Mitsuko Hayashi

An enzymatic cascade of Rab5 effectors regulates phosphoinositide turnover in the endocytic pathway

Generation and turnover of phosphoinositides (PIs) must be coordinated in a spatial- and temporal-restricted manner. The small GTPase Rab5 interacts with two PI 3-kinases, Vps34 and PI3Kβ, suggesting that it regulates the production of 3-PIs at various stages of the early endocytic pathway. Here, we discovered that Rab5 also interacts directly with PI 5- and PI 4-phosphatases and stimulates their activity. Rab5 regulates the production of phosphatidylinositol 3-phosphate (PtdIns[3]P) through a dual mechanism, by directly phosphorylating phosphatidylinositol via Vps34 and by a hierarchical enzymatic cascade of phosphoinositide-3-kinaseβ (PI3Kβ), PI 5-, and PI 4-phosphatases. The functional importance of such an enzymatic pathway is demonstrated by the inhibition of transferrin uptake upon silencing of PI 4-phosphatase and studies in weeble mutant mice, where deficiency of PI 4-phosphatase causes an increase of PtdIns(3,4)P2 and a reduction in PtdIns(3)P. Activation of PI 3-kinase at the plasma membrane is accompanied by the recruitment of Rab5, PI 4-, and PI 5-phosphatases to the cell cortex. Our data provide the first evidence for a dual role of a Rab GTPase in regulating both generation and turnover of PIs via PI kinases and phosphatases to coordinate signaling functions with organelle homeostasis.

Cell- and stimulus-dependent heterogeneity of synaptic vesicle endocytic recycling mechanisms revealed by studies of dynamin 1-null neurons.

Mice lacking expression of dynamin 1, a GTPase implicated in the fission reaction of synaptic vesicle endocytosis, fail to thrive and exhibit severe activity-dependent endocytic defects at their synapses. Here, we have used electron tomography to investigate the massive increase in clathrin-coated pit abundance that is selectively observed at a subset of synapses in dynamin 1 KO primary neuron cultures under conditions of spontaneous network activity. This increase, leading to branched tubular plasma membrane invaginations capped by clathrin-coated buds, occurs selectively at inhibitory synapses. A similar massive increase of clathrin-coated profiles (in this case, of clathrin-coated vesicles) is observed at inhibitory synapses of neurons that lack expression of synaptojanin 1, a phosphoinositide phosphatase involved in clathrin-coated vesicle uncoating. Thus, although excitatory synapses are largely spared under these conditions, inhibitory synapses are uniquely sensitive to perturbation of endocytic proteins, probably as a result of their higher levels of tonic activity leading to a buildup of clathrin-coated intermediates in these synapses. In contrast, the predominant endocytic structures observed at the majority of dynamin 1 KO synapses after acute stimulation are endosome-like intermediates that originate by a dynamin 1-independent form of endocytosis. These findings reveal a striking heterogeneity in the mode of synaptic vesicle recycling in different synapses and functional states.

Differentiation-associated Na+-dependent Inorganic Phosphate Cotransporter (DNPI) Is a Vesicular Glutamate Transporter in Endocrine Glutamatergic Systems

Vesicular glutamate transporter is present in neuronal synaptic vesicles and endocrine synaptic-like microvesicles and is responsible for vesicular storage ofl-glutamate. A brain-specific Na+-dependent inorganic phosphate transporter (BNPI) functions as a vesicular glutamate transporter in synaptic vesicles, and the expression of this BNPI defines the glutamatergic phenotype in the central nervous system (Bellocchio, E. E., Reimer, R. J., Fremeau, R. T., Jr., and Edwards, R. H. (2000) Science 289, 957–960; Takamori, S., Rhee, J. S., Rosenmund, C., and Jahn, R. (2000) Nature 407, 189–194). However, since not all glutamatergic neurons contain BNPI, an additional transporter(s) responsible for vesicular glutamate uptake has been postulated. Here we report that differentiation-associated Na+-dependent inorganic phosphate cotransporter (DNPI), an isoform of BNPI (Aihara, Y., Mashima, H., Onda, H., Hisano, S., Kasuya, H., Hori, T., Yamada, S., Tomura, H., Yamada, Y., Inoue, I., Kojima, I., and Takeda, J. (2000) J. Neurochem.74, 2622–2625), also transports l-glutamate at the expense of an electrochemical gradient of protons established by the vacuolar proton pump when expressed in COS7 cells. Molecular, biological, and immunohistochemical studies have indicated that besides its presence in neuronal cells DNPI is preferentially expressed in mammalian pinealocytes, αTC6 cells, clonal pancreatic α cells, and α cells of Langerhans islets, these cells being proven to secretel-glutamate through Ca2+-dependent regulated exocytosis followed by its vesicular storage. Pancreatic polypeptide-secreting F cells of Langerhans islets also expressed DNPI. These results constitute evidence that DNPI functions as another vesicular transporter in glutamatergic endocrine cells as well as in neurons.

Secretory granule-mediated co-secretion of L-glutamate and glucagon triggers glutamatergic signal transmission in islets of Langerhans.

l-Glutamate is believed to function as an intercellular transmitter in the islets of Langerhans. However, critical issues, i.e. where, when and howl-glutamate appears, and what happens upon stimulation of glutamate receptors in the islets, remain unresolved. Vesicular glutamate transporter 2 (VGLUT2), an isoform of the vesicular glutamate transporter essential for neuronal storage of l-glutamate, is expressed in α cells (Hayashi, M., Otsuka, M., Morimoto, R., Hirota, S., Yatsushiro, S., Takeda, J., Yamamoto, A., and Moriyama, Y. (2001) J. Biol. Chem. 276, 43400–43406). Here we show that VGLUT2 is specifically localized in glucagon-containing secretory granules but not in synaptic-like microvesicles in αTC6 cells, clonal α cells, and islet α cells. VGLUT1, another VGLUT isoform, is also expressed and localized in secretory granules in α cells. Low glucose conditions triggered co-secretion of stoichiometric amounts ofl-glutamate and glucagon from αTC6 cells and isolated islets, which is dependent on temperature and Ca2+ and inhibited by phentolamine. Similar co-secretion ofl-glutamate and glucagon from islets was observed upon stimulation of β-adrenergic receptors with isoproterenol. Under low glucose conditions, stimulation of glutamate receptors facilitates secretion of γ-aminobutyric acid from MIN6 m9, clonal β cells, and isolated islets. These results indicate that co-secretion ofl-glutamate and glucagon from α cells under low glucose conditions triggers GABA secretion from β cells and defines the mode of action of l-glutamate as a regulatory molecule for the endocrine function. To our knowledge, this is the first example of secretory granule-mediated glutamatergic signal transmission.

The Zebrafish nrc Mutant Reveals a Role for the Polyphosphoinositide Phosphatase Synaptojanin 1 in Cone Photoreceptor Ribbon Anchoring

Visual, vestibular, and auditory neurons rely on ribbon synapses for rapid continuous release and recycling of synaptic vesicles. Molecular mechanisms responsible for the properties of ribbon synapses are mostly unknown. The zebrafish vision mutant nrc has unanchored ribbons and abnormal synaptic transmission at cone photoreceptor synapses. We used positional cloning to identify the nrc mutation as a premature stop codon in the synaptojanin1 (synj1) gene. Synaptojanin 1 (Synj1) is undetectable in nrc extracts, and biochemical activities associated with it are reduced. Furthermore, morpholinos directed against synj1 phenocopy the nrc mutation. Synj1 is a polyphosphoinositide phosphatase important at conventional synapses for clathrin-mediated endocytosis and actin cytoskeletal rearrangement. In the nrc cone photoreceptor pedicle, not only are ribbons unanchored, but synaptic vesicles are reduced in number, abnormally distributed, and interspersed within a dense cytoskeletal matrix. Our findings reveal a new role for Synj1 and link phosphoinositide metabolism to ribbon architecture and function at the cone photoreceptor synapse.

Vacuolar H+-ATPase localized in plasma membranes of malaria parasite cells, Plasmodium falciparum, is involved in regional acidification of parasitized erythrocytes

Recent biochemical studies involving 2′,7′-bis-(2-carboxyethyl)-5,6-carboxylfluorescein (BCECF)-labeled saponin-permeabilized and parasitized erythrocytes indicated that malaria parasite cells maintain the resting cytoplasmic pH at about 7.3, and treatment with vacuolar proton-pump inhibitors reduces the resting pH to 6.7, suggesting proton extrusion from the parasite cells via vacuolar H+-ATPase (Saliba, K. J., and Kirk, K. (1999) J. Biol. Chem. 274, 33213–33219). In the present study, we investigated the localization of vacuolar H+-ATPase in Plasmodium falciparum cells infecting erythrocytes. Antibodies against vacuolar H+-ATPase subunit A and Bspecifically immunostained the infecting parasite cells and recognized a single 67- and 55-kDa polypeptide, respectively. Immunoelectron microscopy indicated that the immunological counterpart of V-ATPase subunits A and B is localized at the plasma membrane, small clear vesicles, and food vacuoles, a lower extent being detected at the parasitophorus vacuolar membrane of the parasite cells. We measured the cytoplasmic pH of both infected erythrocytes and invading malaria parasite cells by microfluorimetry using BCECF fluorescence. It was found that a restricted area of the erythrocyte cytoplasm near a parasite cell is slightly acidic, being about pH 6.9. The pH increased to pH 7.3 upon the addition of either concanamycin B or bafilomycin A1, specific inhibitors of vacuolar H+-ATPase. Simultaneously, the cytoplasmic pH of the infecting parasite cell decreased from pH 7.3 to 7.1. Neither vanadate at 0.5 mm, an inhibitor of P-type H+-ATPase, nor ethylisopropylamiloride at 0.2 mm, an inhibitor of Na+/H+-exchanger, affected the cytoplasmic pH of erythrocytes or infecting parasite cells. These results constitute direct evidence that plasma membrane vacuolar H+-ATPase is responsible for active extrusion of protons from the parasite cells.

Secretion of L-glutamate from osteoclasts through transcytosis

Osteoclasts are involved in the catabolism of the bone matrix and eliminate the resulting degradation products through transcytosis, but the molecular mechanism and regulation of transcytosis remain poorly understood. Upon differentiation, osteoclasts express vesicular glutamate transporter 1 (VGLUT1), which is essential for vesicular storage and subsequent exocytosis of glutamate in neurons. VGLUT1 is localized in transcytotic vesicles and accumulates L‐glutamate. Osteoclasts secrete L‐glutamate and the bone degradation products upon stimulation with KCl or ATP in a Ca2+‐dependent manner. KCl‐ and ATP‐dependent secretion of L‐glutamate was absent in osteoclasts prepared from VGLUT1−/− knockout mice. Osteoclasts express mGluR8, a class III metabotropic glutamate receptor. Its stimulation by a specific agonist inhibits secretion of L‐glutamate and bone degradation products, whereas its suppression by a specific antagonist stimulates bone resorption. Finally, it was found that VGLUT1−/− mice develop osteoporosis. Thus, in bone‐resorbing osteoclasts, L‐glutamate and bone degradation products are secreted through transcytosis and the released L‐glutamate is involved in autoregulation of transcytosis. Glutamate signaling may play an important role in the bone homeostasis.

Expression and Localization of Vesicular Glutamate Transporters in Pancreatic Islets, Upper Gastrointestinal Tract, and Testis

The wide-ranging expression of glutamate receptors in peripheral tissues suggests an unexpectedly wider role(s) of l-glutamate as an intercellular signaling molecule. However, the peripheral glutamatergic system is poorly understood, partly because the sites of l-glutamate signal appearance are less well characterized. Vesicular glutamate transporters (VGLUTs) are potential probes for the sites of vesicular storage and subsequent secretion of l-glutamate. In this study we raised specific polyclonal antibodies against two VGLUT isoforms, VGLUT1 and VGLUT2, and investigated their localization in peripheral tissues of rat. We detected the expression of either VGLUT1 or VGLUT2, or both, in pancreas, stomach, intestine, and testis. In pancreas, VGLUT1 and VGLUT2 are present in pancreatic polypeptide-containing secretory granules in F-cells in the islets of Langerhans. In stomach, VGLUT2 is abundant in the antrum and pylorus and is present in a subset of pancreatic polypeptide-containing cells. In intestine, VGLUT2 is abundant in the ileum and is co-localized with glucagon-like immunoreactive peptide and polypeptide YY (PYY). In testis, VGLUT2 is expressed and localized in the outer acrosomal membrane of spermatids, where KA1 and GluR5, kainate receptor subunits, are almost always localized. Taken together, these results strongly suggest the occurrence of a peripheral glutamatergic system in the gastroenteropancreatic system and testis.

Co-expression of vesicular glutamate transporters (VGLUT1 and VGLUT2) and their association with synaptic-like microvesicles in rat pinealocytes

A vesicular glutamate transporter (VGLUT) is responsible for the accumulation of l‐glutamate in synaptic vesicles in glutamatergic neurons. Two isoforms, VGLUT1 and VGLUT2, have been identified, which are complementarily expressed in these neurons. Mammalian pinealocytes, endocrine cells for melatonin, are also glutamatergic in nature, accumulate l‐glutamate in synaptic‐like microvesicles (SLMVs), and secrete it through exocytosis. Although the storage of l‐glutamate in SLMVs is mediated through a VGLUT, the molecular nature of the transporter is less understood. We recently observed that VGLUT2 is expressed in pinealocytes. In the present study, we show that pinealocytes also express VGLUT1. RT–PCR and northern blot analyses indicated expression of the VGLUT1 gene in pineal gland. Western blotting with specific antibodies against VGLUT1 indicated the presence of VGLUT1 in pineal gland. Indirect immunofluorescence microscopy with a section of pineal gland and cultured cells indicated that VGLUT1 and VGLUT2 are co‐localized with process terminal regions of pinealocytes. Furthermore, immunoelectronmicroscopy as well as subcellular fractionation studies revealed that both VGLUT1 and VGLUT2 are specifically associated with SLMVs. These results indicate that both VGLUTs are responsible for storage of l‐glutamate in SLMVs in pinealocytes. Pinealocytes are the first exception as to complementary expression of VGLUT1 and VGLUT2.

Vesicular Monoamine Transporter 1 Is Responsible for Storage of 5-Hydroxytryptamine in Rat Pinealocytes

Abstract : Vesicular monoamine transporters (VMATs) are involved in chemical transduction in monoaminergic neurons and various endocrine cells through the storage of monoamines in secretory vesicles. Mammalian pinealocytes contain more 5‐hydroxytryptamine (5‐HT) than any other cells and are expected on contain VMAT, although no information is available so far. Upon the addition of ATP, radiolabeled 5‐HT was taken up by a particulate fraction prepared from cultured rat pinealocytes. The 5‐HT uptake was inhibited significantly by bafilomycin A1 (an inhibitor of vacuolar H+‐ATPase), 3,5‐di‐tert‐butyl‐4‐hydroxybenzyli‐denemalononitrile (a proton conductor), or reserpine (an inhibitor of VMAT). RT‐PCR analysis suggested that VMAT type 1 (VMAT1), but not type 2, is expressed. Antibodies against VMAT1 recognized a single polypeptide with an apparent molecular mass of ~55 kDa, and specifically immunostained pinealocytes. VMAT1 immunoreactivity was high in the vesicular structures in the varicosities of long branching processes and was associated with 5‐HT, but not with synaptophysin, a marker protein for microvesicles. The 5‐HT immunoreactivity in the long branching processes disappeared upon incubation with reserpine. These results indicate that 5‐HT, at least in part, is stored in vesicles other than microvesicles in pinealocytes through a mechanism similar to that of various secretory vesicles.