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Organelle-localizable small-molecule ligands are valuable tools for spatiotemporally controlling protein localization and signaling processes in living cells. Here, we present synthetic ligands that specifically localize to the Golgi surface. The ligands can rapidly recruit their target proteins from the cytoplasm to the Golgi and be applied to manipulate signaling proteins and lipids on the Golgi membrane, offering a new useful chemical tool for the study and control of Golgi/cell functions.

DOI： 10.1039/d0cc06908f

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The growth cone is an essential structure for nerve growth. Although its membrane and cytoskeleton are likely to interact coordinately during nerve growth, the mechanisms are unknown due to their close proximity. Here, we used superresolution microscopy to simultaneously observe vesicles and F-actin in growth cones. We identified a novel vesicular generation mechanism that is independent of clathrin and dependent on endophilin-3-and dynamin-1 and that occurs proximal to the leading edge simultaneously with fascin-1-dependent F-actin bundling. In contrast to conventional clathrin-dependent endocytosis, which occurs distal from the leading edge at the basal surfaces of growth cones, this mechanism was distinctly observed at the apical surface using 3D imaging and was involved in mediating axon growth. Reduced endophilin or fascin inhibited this endocytic mechanism. These results suggest that, at the leading edge, vesicles are coordinately generated and transported with actin bundling during nerve growth.

DOI： 10.1016/j.celrep.2017.02.008

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Rhythmicity is prevalent in the cortical dynamics of diverse single and multicellular systems. Current models of cortical oscillations focus primarily on cytoskeleton-based feedbacks, but information on signals upstream of the actin cytoskeleton is limited. In addition, inhibitory mechanisms-especially local inhibitory mechanisms, which ensure proper spatial and kinetic controls of activation-are not well understood. Here, we identified two phosphoinositide phosphatases, synaptojanin 2 and SHIP1, that function in periodic traveling waves of rat basophilic leukemia (RBL) mast cells. The local, phase-shifted activation of lipid phosphatases generates sequential waves of phosphoinositides. By acutely perturbing phosphoinositide composition using optogenetic methods, we showed that pulses of PtdIns(4,5)P-2 regulate the amplitude of cyclic membrane waves while PtdIns(3,4)P-2 sets the frequency. Collectively, these data suggest that the spatiotemporal dynamics of lipid metabolism have a key role in governing cortical oscillations and reveal how phosphatidylinositol 3-kinases (PI3K) activity could be frequency encoded by a phosphatase-dependent inhibitory reaction.

DOI： 10.1038/NCHEMBIO.2000

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How phosphoinositide metabolism is coupled to primary cilia physiology is poorly understood. Reporting recently in Developmental Cell, Chavez et al. (2015) and Garcia-Gonzalo et al. (2015) show that INPP5E-mediated phosphoinositide metabolism, which creates a specific phosphoinositide distribution, ensures proper protein trafficking and Hh signaling in primary cilia.

DOI： 10.1016/j.devcel.2015.08.008

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Axonal growth and neuronal rewiring facilitate functional recovery after spinal cord injury. Known interventions that promote neural repair remain limited in their functional efficacy. To understand genetic determinants of mammalian CNS axon regeneration, we completed an unbiased RNAi gene-silencing screen across most phosphatases in the genome. We identified one known and 17 previously unknown phosphatase suppressors of injury-induced CNS axon growth. Silencing Inpp5f (Sac2) leads to robust enhancement of axon regeneration and growth cone reformation. Results from cultured Inpp5f(-/-) neurons confirm lentiviral shRNA results from the screen. Consistent with the nonoverlapping substrate specificity between Inpp5f and PTEN, rapamycin does not block enhanced regeneration in Inpp5f(-/-) neurons, implicating mechanisms independent of the PI3K/AKT/mTOR pathway. Inpp5f(-/-) mice develop normally, but show enhanced anatomical and functional recovery after mid-thoracic dorsal hemisection injury. More serotonergic axons sprout and/or regenerate caudal to the lesion level, and greater numbers of corticospinal tract axons sprout rostral to the lesion. Functionally, Inpp5f-null mice exhibit enhanced recovery of motor functions in both open-field and rotarod tests. This study demonstrates the potential of an unbiased high-throughput functional screen to identify endogenous suppressors of CNS axon growth after injury, and reveals Inpp5f (Sac2) as a novel suppressor of CNS axon repair after spinal cord injury.

DOI： 10.1523/JNEUROSCI.1718-15.2015

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Lipid transfer between cell membrane bilayers at contacts between the endoplasmic reticulum (ER) and other membranes help to maintain membrane lipid homeostasis. We found that two similar ER integral membrane proteins, oxysterol-binding protein (OSBP)-related protein 5 (ORP5) and ORP8, tethered the ER to the plasma membrane (PM) via the interaction of their pleckstrin homology domains with phosphatidylinositol 4-phosphate (PI4P) in this membrane. Their OSBP-related domains (ORDs) harbored either PI4P or phosphatidylserine (PS) and exchanged these lipids between bilayers. Gain-and loss-of-function experiments showed that ORP5 and ORP8 could mediate PI4P/PS countertransport between the ER and the PM, thus delivering PI4P to the ER-localized PI4P phosphatase Sac1 for degradation and PS from the ER to the PM. This exchange helps to control plasma membrane PI4P levels and selectively enrich PS in the PM.

DOI： 10.1126/science.aab1370

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The recruitment of inositol phosphatases to endocytic membranes mediates dephosphorylation of PI(4,5)P-2, a phosphoinositide concentrated in the plasma membrane, and prevents its accumulation on endosomes. The importance of the conversion of PI(4,5)P-2 to PtdIns during endocytosis is demonstrated by the presence of both a 5-phosphatase and a 4-phosphatase (Sac domain) module in the synaptojanins, endocytic PI(4,5)P-2 phosphatases conserved from yeast to humans and the only PI(4,5)P-2 phosphatases in yeast. OCRL, another 5-phosphatase that couples endocytosis to PI(4,5)P-2 dephosphorylation, lacks a Sac domain. Here we show that Sac2/INPP5F is a PI4P phosphatase that colocalizes with OCRL on endocytic membranes, including vesicles formed by clathrin-mediated endocytosis, macropinosomes, and Rab5 endosomes. An OCRL-Sac2/INPP5F interaction could be demonstrated by coimmunoprecipitation and was potentiated by Rab5, whose activity is required to recruit Sac2/INPP5F to endosomes. Sac2/INPP5F and OCRL may cooperate in the sequential dephosphorylation of PI(4,5)P-2 at the 5 and 4 position of inositol in a partnership that mimics that of the two phosphatase modules of synaptojanin.

DOI： 10.1083/jcb.201409064

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Plasma membrane PI4P is an important direct regulator of many processes that occur at the plasma membrane and also a biosynthetic precursor of PI(4,5)P2 and its downstream metabolites. The majority of this PI4P pool is synthesized by an evolutionarily conserved complex, which has as its core the PI 4-kinase PI4KIIIalpha (Stt4 in yeast) and also comprises TTC7 (Ypp1 in yeast) and the peripheral plasma membrane protein EFR3. While EFR3 has been implicated in the recruitment of PI4KIIIalpha via TTC7, the plasma membrane protein Sfk1 was also shown to participate in this targeting and activity in yeast. Here, we identify a member of the TMEM150 family as a functional homologue of Sfk1 in mammalian cells and demonstrate a role for this protein in the homeostatic regulation of PI(4,5)P2 at the plasma membrane. We also show that the presence of TMEM150A strongly reduces the association of TTC7 with the EFR3-PI4KIIIalpha complex, without impairing the localization of PI4KIIIalpha at the plasma membrane. Collectively our results suggest a plasticity of the molecular interactions that control PI4KIIIalpha localization and function.

DOI： 10.15252/embr.201439151

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The selective transport of proteins or lipids by vesicular transport is a fundamental process supporting cellular physiology. The budding process involves cargo sorting and vesicle formation at the donor membrane and constitutes an important process in vesicular transport. This process is particularly important for the polarized sorting in epithelial cells, in which the cargo molecules need to be selectively sorted and transported to two distinct destinations, the apical or basolateral plasma membrane. Adaptor protein (AP)-1, a member of the AP complex family, which includes the ubiquitously expressed AP-1A and the epithelium-specific AP-1B, regulates polarized sorting at the trans-Golgi network and/or at the recycling endosomes. A growing body of evidence, especially from studies using model organisms and animals, demonstrates that the AP-1-mediated polarized sorting supports the development and physiology of multi-cellular units as functional organs and tissues (e.g., cell fate determination, inflammation and gut immune homeostasis). Furthermore, a possible involvement of AP-1B in the pathogenesis of human diseases, such as Crohn’s disease and cancer, is now becoming evident. These data highlight the significant contribution of AP-1 complexes to the physiology of multicellular organisms, as master regulators of polarized sorting in epithelial cells.

DOI： 10.3390/membranes4040747

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The Dot/Icm system of the intracellular pathogen Legionella pneumophila has the capacity to deliver over 270 effector proteins into host cells during infection. Important questions remain as to spatial and temporal mechanisms used to regulate such a large array of virulence determinants after they have been delivered into host cells. Here we investigated several L. pneumophila effector proteins that contain a conserved phosphatidylinositol-4-phosphate (PI4P)-binding domain first described in the effector DrrA (SidM). This PI4P binding domain was essential for the localization of effectors to the early L. pneumophila-containing vacuole (LCV), and DrrA-mediated recruitment of Rab1 to the LCV required PI4P-binding activity. It was found that the host cell machinery that regulates sites of contact between the plasma membrane (PM) and the endoplasmic reticulum (ER) modulates PI4P dynamics on the LCV to control localization of these effectors. Specifically, phosphatidylinositol-4-kinase III alpha (PI4KIII alpha) was important for generating a PI4P signature that enabled L. pneumophila effectors to localize to the PM-derived vacuole, and the ER-associated phosphatase Sac1 was involved in metabolizing the PI4P on the vacuole to promote the dissociation of effectors. A defect in L. pneumophila replication in macrophages deficient in PI4KIII alpha was observed, highlighting that a PM-derived PI4P signature is critical for biogenesis of a vacuole that supports intracellular multiplication of L. pneumophila. These data indicate that PI4P metabolism by enzymes controlling PM-ER contact sites regulate the association of L. pneumophila effectors to coordinate early stages of vacuole biogenesis.

DOI： 10.1371/journal.ppat.1004222

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The Dot/Icm system of the intracellular pathogen Legionella pneumophila has the capacity to deliver over 270 effector proteins into host cells during infection. Important questions remain as to spatial and temporal mechanisms used to regulate such a large array of virulence determinants after they have been delivered into host cells. Here we investigated several L. pneumophila effector proteins that contain a conserved phosphatidylinositol-4-phosphate (PI4P)-binding domain first described in the effector DrrA (SidM). This PI4P binding domain was essential for the localization of effectors to the early L. pneumophila-containing vacuole (LCV), and DrrA-mediated recruitment of Rab1 to the LCV required PI4P-binding activity. It was found that the host cell machinery that regulates sites of contact between the plasma membrane (PM) and the endoplasmic reticulum (ER) modulates PI4P dynamics on the LCV to control localization of these effectors. Specifically, phosphatidylinositol-4-kinase IIIα (PI4KIIIα) was important for generating a PI4P signature that enabled L. pneumophila effectors to localize to the PM-derived vacuole, and the ER-associated phosphatase Sac1 was involved in metabolizing the PI4P on the vacuole to promote the dissociation of effectors. A defect in L. pneumophila replication in macrophages deficient in PI4KIIIα was observed, highlighting that a PM-derived PI4P signature is critical for biogenesis of a vacuole that supports intracellular multiplication of L. pneumophila. These data indicate that PI4P metabolism by enzymes controlling PM-ER contact sites regulate the association of L. pneumophila effectors to coordinate early stages of vacuole biogenesis. © 2014 Hubber et al.

DOI： 10.1371/journal.ppat.1004222

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Background &amp
Aims In epithelial cells, protein sorting mechanisms regulate localization of plasma membrane proteins that generate and maintain cell polarity. The clathrin-adaptor protein (AP) complex AP-1B is expressed specifically in polarized epithelial cells, where it regulates basolateral sorting of membrane proteins. However, little is known about its physiological significance. Methods We analyzed the intestinal epithelia of mice deficient in Ap1m2 (Ap1m2-/- mice), which encodes the AP-1B μ1B subunit, and compared it with 129/B6/CD1 littermates (controls). Notch signaling was inhibited by intraperitoneal injection of dibenzazepine, and β-catenin signaling was inhibited by injection of IWR1. Intestinal tissue samples were collected and analyzed by immunofluorescence analysis. Results Ap1m2 -/- mice developed intestinal epithelial cell hyperplasia. The polarity of intestinal epithelial cells was disrupted, as indicated by the appearance of ectopic microvilli-like structures on the lateral plasma membrane and mislocalization of basolateral membrane proteins, including the low-density lipoprotein receptor and E-cadherin. The E-cadherin-β-catenin complex therefore was disrupted at the adherens junction, resulting in nuclear translocation of β-catenin. This resulted in up-regulation of genes regulated by β-catenin/transcription factor 4 (Tcf4) complex, and increased the proliferation of intestinal epithelial cells. Conclusions AP-1B is required for protein sorting and polarization of intestinal cells in mice. Loss of AP-1B in the intestinal epithelia results in mislocalization of E-cadherin, activation of β-catenin/Tcf4 complex, proliferation, and hyperplasia. © 2013 by the AGA Institute.

DOI： 10.1053/j.gastro.2013.05.013

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DOI： 10.1053/j.gastro.2013.05.013

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Plasma membrane phosphatidylinositol (PI) 4-phosphate (PtdIns4P) has critical functions via both direct interactions and metabolic conversion to PI 4,5-bisphosphate (PtdIns(4,5)P(2)) and other downstream metabolites. However, mechanisms that control this PtdIns4P pool in cells of higher eukaryotes remain elusive. PI4KIIIalpha, the enzyme thought to synthesize this PtdIns4P pool, is reported to localize in the ER, contrary to the plasma membrane localization of its yeast homologue, Stt4. In this paper, we show that PI4KIIIalpha was targeted to the plasma membrane as part of an evolutionarily conserved complex containing Efr3/rolling blackout, which we found was a palmitoylated peripheral membrane protein. PI4KIIIalpha knockout cells exhibited a profound reduction of plasma membrane PtdIns4P but surprisingly only a modest reduction of PtdIns(4,5)P(2) because of robust up-regulation of PtdIns4P 5-kinases. In these cells, however, much of the PtdIns(4,5)P(2) was localized intracellularly, rather than at the plasma membrane as in control cells, along with proteins typically restricted to this membrane, revealing a major contribution of PI4KIIIalpha to the definition of plasma membran

DOI： 10.1083/jcb.201206095

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BACKGROUND & AIMS: Epithelial cells that cover the intestinal mucosal surface maintain immune homeostasis and tolerance in the gastrointestinal tract. However, little is known about the molecular mechanisms that regulate epithelial immune functions. Epithelial cells are distinct in that they are highly polarized; this polarity is, at least in part, established by the epithelium-specific polarized sorting factor adaptor protein (AP)-1B. We investigated the role of AP-1B-mediated protein sorting in the maintenance of gastrointestinal immune homeostasis. METHODS: The role of AP-1B in intestinal immunity was examined in AP-1B-deficient mice (Ap1m2(-/-)) by monitoring their phenotypes, intestinal morphology, and epithelial barrier functions. AP-1B-mediated protein sorting was examined in polarized epithelial cells from AP-1B knockdown and Ap1m2(-/-) mice. RESULTS: Ap1m2(-/-) mice developed spontaneous chronic colitis, characterized by accumulation of interleukin-17A-producing, T-helper 17 cells. Deficiency of AP-1B caused epithelial immune dysfunction, such as reduced expression of antimicrobial proteins and impaired secretion of immunoglobulin A. These defects promoted intestinal dysbiosis and increased bacterial translocation within the mucosa. Importantly, AP-1B deficiency led to mistargeting of a subset of basolateral cytokine receptors to the apical plasma membrane in a polarized epithelial cell line and in colonic epithelial cells from mice. AP1M2 expression was reduced significantly in colonic epithelium samples from patients with Crohn's disease. CONCLUSIONS: AP-1B is required for proper localization of a subset of cytokine receptors in polarized epithelial cells, which allows them to respond to cytokine signals from underlying lamina propria cells. The AP-1B-mediated protein sorting machinery is required for maintenance of immune homeostasis and prevention of excessive inflammation.

DOI： 10.1053/j.gastro.2011.04.056

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DOI： 10.1053/j.gastro.2011.04.056

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Phosphatidylinositol (PI) 4,5-bisphosphate (PI(4,5)P(2)) and its phosphorylated product PI 3,4,5-triphosphate (PI(3,4,5)P(3)) are two major phosphoinositides concentrated at the plasma membrane. Their levels, which are tightly controlled by kinases, phospholipases, and phosphatases, regulate a variety of cellular functions, including clathrin-mediated endocytosis and receptor signaling. In this study, we show that the inositol 5-phosphatase SHIP2, a negative regulator of PI(3,4,5)P(3)-dependent signaling, also negatively regulates PI(4,5)P(2) levels and is concentrated at endocytic clathrin-coated pits (CCPs) via interactions with the scaffold protein intersectin. SHIP2 is recruited early at the pits and dissociates before fission. Both knockdown of SHIP2 expression and acute production of PI(3,4,5)P(3) shorten CCP lifetime by enhancing the rate of pit maturation, which is consistent with a positive role of both SHIP2 substrates, PI(4,5)P(2) and PI(3,4,5)P(3), on coat assembly. Because SHIP2 is a negative regulator of insulin signaling, our findings suggest the importance of the phosphoinositide metabolism at CCPs in the regulation of insulin signal output.

DOI： 10.1083/jcb.201005018

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Phosphatidylinositol (PI) 4,5-bisphosphate (PI(4,5)P(2)) and its phosphorylated product PI 3,4,5-triphosphate (PI(3,4,5)P(3)) are two major phosphoinositides concentrated at the plasma membrane. Their levels, which are tightly controlled by kinases, phospholipases, and phosphatases, regulate a variety of cellular functions, including clathrin-mediated endocytosis and receptor signaling. In this study, we show that the inositol 5-phosphatase SHIP2, a negative regulator of PI(3,4,5)P(3)-dependent signaling, also negatively regulates PI(4,5)P(2) levels and is concentrated at endocytic clathrin-coated pits (CCPs) via interactions with the scaffold protein intersectin. SHIP2 is recruited early at the pits and dissociates before fission. Both knockdown of SHIP2 expression and acute production of PI(3,4,5)P(3) shorten CCP lifetime by enhancing the rate of pit maturation, which is consistent with a positive role of both SHIP2 substrates, PI(4,5)P(2) and PI(3,4,5)P(3), on coat assembly. Because SHIP2 is a negative regulator of insulin signaling, our findings suggest the importance of the phosphoinositide metabolism at CCPs in the regulation of insulin signal output.

DOI： 10.1083/jcb.201005018

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BFNC is an autosomal dominant epileptic disorder caused by mutations of KCNQ2 or KCNQ3 potassium channel gene. W309R missense mutation in KCNQ3 gene was previously reported in a family with BFNC. In this study, potassium currents were recorded from HEK293 cells expressing both W309R mutant KCNQ3 and wild type KCNQ2 channels. We found a lack of potassium current in W309R mutant KCNQ3 and KCNQ2 channels, which can explain the hyper-excitability of CNS in patients with BFNC.

DOI： 10.1016/j.eplepsyres.2008.12.003

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DOI： 10.1016/j.eplepsyres.2008.12.003

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The high-affinity choline transporter (CHT) is expressed in cholinergic neurons and efficiently transported to axon terminals where it controls the rate-limiting step in acetylcholine synthesis. Recent studies have shown that the majority of CHT is unexpectedly localized on synaptic vesicles (SV) rather than the presynaptic plasma membrane, establishing vesicular CHT trafficking as a basis for activity-dependent CHT regulation. Here, we analyse the intracellular distribution of CHT in the adaptor protein-3 (AP-3)-deficient mouse model mocha. In the mocha mouse, granular structures in cell bodies are intensely labelled with CHT antibody, indicating possible deficits in CHT trafficking from the cell body to the axon terminal. Western blot analyses reveal that CHT on SV in mocha mice is decreased by 30% compared with wild-type mice. However, no significant difference in synaptosomal choline uptake activity is detected, consistent with the existence of a large reservoir pool for CHT. To further characterize CHT trafficking, we established a PC12D-CHT cell line. In this line, CHT is found associated with a subpopulation of synaptophysin-positive synaptic-like microvesicles (SLMV). The amounts of CHT detected on SLMV are greatly reduced by treating the cell with agents that halt AP-dependent membrane trafficking. These results demonstrate that APs have important functions for CHT trafficking in neuronal cells. © The Authors (2008).

DOI： 10.1111/j.1460-9568.2008.06268.x

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<p>The high-affinity choline transporter (CHT) is expressed in cholinergic neurons and efficiently transported to axon terminals where it controls the rate-limiting step in acetylcholine synthesis. Recent studies have shown that the majority of CHT is unexpectedly localized on synaptic vesicles (SV) rather than the presynaptic plasma membrane, establishing vesicular CHT trafficking as a basis for activity-dependent CHT regulation. Here, we analyse the intracellular distribution of CHT in the adaptor protein-3 (AP-3)-deficient mouse model mocha. In the mocha mouse, granular structures in cell bodies are intensely labelled with CHT antibody, indicating possible deficits in CHT trafficking from the cell body to the axon terminal. Western blot analyses reveal that CHT on SV in mocha mice is decreased by 30% compared with wild-type mice. However, no significant difference in synaptosomal choline uptake activity is detected, consistent with the existence of a large reservoir pool for CHT. To further characterize CHT trafficking, we established a PC12D-CHT cell line. In this line, CHT is found associated with a subpopulation of synaptophysin-positive synaptic-like microvesicles (SLMV). Th

DOI： 10.1111/j.1460-9568.2008.06268.x

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Evidence that some types of epilepsies show strong genetic predisposition has been well documented. AP3M2 is considered to be an epileptogenic gene because AP3M2 knockout mice exhibit symptoms of spontaneous epileptic seizures. In order to investigate whether the AP3M2 gene causes susceptibility to epilepsy, we performed mutation screening of the genomic DNA of 190 patients with six epilepsy types; this screening involved all the 9 exons and the relevant exon-intron boundaries of AP3M2. Although neither missense nor nonsense mutations were detected, we identified 21 sequence variations, of which 16 variations were novel. Of the 21 variations, I I were detected in 5' and 3' UTRs, while the remaining variations were detected in introns. Although the present study failed to identify the possible AP3M2 mutations that may cause epilepsy, our results suggest that some AP3M2 mutations still remain candidates for unmapped disorders including epilepsy, febrile seizure, and other neuronal developmental disorders associated with functional abnormalities of GABAergic transmission. (c) 2007 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.braindev.2006.12.004

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Phosphaticlylinositol 4,5-bisphosphate [PI(4,5)P-2], a phosphoinositide concentrated predominantly in the plasma membrane, binds endocytic clathrin adaptors, many of their accessory factors, and a variety of actin-regulatory proteins. Here we have used fluorescent fusion proteins and total internal reflection fluorescence microscopy to investigate the effect of acute PI(4,5)P-2 breakdown on the dynamics of endocytic clathrin-coated pit components and of the actin regulatory complex, Arp2/3. PI(4,5)P-2 breakdown was achieved by the inducible recruitment to the plasma membrane of an inositol 5-phosphatase module through the rapamycin/FRB/FKBP system or by treatment with ionomycin. PI(4,5)P-2 depletion resulted in a dramatic loss of clathrin puncta, which correlated with a massive dissociation of endocytic adaptors from the plasma membrane. Remaining clathrin spots at the cell surface had only weak fluorescence and were static over time. Dynamin and the p20 subunit of the Arp2/3 actin regulatory complex, which were concentrated at late-stage clathrin-coated pits and in lamellipodia, also dissociated from the plasma membrane, and these changes correlated with an arrest of motility at the cell edge. These findings demonstrate the critical importance of PI(4,5)P-2 in clathrin coat dynamics and Arp2/3-dependent actin regulation.

DOI： 10.1073/pnas.0611733104

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Newly synthesized membrane proteins are sorted in the trans-Golgi network (TGN) on the basis of sorting signals carried in their cytoplasmic domains and delivered to their final destinations in the secretory and endocytic pathways. Although previous studies have suggested the involvement of early endosomes in the biosynthetic pathway of transmembrane proteins, the precise trafficking routes followed by the newly synthesized plasma membrane proteins, such as transferrin receptors (TfRs), after exit from the TGN remain unclear. In this report, first, we demonstrated the advantages of photoactivating PA-GFP, a variant of the Aequorea victoria green fluorescent protein (GFP), with multiphoton laser light rather than single-photon laser light, in terms of photoactivation efficiency and spatial resolution. We then applied the multiphoton photoactivation technique to selectively photoactivate the TfR tagged with PA-GFP (PA-GFP-TfR) at the TGN, and monitored the movement of the photoactivated PA-GFP-TfR in live cells. We observed that the PA-GFP-TfR photoactivated at the TGN are transported to the Tfn+EEA1+ endosomal compartments after exiting the TGN. These data support the notion that early endosomes can serve as a sorting station for not only internalized plasma membrane proteins in the endocytic pathway but also newly synthesized membrane proteins in the post-Golgi secretory pathway. © 2006 by Japan Society for Cell Biology.

DOI： 10.1247/csf.31.63

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DOI： 10.1247/csf.31.63

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The heterotetrameric adaptor protein (AP) complexes AP-1, AP-2, AP-3, and AP-4 play key roles in transport vesicle formation and cargo sorting in post-Golgi trafficking pathways. Studies on cultured mammalian cells have shown that AP-2 mediates rapid endocytosis of a subset of plasma membrane receptors. To determine whether this function is essential in the context of a whole mammalian organism, we carried out targeted disruption of the gene encoding the mu 2 subunit of AP-2 in the mouse. We found that mu 2 heterozygous mutant mice were viable and had an apparently normal phenotype. In contrast, no mu 2 homozygous mutant embryos were identified among blastocysts from intercrossed heterozygotes, indicating that mu 2-deficient embryos die before day 3.5 postcoitus (E3.5). These results indicate that AP-2 is indispensable for early embryonic development, which might be due to its requirement for cell viability.

DOI： 10.1128/MCB.25.21.9318-9323.2005

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A P-3 is a member of the adaptor protein (AP) complex family that regulates the vesicular transport of cargo proteins in the secretory and endocytic pathways. There are two isoforms of AP-3: the ubiquitously expressed AP-3A and the neuron-specific AP-3B. Although the physiological role of AP-3A has recently been elucidated, that of AP-3B remains unsolved. To address this question, we generated mice lacking mu3B, a subunit of AP-3B. mu3B(-/-) mice suffered from spontaneous epileptic seizures. Morphological abnormalities were observed at synapses in these mice. Biochemical studies demonstrated the impairment of gamma-aminobutyric acid (GABA) release because of, at least in part, the reduction of vesicular GABA transporter in mu3B(-/-) mice. This facilitated the induction of long-term potentiation in the hippocampus and the abnormal propagation of neuronal excitability via the temporoammonic pathway. Thus, AP-3B plays a critical role in the normal formation and function of a subset of synaptic vesicles. This work adds a new aspect to the pathogenesis of epilepsy.

DOI： 10.1083/jcb.200405032

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Adaptor protein (AP) complexes are cytosolic heterotetramers that mediate the sorting of membrane proteins in the secretory and endocytic pathways. AP complexes are involved in the formation of clathrin-coated vesicles (CCVs) by recruiting the scaffold protein, clathrin. AP complexes also play a pivotal role in the cargo selection by recognizing the sorting signals within the cytoplasmic tail of integral membrane proteins. Six distinct AP complexes have been identified. AP-2 mediates endocytosis from the plasma membrane, while AP-1, AP-3 and AP-4 play a role in the endosomall/lysosomal sorting pathways. Moreover, tissue-specific sorting events such as the basolateral sorting in polarized epithelial cells and the biogenesis of specialized organelles including melanosomes and synaptic vesicles are also regulated by members of AP complexes. The application of a variety of methodologies have gradually revealed the physiological role of AP complexes.

DOI： 10.1247/csf.28.419

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC CELL BIOLOGY  

To investigate the importance of tyrosine recognition by the AP-1B clathrin adaptor subunit mu1B for basolateral sorting of integral membrane proteins in polarized epithelial cells, we have produced and characterized a mutant form of mu1B. The mutant (M-mu1B) contains alanine substitutions of each of the four conserved residues, which in the AP-2 adaptor subunit mu2 are critical for interacting with tyrosine-based endocytosis signals. We show M-mu1B is defective for tyrosine binding in vitro, but is nevertheless incorporated into AP-1 complexes in transfected cells. Using LLC-PK1 cells expressing either wild type or M-mu1B, we find that there is inefficient basolateral expression of membrane proteins whose basolateral targeting signals share critical tyrosines with signals for endocytosis. In contrast, membrane proteins whose basolateral targeting signals are distinct from their endocytosis signals (transferrin and low-density lipoprotein receptors) accumulate at the basolateral domain normally, although in a manner that is strictly dependent on mu1B or M-mu1B expression. Our results suggest that mu1B interacts with different classes of basolateral targeting signals in distinct ways.

DOI： 10.1091/mbc.E01-10-0096

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING LTD  

Background: S-Adenosylmethionine decarboxylase (AdoMetDC) is one of the key enzymes involved in the biosynthesis of spermidine and spermine, which are essential for normal cell growth. To examine the role of polyamines in embryogenesis, we carried out targeted disruption of the mouse Amd1 gene, encoding AdoMetDC, to generate mice that can not synthesize spermidine and spermine.
Results: Amd1 heterozygous mice were viable, normal and fertile. However, homozygous Amd1(-/-) embryos died early in embryonic development, between E3.5 and E6.5 days post-coitus. Homozygous (Amd1(-/-)) blastocysts at E3.5 arrested cell proliferation immediately after the onset of cell culture, and this arrest was rescued by the addition of spermidine. Chromosomal DNA breakage did not occur in Amd1(-/-) blastocysts at E3.5, as determined by TUNEL assay.
Conclusions: These results indicate that AdoMetDC plays an essential role in embryonic development and that polyamines are required for cell proliferation in the embryo after E3.5.

DOI： 10.1046/j.1356-9597.2001.00494.x

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

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC IMMUNOLOGISTS  

The pre-TCR alpha (pT alpha) is exclusively expressed in immature thymocytes and constitutes the pre-TCR complex with TCR beta, which regulates early T cell differentiation. Despite the recent identification of the pT alpha enhancer, the contribution of the promoter region, the direct DNA-protein interaction, and the regulation of such interaction along with T cell development have not been investigated. We analyzed the pT alpha promoter region and identified the critical elements for transcription of the pT alpha gene. The pT alpha promoter was found to contain two consecutive E-box elements that are critical for pT alpha transcription. The E-box elements in the promoter region formed the specific DNA-protein complex that was exclusively observed in immature thymocytes, not in mature thymocytes and T cells. The E proteins in this complex were identified as E2A and HeLa E-box binding protein (HEB), and overexpression of E2A and HEB resulted in activation of the pT alpha promoter. The binding complex in the consecutive E-boxes in the pTa promoter changed along with T cell development, as a distinct DNA-binding complex was observed in mature T cells. Comparing the E-box regions in the enhancer and the promoter, those in the promoter appear to make a greater contribution to pT alpha gene transcription.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Jak3 is responsible for growth signals by various cytokines such as interleukin (IL)-2, IL-4, and IL-7 through association with the common gamma chain (gammac) in lymphocytes. We found that T cells from Jak3-deficient mice exhibit impairment of not only cytokine signaling but also early activation signals and that Jak3 is phosphorylated upon T cell receptor (TCR) stimulation. TCR-mediated phosphorylation of Jak3 is independent of IL-2 receptor/gammac but is dependent on Lck and ZAP-70. Jak3 was found to be assembled with the TCR complex, particularly through direct association with CD3 zeta via its JH4 region, which is a different region from that for gammac association. These results suggest that Jak3 plays a role not only in cell growth but also in T cell activation and represents cross-talk of a signaling molecule between TCR and growth signals.

DOI： 10.1074/jbc.M011363200,

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Some plasma membrane receptors in yeast are known to be internalized and degraded in lysosomes up on ligand-dependent ubiquitination. However, the role of ubiquitination in endocytosis and lysosomal degradation in higher eukaryotes has been controversial. In order to directly assess this question, we investigated the fate of chimeric molecules in which ubiquitin moiety was fused in-frame to the cytoplasmic region of membrane proteins, The chimeric proteins with the wild-type ubiquitin were endocytosed and delivered to lysosomes efficiently. Mutant ubiquitin with lysine-to-arginine substitution could still mediate endocytosis, suggesting that polyubiquitination is not required for the endocytosis. We next searched for the existence of an endocytosis signal(s) in the ubiquitin moiety, and identified a di-leucine signal, Leu(43)-Ile(44). The Leu(43)-Ile(44) sequence mediated endocytosis and lysosomal sorting in a Leu(43)-dependent manner. These results suggest that the di-leucine signal in ubiquitin can be involved in ubiquitination-mediated endocytosis and lysosomal targeting of membrane proteins.

DOI： 10.1074/jbc.M907720199,

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The apical and basolateral plasma membrane domains of polarized epithelial cells contain distinct sets of integral membrane proteins. Biosynthetic targeting of proteins to the basolateral plasma membrane is mediated by cytosolic tail determinants, many of which resemble signals involved in the rapid endocytosis or lysosomal targeting, Since these signals are recognized by adaptor proteins, we hypothesized that there could be epithelial-specific adaptors involved in polarized sorting. Here, we report the identification of a novel member of the adaptor medium chain family, named mu 1B, which is closely related to the previously described mu 1A (79% amino acid sequence identity). Northern blotting and in situ hybridization analyses reveal the specific expression of mu 1B mRNA in a subset of polarized epithelial and exocrine cells. Yeast two-hybrid analyses show that mu 1B is capable of interacting with generic tyrosine-based sorting signals. These observations suggest that mu 1B may be involved in protein sorting events specific to polarized cells. (C) 1999 Federation of European Biochemical Societies.

DOI： 10.1016/S0014-5793(99)00432-9

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KARGER  

The protein mu 1B is a member of the medium chain family of the clathrin-associated adaptor complex and is expressed exclusively in epithelial cells. We determined the genomic structure of previously cloned murine genes for mu 1B (Ap1m2) and its closely related homolog, mu 1A (Ap1m1). Comparison of their genomic structures revealed that the positions of introns are identical between these two genes, except for the insertion of an additional intron in Ap1m1 (intron 4). By contrast, these structures are different from that of the more distantly related Ap2m1 gene encoding mu 2. Taken together with the similarity of amino acid sequences among these genes, the data presented in this study suggest that Ap1m1/2 and Ap2m1 diverged long before the separation of Ap1m1 and Ap1m2, which most likely resulted from a relatively recent gene duplication. We also mapped AP1M2 to human chromosome 19p13.2 and Ap1m2 to the proximal region of mouse chromosome 9. The results are consistent with the fact that these regions are syntenic. Copyright (C) 1999 S. Karger AG, Basel.

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Language：Japanese   Publisher：(一社)日本神経学会  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：JAPAN SOC CELL BIOLOGY  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：JAPAN SOC CELL BIOLOGY  

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Language：Japanese   Publisher：(公財)先進医薬研究振興財団  

他の疾患標的蛋白として細胞内小胞輸送障害が中枢神経系機能性疾患発現に関与している可能性を,二種類の遺伝子ノックアウトマウスを用いて検証した.AP3M2遺伝子ノックアウトマウスは8週以降で自発性けいれんが生じ,この時点でGABA開口分泌機能の低下とtemporoammonic pathwayの興奮性伝播が過剰亢進していた.PRIP-1遺伝子ノックアウトマウスは自発性けいれんの発現率は5%と低いものの,この痙攣発作は致死性で,けいれん発作後の海馬CA3の好酸化性細胞死を確認した.加えてGABAとglutamateの開口分泌が亢進していた.よって,細胞内蛋白輸送障害が新たな中枢神経系機能性疾患の病態に関与している可能性が示唆された

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Language：Japanese   Publisher：(一社)日本てんかん学会  

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Language：Japanese   Publisher：てんかん治療研究振興財団  

:アダプター複合体(AP)はシナプス小胞の生合成を担う機能蚤白質であるが,そのサブタイプのひとつであるAP･3Bは神経系に特異的な分布を示す｡てんかんの分子基盤におけるAP13Bの役割を検討するため,μ3B遺伝子ノックアウトマウス(μ3BKO)を用い,キンドリングによる行動学的,脳波学的解析を行った｡μ3BKOではキンドリング完成までの期間の著明な短縮,および後発射持続時間の延長が認められた｡キンドリングによって誘発された発作型は,無動状態と引き続く全般性強直間代発作であった｡また,後発射の相関次元およおよび高次スペクトル解析の結果から.きわめて複雑かつランダムな神経活動の発現と,神経回路網における相互作用の消失が引き蒔こされていることが予想された｡以上より,μ3BKOのけいれん準備性の著明な先進はキンドリングによって獲得されたものでなく,geneticな因子等によって内在的に惹起されている神経機能不全に帰因する可能性が推察された。

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER SOC CELL BIOLOGY  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER SOC CELL BIOLOGY  

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Language：Japanese   Publisher：(公社)日本薬理学会  

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Language：Japanese   Publisher：(一社)日本細胞生物学会  

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