The fluidic channel in the flexible probe has three functions: (i) to inject chemicals into the tissues, (ii) to measure the neural activities from the tissues, and (iii) to improve the mechanical stiffness of the probe by filling the channel with a solid material. A 10-microm-thick microfluidic channel was embedded into the probe by using sacrificial photoresist patterns. Polyethylene glycol (PEG), which is solid at room temperature and dissolves when in contact with water, was used to fill the channel and increase the stiffness of the probe before insertion into the tissue. The impedance of the electrode inside the fluidic channel was around 100 kOmega at 1 kHz when the channel was filled with saline solution. We were able to insert the probe into a rat's brain and measure the neural signals with the electrode.

Vesicular glutamate transporters (VGLUTs) are responsible for the vesicular storage of L-glutamate and play an essential role in glutamatergic signal transmission in the central nervous system. The molecular mechanism of the transport remains unknown. Here, we established a novel in vitro assay procedure, which includes purification of wild and mutant VGLUT2 and their reconstitution with purified bacterial F o F 1 -ATPase (F-ATPase) into liposomes. Upon the addition of ATP, the proteoliposomes facilitated L-glutamate uptake in a membrane potential (⌬)-dependent fashion. The ATP-dependent L-glutamate uptake exhibited an absolute requirement for ϳ4 mM Cl ؊ , was sensitive to Evans blue, but was insensitive to D,L-aspartate. VGLUT2s with mutations in the transmembrane-located residues Arg 184 , His 128 , and Glu 191 showed a dramatic loss in L-glutamate transport activity, whereas Na ؉ -dependent inorganic phosphate (P i ) uptake remained comparable to that of the wild type. Furthermore, P i transport did not require Cl ؊ and was not inhibited by Evans blue. Thus, VGLUT2 appears to possess two intrinsic transport machineries that are independent of each other: a ⌬-dependent L-glutamate uptake and a Na ؉ -dependent P i uptake.Vesicular storage and subsequent exocytosis of L-glutamate is the major pathway for excitatory signal transmission in the central nervous system (1-3). Vesicular glutamate transporters (VGLUTs) 2 are essential for the vesicular storage of L-glutamate through active transport of L-glutamate into synaptic vesicles at the expense of ⌬H ϩ established by vacuolar H ϩ -ATPase (V-ATPase) (1). There are three isoforms of VGLUT, denoted VGLUT1, VGLUT2, and VGLUT3 on the basis of the order of their discovery (2, 4 -6). VGLUT1 and VGLUT2 show a complementary expression pattern in essentially all known glutamatergic neurons, suggesting that the two VGLUTs are involved in glutamatergic neurotransmission (7-9). In fact, VGLUT1 knock-out mice exhibit a loss of secretion of L-glutamate and glutamatergic neurotransmission in neurons that normally express VGLUT1 (4, 10). In contrast, VGLUT3 is expressed in neurons that are usually classified as non-glutamatergic neurons and astrocytes suggesting the involvement of VGLUT3 in a novel mode of L-glutamate signaling (11-13). VGLUTs are also expressed in peripheral nonneuronal cells, associated with a wide variety of secretory vesicles and are responsible for glutamate-mediated regulation in various cellular processes (5).VGLUTs belong to the SLC17/type I anion transport family, one of the major facilitator superfamilies (MFS), and are not related to other neurotransmitter transporters such as vesicular acetylcholine transporter and vesicular monoamine transporter (2, 14). VGLUT exhibits unique transport properties when compared with other vesicular neurotransmitter transporters. For one, VGLUT is activated by low concentrations of Cl Ϫ (ϳ4 mM) through a putative Cl Ϫ binding site (15-18). Furthermore, VGLUT requires membrane potential (positive inside) as a driving...

Amphiphysin is a major dynamin-binding partner at the synapse; however, its function in fission is unclear. Incubation of large unilamellar liposomes with mice brain cytosol led to massive formation of small vesicles, whereas cytosol of amphiphysin 1 knockout mice was much less efficient in this reaction. Vesicle formation from large liposomes by purified dynamin was also strongly enhanced by amphiphysin. In the presence of liposomes, amphiphysin strongly affected dynamin GTPase activity and the recruitment of dynamin to the liposomes, but this activity was highly dependent on liposome size. Deletion from amphiphysin of its central proline-rich stretch dramatically potentiated its effect on dynamin, possibly by relieving an inhibitory intramolecular interaction. These results suggest a model in which maturation of endocytic pits correlates with the oligomerization of dynamin with either amphiphysin or other proteins with similar domain structure. Formation of these complexes is coupled to the activation of dynamin GTPase activity, thus explaining how deep invagination of the pit leads to fission.

Dynamin GTPase, a key molecule in endocytosis, mechanically severs the invaginated membrane upon GTP hydrolysis. Dynamin functions also in regulating actin cytoskeleton, but the mechanisms are yet to be defined. Here we show that dynamin 1, a neuronal isoform of dynamin, and cortactin form ring complexes, which twine around F-actin bundles and stabilize them. By negative-staining EM, dynamin 1-cortactin complexes appeared as "open" or "closed" rings depending on guanine nucleotide conditions. By pyrene actin assembly assay, dynamin 1 stimulated actin assembly in mouse brain cytosol. In vitro incubation of F-actin with both dynamin 1 and cortactin led to the formation of long and thick actin bundles, on which dynamin 1 and cortactin were periodically colocalized in puncta. A depolymerization assay revealed that dynamin 1 and cortactin increased the stability of actin bundles, most prominently in the presence of GTP. In rat cortical neurons and human neuroblastoma cell line, SH-SY5Y, both dynamin 1 and cortactin localized on actin filaments and the bundles at growth cone filopodia as revealed by immunoelectron microscopy. In SH-SY5Y cell, acute inhibition of dynamin 1 by application of dynamin inhibitor led to growth cone collapse. Cortactin knockdown also reduced growth cone filopodia. Together, our results strongly suggest that dynamin 1 and cortactin ring complex mechanically stabilizes F-actin bundles in growth cone filopodia. Thus, the GTPase-dependent mechanochemical enzyme property of dynamin is commonly used both in endocytosis and regulation of F-actin bundles by a dynamin 1-cortactin complex.

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SummaryPeroxisome division is regulated by several factors, termed fission factors, as well as the conditions of the cellular environment. Over the past decade, the idea of metabolic control of peroxisomal morphogenesis has been postulated, but remains largely undefined to date. In the current study, docosahexaenoic acid (DHA, C22:6n-3) was identified as an inducer of peroxisome division. In fibroblasts isolated from patients that carry defects in peroxisomal fatty acid b-oxidation, peroxisomes are much less abundant than normal cells. Treatment of these patient fibroblasts with DHA induced the proliferation of peroxisomes to the level seen in normal fibroblasts. DHA-induced peroxisomal proliferation was abrogated by treatment with a small inhibitory RNA (siRNA) targeting dynamin-like protein 1 and with dynasore, an inhibitor of dynamin-like protein 1, which suggested that DHA stimulates peroxisome division. DHA augmented the hyperoligomerization of Pex11pb and the formation of Pex11pb-enriched regions on elongated peroxisomes. Time-lapse imaging analysis of peroxisomal morphogenesis revealed a sequence of steps involved in peroxisome division, including elongation in one direction followed by peroxisomal fission. DHA enhanced peroxisomal division in a microtubule-independent manner. These results suggest that DHA is a crucial signal for peroxisomal elongation, a prerequisite for subsequent fission and peroxisome division.

This article introduces DoBISCUIT (Database of BIoSynthesis clusters CUrated and InTegrated, http://www.bio.nite.go.jp/pks/), a literature-based, manually curated database of gene clusters for secondary metabolite biosynthesis. Bacterial secondary metabolites often show pharmacologically important activities and can serve as lead compounds and/or candidates for drug development. Biosynthesis of each secondary metabolite is catalyzed by a number of enzymes, usually encoded by a gene cluster. Although many scientific papers describe such gene clusters, the gene information is not always described in a comprehensive manner and the related information is rarely integrated. DoBISCUIT integrates the latest literature information and provides standardized gene/module/domain descriptions related to the gene clusters.