transmission by controlling the reserve pool of synaptic vesicles. Each of the three mammalian synapsin genes is subject to alternative splicing, yielding several isoforms whose roles are unknown. To investigate the function of these isoforms, we examined the synaptic effects of introducing each isoform into glutamatergic cultured hippocampal neurons from synapsin triple knock-out mice. Remarkably, we found that synapsin IIa was the only isoform that could rescue the synaptic depression phenotype of the triple knock-out mice; other isoforms examined, including the well-studied synapsin Ia isoform, had no significant effect on the kinetics of synaptic depression. The slowing of synaptic depression by synapsin IIa was quantitatively paralleled by an increase in the density of reserve pool synaptic vesicles, as measured either by fluorescent tagging of the vesicle protein synaptobrevin-2 or by staining with the styryl dye FM4-64 [N-(3-triethylammoniumpropyl)-4-(6-(4-diethylamino)phenyl)-hexatrienyl)pyridinium dibromide]. Our results provide further support for the hypothesis that synapsins define the kinetics of synaptic depression at glutamatergic synapses by controlling the size of the vesicular reserve pool and identify synapsin IIa as the isoform primarily responsible for this task.

Heterogeneity of milk fat globule membrane structure and composition as observed using fluorescence microscopy techniques

Fluorescence microscopy studies of fat globules in milk and milk products using fat-soluble stains (Nile Blue or Nile Red) allow visualisation of the neutral fat (core) of the globules, but provide no information about the milk fat globule membrane (MFGM). We applied the lipophilic probes 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine-5,5′-disulfonic acid (DiIC 18(3)-DS) and N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM4-64), as well as fluorescent conjugates of the lectin wheat germ agglutinin (WGA488, WGA594 and WGA647), to milk to stain specifically the MFGM in its native environment. Using fluorescence microscopy, we observed various distribution patterns of the probes. This demonstrates that the MFGM of fat globules in harvested milk is structurally and chemically heterogeneous both within and among globules from the same species and probably between species. We propose that the use of membrane-specific fluorescent probes has significant potential for providing real time structural and chemical information about the MFGM in matrices such as mammary gland tissue, harvested milk, and milk products.

Release of the Styryl Dyes from Single Synaptic Vesicles in Hippocampal Neurons

In small presynaptic boutons in brain, synaptic vesicles are thought not to merge with the plasma membrane when they release transmitter, but instead to close their fusion pores and survive intact for future use (kiss-and-run exocytosis). The strongest evidence for this idea is the slow and incomplete release of the fluorescent membrane marker, FM1-43 [N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide], from single vesicles. We investigated the release of FM1-43 from sparse cultures of hippocampal neurons grown on coverslips with no glia. This allowed presynaptic boutons to be imaged at favorable signal-to-noise ratio. Sparingly stained boutons were imaged at high time resolution, while high-frequency electrical stimulation caused exocytosis. The release of FM1-43 was quantal and occurred in abrupt steps, each representing a single fusion event. The fluorescence of vesicle clusters traveling along axons had a distribution with the same quantal size, indicating that a vesicle releases all the dye it contains. In most fusion events, the time constant of dye release was <100 ms, and slower release was rarely observed. After exocytosis, no FM1-43 could be detected in the axon to either side of a bouton, indicating that dye was released before it could spread. Our results are consistent with synaptic vesicles fusing fully with the plasma membrane during high-frequency stimulation.

Synaptic Vesicle Mobility in Mouse Motor Nerve Terminals with and without Synapsin

We measured synaptic vesicle mobility using fluorescence recovery after photobleaching of FM 1-43 [N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide] stained mouse motor nerve terminals obtained from wild-type (WT) and synapsin triple knock-out (TKO) mice at room temperature and physiological temperature. Vesicles were mobile in resting terminals at physiological temperature but virtually immobile at room temperature. Mobility was increased at both temperatures by blocking phosphatases with okadaic acid, decreased at physiological temperature by blocking kinases with staurosporine, and unaffected by disrupting actin filaments with latrunculin A or reducing intracellular calcium concentration with BAPTA-AM. Synapsin TKO mice showed reduced numbers of synaptic vesicles and reduced FM 1-43 staining intensity. Synaptic transmission, however, was indistinguishable from WT, as was synaptic vesicle mobility under all conditions tested. Thus, in TKO mice, and perhaps WT mice, a phospho-protein different from synapsin but otherwise of unknown identity is the primary regulator of synaptic vesicle mobility.

Vesicle Pool Heterogeneity at Hippocampal Glutamate and GABA Synapses

Glutamate and GABA are the major fast excitatory and inhibitory neurotransmitters, respectively, in the CNS. Although glutamate and GABA have clearly distinct postsynaptic actions, we are just beginning to appreciate that presynaptic differences between glutamatergic and GABAergic neurons may contribute to distinct functions of these transmitter systems. We therefore probed possible differences between the functional synaptic vesicle populations of glutamatergic and GABAergic neurons. We examined superecliptic synaptopHluorin (SpH) fluorescence during 20 Hz electrical stimulation in transfected hippocampal neurons and identified the phenotype of SpH-fluorescent synapses with post hoc immunostaining. With 200 stimuli (10 s), individual glutamate synapses displayed considerably more variability in peak SpH fluorescence than GABA synapses, without a strong difference in the mean SpH fluorescence increase. This spatial heterogeneity could not be accounted for by differences in endocytosis, which was nearly constant over these short time periods across glutamate and GABA synapses. Instead, variability in vesicle exocytosis correlated with variability in total vesicle staining and in measures of the total recycling pool size. Differences were also evident using FM1-43 [N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide] uptake. These data support the idea that the population of glutamate synapses exhibits more heterogeneity in release properties than the population of GABA synapses, possibly correlated with glutamatergic synaptic malleability.

New aspects of membrane dynamics of Amoeba proteus contractile vacuole revealed by vital staining with FM 4-64

The contractile vacuole (CV) cycle of Amoeba proteus has been studied by phase contrast and electron microscopy. However, the understanding of membrane dynamics in this cycle is still poor. In this study, we used live imaging by fluorescence microscopy to obtain new insights. We succeeded in staining the CV with a styryl dye, FM 4-64 (N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide), and obtained the following results. (1) The CV membrane was directly stained with the dye in the external medium when the CV pore opened upon contraction. This indicates that transfer of plasma membrane to the CV does not occur. (2) The membrane dynamics during the CV cycle were elucidated. In particular, the fluorescent CV membrane was maintained as an aggregate just after contraction and the vacuole re-formed from the aggregate. Staining was maintained during continued contraction cycles. We conclude that the CV membrane is maintained during the CV cycle.

High-Content Screening

High-Content Screening

UNC-31 (CAPS) Is Required for Dense-Core Vesicle But Not Synaptic Vesicle Exocytosis in Caenorhabditis elegans

Previous studies indicated that CAPS (calcium-dependent activator protein for secretion) functions as an essential component for the Ca2+-dependent exocytosis of dense-core vesicles in neuroendocrine cells. However, recent mouse knock-out studies suggested an alternative role in the vesicular uptake or storage of catecholamines. To genetically assess the functional role of CAPS, we characterized the sole Caenorhabditis elegans CAPS ortholog UNC-31 (uncoordinated family member) and determined its role in dense-core vesicle-mediated peptide secretion and in synaptic vesicle recycling. Novel assays for dense-core vesicle exocytosis were developed by expressing a prepro-atrial natriuretic factor-green fluorescent protein fusion protein in C. elegans. unc-31 mutants exhibited reduced peptide release in vivo and lacked evoked peptide release in cultured neurons. In contrast, cultured neurons from unc-31 mutants exhibited normal stimulated synaptic vesicle recycling measured by FM4-64 [N-(3-triethylammoniumpropyl)-4-(6-(4-diethylamino)phenyl)hexatrienyl)pyridinium dibromide] dye uptake. Conversely, UNC-13, which exhibits sequence homology to CAPS/UNC-31, was found to be essential for synaptic vesicle but not dense-core vesicle exocytosis. These findings indicate that CAPS/UNC-31 function is not restricted to catecholaminergic vesicles but is generally required for and specific to dense-core vesicle exocytosis. Our results suggest that CAPS/UNC-31 and UNC-13 serve parallel and dedicated roles in dense-core vesicle and synaptic vesicle exocytosis, respectively, in the C. elegans nervous system.

Dopaminergic Modulation of Local Network Activity in Rat Prefrontal Cortex

Dopamine modulates prefrontal cortex excitability in complex ways. Dopamine's net effect on local neuronal networks is therefore difficult to predict based on studies on pharmacologically isolated excitatory or inhibitory connections. In the present work, we have studied the effects of dopamine on evoked activity in acute rat brain slices when both excitation and inhibition are intact. Whole cell recordings from layer II/III pyramidal cells under conditions of normal synaptic transmission showed that bath-applied dopamine (30 microM) increased the outward inhibitory component of composite postsynaptic currents, whereas inward excitatory currents were not significantly affected. Optical imaging with the voltage-sensitive dye N-(3-(triethylammonium)propyl)-4-(4-(p-diethylaminophenyl)buta-dienyl)pyridinium dibromide revealed that bath application of dopamine significantly decreased the amplitude, duration, and lateral spread of activity in local cortical networks. This effect of dopamine was observed both with single and train (5 at 20 Hz) stimuli. The effect was mimicked by the D1-like receptor agonistR(+)-6-chloro-7,8-dihydroxy-1-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (1 microM) and was blocked by R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (10 microM), a selective antagonist for D1-like receptors. The D2-like receptor agonist quinpirole (10 microM) had no significant effect on evoked dye signals. Our results suggest that dopamine's effect on inhibition dominates over that on excitation under conditions of normal synaptic transmission. Such neuromodulation by dopamine may be important for maintenance of stability in local neuronal networks in the prefrontal cortex.

Synaptic vesicle pools at diaphragm neuromuscular junctions vary with motoneuron soma, not axon terminal, inactivity

Both spinal hemisection (SH) at C 2 and tetrodotoxin (TTX) phrenic nerve blockade result in diaphragm muscle paralysis and inactivity of the phrenic axon terminals. However, phrenic motoneuron somata are inactive with SH but remain active with TTX phrenic nerve blockade. Neuromuscular transmission failure with repeated activation decreases following SH and increases following TTX phrenic nerve blockade, suggesting that matching (or mismatching) of somal and synaptic inactivities of phrenic motoneurons differentially regulates synaptic vesicle pools at diaphragm neuromuscular junctions. At individual type-identified rat diaphragm presynaptic terminals, the size of the releasable pool of synaptic vesicles was analyzed by fluorescence confocal microscopy of N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl) pyridinium dibromide (FM4-64) uptake and synaptic vesicle density at active zones was determined using transmission electron microscopy. After 14 days of SH and TTX-induced diaphragm muscle inactivity, neuromuscular junction size was not different at type I or IIa fibers, but increased at type IIx and/or IIb fibers (by 51% in SH and 35% in TTX) compared with control. With SH, synaptic vesicle pool size and density increased at presynaptic terminals innervating type I or IIa fibers (17 and 63%, respectively; P<0.001) and type IIx and/or IIb fibers (41 and 31%, respectively; P<0.001) when compared with controls. Following TTX, synaptic vesicle pool size and density decreased by 64 and 17%, respectively, at presynaptic terminals innervating type I or IIa fibers, and by 50 and 36%, respectively, at type IIx and/or IIb fibers ( P<0.001, for all comparisons). Thus, matching motoneuron soma and axon terminal inactivity (SH) increases the size and density of releasable synaptic vesicle pools at adult rat diaphragm neuromuscular junctions. Mismatching motoneuron soma and axon terminal inactivities (TTX) results in converse presynaptic adaptations. Inactivity-induced neuromuscular plasticity reflects specific adaptations in the size and density of synaptic vesicle pools that depend on motoneuron soma rather than axon terminal (or muscle fiber) inactivity.

A plasmodesmata‐associated β‐1,3‐glucanase in Arabidopsis

Plasmodesmal conductivity is regulated in part by callose turnover, which is hypothesized to be determined by beta-1,3-glucan synthase versus glucanase activities. A proteomic analysis of an Arabidopsis thaliana plasmodesmata (Pd)-rich fraction identified a beta-1,3-glucanase as present in this fraction. The protein encoded by the putative plasmodesmal associated protein (ppap) gene, termed AtBG_ppap, had previously been found to be a post-translationally modified glycosylphosphatidylinositol (GPI) lipid-anchored protein. When fused to green fluorescent protein (GFP) and expressed in tobacco (Nicotiana tabacum) or Nicotiana benthamiana epidermal cells, this protein displays fluorescence patterns in the endoplasmic reticulum (ER) membrane system, along the cell periphery and in a punctate pattern that co-localizes with aniline blue-stained callose present around the Pd. Plasma membrane localization was verified by co-localization of AtBG_ppap:GFP together with a plasma membrane marker N-[3-triethylammoniumpropyl]-4-[p-diethylaminophenylhexatrienyl] pyridinium dibromide (FM4-64) in plasmolysed cells. In Arabidopsis T-DNA insertion mutants that do not transcribe AtBG_ppap, functional studies showed that GFP cell-to-cell movement between epidermal cells is reduced, and the conductivity coefficient of Pd is lower. Measurements of callose levels around Pd after wounding revealed that callose accumulation in the mutant plants was higher. Taken together, we suggest that AtBG_ppap is a Pd-associated membrane protein involved in plasmodesmal callose degradation, and functions in the gating of Pd.

Two types of neurotransmitter release patterns in isolectin B4-positive and negative trigeminal ganglion neurons

Mammalian nociceptors have been classified into subclasses based on differential neurotrophin sensitivity and binding of the plant isolectin B4 (IB4). Most of the nerve growth factor-responsive IB4-negative (IB4 (−)) nociceptors contain neuropeptides such as substance P and calcitonin gene-related peptide, whereas the glial-derived neurotrophic factor-responsive IB4-positive (IB4 (+)) neurons predominantly lack such neuropeptides. We hypothesized that the differences in neuropeptide content between IB4 (+) and (−) neurons might be reflected in differences in stimulated exocytosis and/or endocytosis. To address this, we monitored the secretory activity of acutely dissociated neurons from adult rat trigeminal ganglia (TRG) using cell membrane capacitance (Cm) measurements and the fluorescent membrane-uptake marker N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM4-64). Cm measurements were performed under whole-cell voltage clamp and neurons were depolarized from −75 mV to +10 mV to elicit exocytosis. Both types of TRG neurons showed similarly-sized, calcium-dependent increases in Cm, demonstrating that both IB4 (+) and (−) TRG neurons are capable of stimulated exocytosis. However, the peak Cm of IB4 (+) neurons decayed faster toward baseline than that of IB4 (−) neurons. Also, IB4 (+) neurons had stable Cm responses to repeated stimuli whereas IB4 (−) neurons loss their secretory response during repeated stimulation. These data suggested that the IB4 (+) neurons possess a faster rate of endocytosis and vesicle replenishment than IB4 (−) neurons. To test this, we measured vesicle trafficking with the fluorescent membrane dye FM4-64. FM4-64 staining showed that IB4 (−) neurons exhibit a larger pool of endocytosed vesicles than IB4 (+) neurons because the peak fluorescence increases in IB4 (−) neurons were larger but slower than in IB4 (+) neurons. However, the recycled vesicles were released faster in IB4 (+) compared with IB4 (−) neurons. Taken together these data suggest that the IB4 (+) TRG neurons have faster exocytosis and endocytosis than the IB4 (−) neurons.

Myosin Light Chain Kinase Is Not a Regulator of Synaptic Vesicle Trafficking during Repetitive Exocytosis in Cultured Hippocampal Neurons

The mechanism by which synaptic vesicles (SVs) are recruited to the release site is poorly understood. One candidate mechanism for trafficking of SVs is the myosin-actin motor system. Myosin activity is modulated by myosin light chain kinase (MLCK), which in turn is activated by calmodulin. Ca(2+) signaling in presynaptic terminals, therefore, may serve to regulate SV mobility along actin filaments via MLCK. Previous studies in different types of synapses have supported such a hypothesis. Here, we further investigated the role of MLCK in neurotransmitter release at glutamatergic synapses in cultured hippocampal neurons by examining the effects of two MLCK inhibitors, 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine.HCl (ML-7) and wortmannin. Bath application of ML-7 enhanced short-term depression of EPSCs to repetitive stimulation, whereas it reduced presynaptic release probability. However, ML-7 also inhibited action potential amplitude and voltage-gated Ca(2+) channel currents. These effects were not mimicked by wortmannin, suggesting that ML-7 was not specific to MLCK in hippocampal neurons. When SV exocytosis was directly triggered by a Ca(2+) ionophore, calcimycin, to bypass voltage-gated Ca(2+) channels, ML-7 had no effect on neurotransmitter release. Furthermore, when SV exocytosis elicited by electrical field stimulation was monitored by styryl dye, FM1-43 [N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide], the unloading kinetics of the dye was not altered in the presence of wortmannin. These data indicate that MLCK is not a major regulator of presynaptic SV trafficking during repetitive exocytosis at hippocampal synapses.

NMDA-Dependent, But Not Group I Metabotropic Glutamate Receptor-Dependent, Long-Term Depression at Schaffer Collateral–CA1 Synapses Is Associated with Long-Term Reduction of Release from the Rapidly Recycling Presynaptic Vesicle Pool

Postsynaptic alterations have been suggested to account for NMDA receptor (NMDAR)-dependent long-term depression (LTD) and long-term potentiation of synaptic strength, although there is substantial evidence supporting changes in presynaptic release. Direct chemical activation of either NMDA or group I metabotropic glutamate receptor (mGluR1) elicits LTD of similar magnitudes, but it is unknown whether they share common expression mechanisms. Using dual-photon laser-scanning microscopy of FM1-43 [N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide] to directly visualize presynaptic vesicular release from the rapidly recycling vesicle pool (RRP) at Schaffer collateral terminals in field CA1 of rat hippocampal slices, we found that a persistent reduction in vesicular release from the RRP is induced by NMDA-LTD but not by mGluR1-LTD. Variance-mean analyses of Schaffer collateral release probability (P(r)) at varying extracellular calcium concentrations confirmed that NMDA-LTD was associated with reduced P(r), whereas mGluR1-LTD was not. Pharmacological isolation of NMDAR-dependent and mGluR-dependent forms of stimulus-evoked LTD revealed that both are composed of a combination of presynaptic and postsynaptic alterations. However, when group I mGluR-dependent LTD was isolated by combining an NMDAR blocker with a group II mGluR antagonist, this form of LTD was purely postsynaptic. The nitric oxide synthase inhibitor N omega-nitro-L-arginine blocked the induction of NMDA-LTD but did not alter mGluR-LTD, consistent with a selective role for nitric oxide as a retrograde messenger mediating NMDA-LTD. These data demonstrate that single synapses can express multiple forms of LTD with different sites of expression, that NMDA-LTD is a combination of presynaptic and postsynaptic alterations, but that group I mGluR-LTD appears to be expressed entirely postsynaptically.

The Very Large G-Protein-Coupled Receptor VLGR1: A Component of the Ankle Link Complex Required for the Normal Development of Auditory Hair Bundles

Sensory hair bundles in the inner ear are composed of stereocilia that can be interconnected by a variety of different link types, including tip links, horizontal top connectors, shaft connectors, and ankle links. The ankle link antigen is an epitope specifically associated with ankle links and the calycal processes of photoreceptors in chicks. Mass spectrometry and immunoblotting were used to identify this antigen as the avian ortholog of the very large G-protein-coupled receptor VLGR1, the product of the Usher syndrome USH2C (Mass1) locus. Like ankle links, Vlgr1 is expressed transiently around the base of developing hair bundles in mice. Ankle links fail to form in the cochleae of mice carrying a targeted mutation in Vlgr1 (Vlgr1/del7TM), and the bundles become disorganized just after birth. FM1-43 [N-(3-triethylammonium)propyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide] dye loading and whole-cell recordings indicate mechanotransduction is impaired in cochlear, but not vestibular, hair cells of early postnatal Vlgr1/del7TM mutant mice. Auditory brainstem recordings and distortion product measurements indicate that these mice are severely deaf by the third week of life. Hair cells from the basal half of the cochlea are lost in 2-month-old Vlgr1/del7TM mice, and retinal function is mildly abnormal in aged mutants. Our results indicate that Vlgr1 is required for formation of the ankle link complex and the normal development of cochlear hair bundles.

Regionalization of Plasma Membrane-Bound Flavoproteins of Cerebellar Granule Neurons in Culture by Fluorescence Energy Transfer Imaging

Flavoproteins are components of plasma membrane redox chains, which have been suggested to play major roles in neuronal activity and survival. We found that the red/orange autofluorescence of mature primary cultures of cerebellar granule neurons (8-9 days in vitro) was largely quenched by millimolar concentrations of dithionite added to the extracellular medium, and pointed out that nearly 50% of this autofluorescence was due to plasma membrane-bound flavoproteins. We report in this work that the lipophilic neuronal plasma membrane markers N-(3-triethylammoniumpropyl)-4-(4-(4-(diethylamino)phenyl)butadienyl)-pyridinium dibromide (RH-414) and N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM4-64) can form fluorescence energy transfer donor-acceptor pairs with flavoproteins with calculated R (0) values between 3.7 and 4.2 nm. The quantification of the efficiency of fluorescence energy transfer with different concentrations of acceptor dyes has been worked out with re-suspended neurons. Using quantitative images of the neurons in culture, acquired with a CCD camera attached to an epifluorescence microscope, regionalization of the plasma membrane-bound flavoproteins of cerebellar granule neurons has been achieved from the quenching by dithionite of the fluorescence of the acceptor dye. The results unraveled that plasma membrane-bound flavoproteins are largely enriched in interneuronal contact sites forming clusters of 0.5-1 microm diameter size, which appears largely regionalized in the neuron's cell body.

A class Vb chitin synthase in Colletotrichum graminicola is localized in the growing tips of multiple cell types, in nascent septa, and during septum conversion to an end wall after hyphal breakage

Previous complementation of a chitin synthase class Vb null mutant (Colletotrichum graminicola chsA) indicated that the encoded protein is responsible for approximately 30% of the conidial chitin, is essential for conidial wall strength in media with high water potential, and contributes to strength of hyphal tips. We complemented a chsA null mutant with chsA fused to the green-fluorescent protein (sgfp) gene driven by a heterologous constitutively expressed promoter. Comparisons of the strain with the ectopic chsA-sgfp to the wild type indicated that ChsA-sGFP serves the same biological functions as ChsA in that like the wild type, the chsADelta chsA::sgfp (EC) had conidia that did not explode and hyphal tips that did not swell. Confocal microscopy of ChsA-sGFP (EC) cells stained with the membrane stain FM 4-64 (N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide) indicated that ChsA is localized in the plasma membrane of the following: growing apices of hyphal branches, conidiophores, and falcate and oval conidia; in nascent septa; and in septa that are being converted to an end wall after hyphal breakage. The data support the hypothesis that chsA either directly or indirectly encodes the information for its localization, that ChsA is localized in the plasma membrane, and that the class Vb enzyme produces chitin synthase in multiple cells and after wall breakage.

Vacuolar membrane dynamics in the filamentous fungus Aspergillus oryzae.

Vacuoles in filamentous fungi are highly pleomorphic and some of them, e.g., tubular vacuoles, are implicated in intra- and intercellular transport. In this report, we isolated Aovam3, the homologue of the Saccharomyces cerevisiae VAM3 gene that encodes the vacuolar syntaxin, from Aspergillus oryzae. In yeast complementation analyses, the expression of Aovam3 restored the phenotypes of both Deltavam3 and Deltapep12 mutants, suggesting that AoVam3p is likely the vacuolar and/or endosomal syntaxin in A. oryzae. FM4-64 [N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenyl-hexatrienyl)pyridinium dibromide] and CMAC (7-amino-4-chloromethylcoumarin) staining confirmed that the fusion protein of enhanced green fluorescent protein (EGFP) with AoVam3p (EGFP-AoVam3p) localized on the membrane of the pleomorphic vacuolar networks, including large spherical vacuoles, tubular vacuoles, and putative late endosomes/prevacuolar compartments. EGFP-AoVam3p-expressing strains allowed us to observe the dynamics of vacuoles with high resolutions, and moreover, led to the discovery of several new aspects of fungal vacuoles, which have not been discovered so far with conventional staining methods, during different developmental stages. In old hyphae, EGFP fluorescence was present in the entire lumen of large vacuoles, which occupied most of the cell, indicating that degradation of cytosolic materials had occurred in such hyphae via an autophagic process. In hyphae that were not in contact with nutrients, such as aerial hyphae and hyphae that grew on a glass surface, vacuoles were composed of small punctate structures and tubular elements that often formed reticulum-like networks. These observations imply the presence of so-far-unrecognized roles of vacuoles in the development of filamentous fungi.

Targeting and Trafficking of the Human Thiamine Transporter-2 in Epithelial Cells

Humans lack biochemical pathways for thiamine synthesis, so cellular requirements are met via specific carrier-mediated uptake pathways. Two proteins from the solute carrier SLC19A gene family have been identified as human thiamine transporters (hTHTRs), SLC19A1 (hTHTR1) and SLC19A2 (hTHTR2). Both of these transporters are co-expressed but are differentially targeted in polarized cell types that mediate vectorial thiamine transport (e.g. renal and intestinal epithelia). It is important to understand the domain structure of these proteins, namely which regions within the polypeptide sequence are important for physiological delivery to the cell surface, in order to understand the impact of clinically relevant mutations on thiamine transport. Here we have characterized the mechanisms regulating hTHTR2 distribution by using live cell imaging methods that resolve the targeting and trafficking dynamics of full-length hTHTR2, a series of hTHTR2 truncation mutants, as well as chimeras comprising the hTHTR1 and hTHTR2 sequence. We showed the following: (i) that the cytoplasmic COOH-tail of hTHTR2 is not essential for apical targeting in polarized cells; (ii) that delivery of hTHTR2 to the cell surface is critically dependent on the integrity of the transmembrane backbone of the polypeptide so that minimal truncations abrogate cell surface expression of hTHTR2; and (iii) video rate images of hTHTR2-containing intracellular vesicles displayed rapid bi-directional trafficking events to and from the cell surface impaired by microtubule-disrupting but not microfilament-disrupting agents as well as by overexpression of the dynactin subunit dynamitin (p50). Finally, we compared the behavior of hTHTR2 with that of hTHTR1 and the human reduced folate carrier (SLC19A1) to underscore commonalities in the cell surface targeting mechanisms of the entire SLC19A gene family.