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Weibel–Palade bodies (WPB) are elongated, rod-like secretory organelles unique to endothelial cells that store the pro-coagulant von-Willebrand factor (VWF) and undergo regulated exocytosis upon stimulation with Ca2+- or cAMP-raising agonists. We show here that WPB preferentially initiate fusion with the plasma membrane at their tips and identify synaptotagmin-like protein 2-a (Slp2-a) as a positive regulator of VWF secretion most likely mediating this topological selectivity. Following secretagogue stimulation, Slp2-a accumulates at one WPB tip before fusion occurs at this site. Depletion of Slp2-a reduces Ca2+-dependent secretion of highly multimeric VWF and interferes with the formation of actin rings at WPB–plasma membrane fusion sites that support the expulsion of the VWF multimers and most likely require a tip-end fusion topology. Phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] binding via the C2A domain of Slp2-a is required for accumulation of Slp2-a at the tip ends of fusing WPB, suggesting that Slp2-a mediates polar exocytosis by initiating contacts between WPB tips and plasma membrane PI(4,5)P2.

Calpain-10 (CAPN10) is the calpain family protease identified as the first candidate susceptibility gene for type 2 diabetes mellitus (T2DM). However, the detailed molecular mechanism has not yet been elucidated. Here we report that CAPN10 processes microtubule associated protein 1 (MAP1) family proteins into heavy and light chains and regulates their binding activities to microtubules and actin filaments. Immunofluorescent analysis of Capn10−/− mouse embryonic fibroblasts shows that MAP1B, a member of the MAP1 family of proteins, is localized at actin filaments rather than at microtubules. Furthermore, fluorescence recovery after photo-bleaching analysis shows that calpain-10 regulates actin dynamics via MAP1B cleavage. Moreover, in pancreatic islets from CAPN10 knockout mice, insulin secretion was significantly increased both at the high and low glucose levels. These findings indicate that deficiency of calpain-10 expression may affect insulin secretion by abnormal actin reorganization, coordination and dynamics through MAP1 family processing.

Synaptotagmin-like protein 2-a (Slp2-a) was originally described as a membrane trafficking protein that consists of a Slp homology domain (SHD), a linker domain, and tandem C2 domains (named the C2A domain and C2B domain). Slp2-a mediates docking of Rab27-bearing vesicles to the plasma membrane through simultaneous interaction with Rab27 and phospholipids in the plasma membrane. We have recently reported that Slp2-a regulates renal epithelial cell size through interaction with Rap1GAP2 via the C2B domain independently of Rab27 and demonstrated the presence of excess activation of ezrin, a membrane-cytoskeleton linker and signal transducer, in Slp2-a-knockdown Madin–Darby canine kidney II (MDCK II) cells. However, the precise mechanism of ezrin inactivation by Slp2-a in cell size control has remained largely unknown. In this study, we investigated the functional relationship between Slp2-a and ezrin in MDCK II cells. The results showed that activation of ezrin in control MDCK II cells either pharmacologically or by overexpression of a constitutively active ezrin mutant caused an increase in cell size, whereas inactivation of ezrin in Slp2-a-knockdown cells by a specific ezrin inhibitor restored them to their normal cell size. We also found that Slp2-a interacts via its previously uncharacterized linker domain with protein phosphatase 1β (PP1β), which inactivates ezrin, and that the interaction is required for the plasma membrane localization of PP1β. These results indicate that Slp2-a inactivates ezrin by recruiting PP1 to the plasma membrane.

Actin dynamics in pancreatic β-cells is involved in insulin secretion. However, the molecular mechanisms of the regulation of actin dynamics by intracellular signals in pancreatic β-cells and its role in phasic insulin secretion are largely unknown. In this study, we elucidate the regulation of actin dynamics by neuronal Wiskott-Aldrich syndrome protein (N-WASP) and cofilin in pancreatic β-cells and demonstrate its role in glucose-induced insulin secretion (GIIS). N-WASP, which promotes actin polymerization through activation of the actin nucleation factor Arp2/3 complex, was found to be activated by glucose stimulation in insulin-secreting clonal pancreatic β-cells (MIN6-K8 β-cells). Introduction of a dominant-negative mutant of N-WASP, which lacks G-actin and Arp2/3 complex-binding region VCA, into MIN6-K8 β-cells or knockdown of N-WASP suppressed GIIS, especially the second phase. We also found that cofilin, which severs F-actin in its dephosphorylated (active) form, is converted to the phosphorylated (inactive) form by glucose stimulation in MIN6-K8 β-cells, thereby promoting F-actin remodeling. In addition, the dominant-negative mutant of cofilin, which inhibits activation of endogenous cofilin, or knockdown of cofilin reduced the second phase of GIIS. However, the first phase of GIIS occurs in the G-actin predominant state, in which cofilin activity predominates over N-WASP activity. Thus, actin dynamics regulated by the balance of N-WASP and cofilin activities determines the biphasic response of GIIS.

Slp2-a is required for targeting of the signaling molecule podocalyxin to the apical membrane in MDCK II cells in a Rab27A-dependent manner. Apical membrane localization of podocalyxin is required for expression of the tight junction protein claudin-2 through modulation of intracellular signals, including MAPK signals., Most cells in tissues are polarized and usually have two distinct plasma membrane domains—an apical membrane and a basolateral membrane, which are the result of polarized trafficking of proteins and lipids. However, the mechanism underlying the cell polarization is not fully understood. In this study, we investigated the involvement of synaptotagmin-like protein 2-a (Slp2-a), an effector molecule for the small GTPase Rab27, in polarized trafficking by using Madin–Darby canine kidney II cells as a model of polarized cells. The results show that the level of Slp2-a expression in MDCK II cells increases greatly as the cells become polarized and that its expression is specifically localized at the apical membrane. The results also reveal that Slp2-a is required for targeting of the signaling molecule podocalyxin to the apical membrane in a Rab27A-dependent manner. In addition, ezrin, a downstream target of podocalyxin, and ERK1/2 are activated in Slp2-a–knockdown cells, and their activation results in a dramatic reduction in the amount of the tight junction protein claudin-2. Because both Slp2-a and claudin-2 are highly expressed in mouse renal proximal tubules, Slp2-a is likely to regulate claudin-2 expression through trafficking of podocalyxin to the apical surface in mouse renal tubule epithelial cells.

The formation of epithelial tissues requires both the generation of apical-basal polarity and the coordination of this polarity between neighbouring cells to form a central lumen. During de novo lumen formation, vectorial membrane transport contributes to the formation of a singular apical membrane, resulting in the contribution of each cell to only a single lumen. Here, from a functional screen for genes required for three-dimensional epithelial architecture, we identify key roles for synaptotagmin-like proteins 2-a and 4-a (Slp2-a/4-a) in the generation of a single apical surface per cell. Slp2-a localizes to the luminal membrane in a PtdIns(4,5)P(2)-dependent manner, where it targets Rab27-loaded vesicles to initiate a single lumen. Vesicle tethering and fusion is controlled by Slp4-a, in conjunction with Rab27/Rab3/Rab8 and the SNARE syntaxin-3. Together, Slp2-a/4-a coordinate the spatiotemporal organization of vectorial apical transport to ensure that only a single apical surface, and thus the formation of a single lumen, occurs per cell.

Polarized epithelial cells have two distinct plasma membrane domains, i.e., an apical membrane domain and a basolateral membrane domain, that are the result of polarized trafficking of proteins and lipids. Several members of the Rab-type small GTPases, which are general regulators of membrane trafficking, have been reported to be involved in the regulation of polarized trafficking in epithelial cells, but their precise role in polarized trafficking is poorly understood. In a recent study we used Madin-Darby canine kidney (MDCK) II cells as a model of polarized cells and concluded from the results that Rab27A and its effector synaptotagmin-like protein 2-a (Slp2-a) regulate apical transport of Rab27-bearing vesicles in polarized epithelial cells. Both Rab27A and Slp2-a are uniformly localized at the plasma membrane in subconfluent, non-polarized MDCK II cells, but their expression increases as the cells become polarized, and they are specifically localized at the apical membrane in polarized MDCK II cells (i.e., two-dimensional cell culture). Slp2-a is also localized at the apical membrane of tubular MDCK II cysts (i.e., three-dimensional cell culture) and promotes the formation of a single apical domain in the cysts by regulating polarized trafficking of Rab27-bearing vesicles. In this chapter we describe the assay procedures for analyzing the expression and localization of Rab27A and Slp2-a in non-polarized and polarized renal epithelial cells.

Polarized epithelial cells have two distinct plasma membrane domains, i.e., an apical membrane domain and a basolateral membrane domain, that are the result of polarized trafficking of proteins and lipids. Several members of the Rab-type small GTPases, which are general regulators of membrane trafficking, have been reported to be involved in the regulation of polarized trafficking in epithelial cells, but their precise role in polarized trafficking is poorly understood. In a recent study we used Madin-Darby canine kidney (MDCK) II cells as a model of polarized cells and concluded from the results that Rab27A and its effector synaptotagmin-like protein 2-a (Slp2-a) regulate apical transport of Rab27-bearing vesicles in polarized epithelial cells. Both Rab27A and Slp2-a are uniformly localized at the plasma membrane in subconfluent, non-polarized MDCK II cells, but their expression increases as the cells become polarized, and they are specifically localized at the apical membrane in polarized MDCK II cells (i.e., two-dimensional cell culture). Slp2-a is also localized at the apical membrane of tubular MDCK II cysts (i.e., three-dimensional cell culture) and promotes the formation of a single apical domain in the cysts by regulating polarized trafficking of Rab27-bearing vesicles. In this chapter we describe the assay procedures for analyzing the expression and localization of Rab27A and Slp2-a in non-polarized and polarized renal epithelial cells.

A highly sensitive analysis method for biological molecules is required because of recent developments in molecular biology. Conventional highly sensitive detection methods are based on labelling techniques using fluorescent dyes or enzymes. However, since those techniques involved some problems with signal instability, a new technology has been sought. Because the superconducting interference device (SQUID) is an extremely highly sensitive magnetic sensor, it can be applied to the highly sensitive detection of DNA labelled with small magnetic particles. The signal from SQUID is stable in contrast to fluorescent dyes and enzymes, therefore it permits highly sensitive measurement over long periods of time. Sample coverslips on which the small magnetic particles were anchored using biotin labelled DNA were prepared to demonstrate the availability of this method. Scanning the high-Tc SQUID sensor on the coverslip demonstrated that the magnetic flux on the coverslip agreed well with the pattern of labelled DNA anchored on the coverslip. This result suggests that SQUID can be applied for the specific detection of DNA molecules, especially for the detection of DNA chips.

Aims/hypothesisMore than 90% of Chinese familial early-onset type 2 diabetes mellitus is genetically unexplained. To investigate the molecular aetiology, we identified and characterised whether mutations in the KCNJ11 gene are responsible for these families.MethodsKCNJ11 mutations were screened for 96 familial early-onset type 2 diabetic probands and their families. Functional significance of the identified mutations was confirmed by physiological analysis, molecular modelling and population survey.ResultsThree novel KCNJ11 mutations, R27H, R192H and S116F117del, were identified in three families with early-onset type 2 diabetes mellitus. Mutated KCNJ11 with R27H or R192H markedly reduced ATP sensitivity (E23K>R27H>C42R>R192H>R201H), but no ATP-sensitive potassium channel currents were detected in the loss-of-function S116F117del channel in vitro. Molecular modelling indicated that R192H had a larger effect on the channel ATP-binding pocket than R27H, which may qualitatively explain why the ATP sensitivity of the R192H mutation is seven times less than R27H. The shape of the S116F117del channel may be compressed, which may explain why the mutated channel had no currents. Discontinuation of insulin and implementation of sulfonylureas for R27H or R192H carriers and continuation/switch to insulin therapy for S116F117del carriers resulted in good glycaemic control.Conclusions/interpretationOur results suggest that genetic diagnosis for the KCNJ11 mutations in familial early-onset type 2 diabetes mellitus may help in understanding the molecular aetiology and in providing more personalised treatment for these specific forms of diabetes in Chinese and other Asian patients.

Synaptotagmin-like protein 2 (Slp2-a/Sytl2) is a Rab27 effector protein that regulates transport of Rab27-bearing vesicles and organelles through its N-terminal Rab27-binding domain and a phospholipid-binding C2A domain. Here we demonstrate a Rab27-independent function of Slp2-a in the control of renal cell size through a previously uncharacterized C2B domain. We found that by recruiting Rap1 GAPs to the plasma membrane of MDCK II cells through the C2B domain, Slp2-a inactivates Rap signaling and modulates the size of the cells. Functional ablation of Slp2-a resulted in an increase in the size of MDCK II cells. Drosophila Slp Bitesize was found to compensate for the function of Slp2-a in MDCK II cells, thereby indicating that the mechanism of the cell size control by Slp proteins has been evolutionarily conserved. Interestingly, blockade of the activity of ezrin, a downstream target of Rap, with the glucosylceramide synthase inhibitor, miglustat, effectively inhibited cell spreading of Slp2-a-knockdown cells. We also discovered aberrant expression of Slp2-a and increased activity of ezrin in pcy ( Nphp3 pcy ) mice, a model of polycystic kidney disease that is characterized by renal cell spreading. Our findings indicate that Slp2-a controls renal cell size through regulation of Rap–ezrin signaling independently of Rab27.

A 74-year-old woman was noted to have a mass lesion near the right elbow joint during medication for pulmonary tuberculosis. After discontinuation of medication, the mass gradually became enlarged with swelling and tenderness of the joint. Radiological evaluation disclosed tenosynovitis with an encapsulated abscess. Microscopic examination and culture of an aspiration biopsy specimen from the abscess showed no microorganisms. However, DNA extracted from the specimen contained mycobacterium tuberculosis DNA, permitting a diagnosis of tuberculous tenosynovitis. Mycobacterium is not always detected in biopsy specimens of tuberculous arthritis and tenosynovitis. In such cases, genetic diagnosis may be of great use.

Autosomal-recessive cerebellar ataxias (ARCAs) are clinically and genetically heterogeneous disorders associated with diverse neurological and nonneurological features that occur before the age of 20. Currently, mutations in more than 20 genes have been identified, but approximately half of the ARCA patients remain genetically unresolved. In this report, we describe a Japanese family in which two siblings have slow progression of a type of ARCA with psychomotor retardation. Using whole-exome sequencing combined with homozygosity mapping, we identified a homozygous missense mutation in SYT14, encoding synaptotagmin XIV (SYT14). Expression analysis of the mRNA of SYT14 by a TaqMan assay confirmed that SYT14 mRNA was highly expressed in human fetal and adult brain tissue as well as in the mouse brain (especially in the cerebellum). In an in vitro overexpression system, the mutant SYT14 showed intracellular localization different from that of the wild-type. An immunohistochemical analysis clearly showed that SYT14 is specifically localized to Purkinje cells of the cerebellum in humans and mice. Synaptotagmins are associated with exocytosis of secretory vesicles (including synaptic vesicles), indicating that the alteration of the membrane-trafficking machinery by the SYT14 mutation may represent a distinct pathomechanism associated with human neurodegenerative disorders.

Insulin secretion is essential for maintenance of glucose homeostasis, but the mechanism of insulin granule exocytosis, the final step of insulin secretion, is largely unknown. Here, we investigated the role of Rim2alpha in insulin granule exocytosis, including the docking, priming, and fusion steps. We found that interaction of Rim2alpha and Rab3A is required for docking, which is considered a brake on fusion events, and that docking is necessary for K(+)-induced exocytosis, but not for glucose-induced exocytosis. Furthermore, we found that dissociation of the Rim2alpha/Munc13-1 complex by glucose stimulation activates Syntaxin1 by Munc13-1, indicating that Rim2alpha primes insulin granules for fusion. Thus, Rim2alpha determines docking and priming states in insulin granule exocytosis depending on its interacting partner, Rab3A or Munc13-1, respectively. Because Rim2alpha(-/-) mice exhibit impaired secretion of various hormones stored as dense-core granules, including glucose-dependent insulinotropic polypeptide, growth hormone, and epinephrine, Rim2alpha plays a critical role in exocytosis of these dense-core granules.

The X-ray molecular structure of the addition product of trans-HPd(PEt3)2(C≡CPh) with dimethyl acetylene-dicarboxylate has been determined to be [trans-{(MeO2C)HC=C(CO2Me)}Pd(PEt3)2(C≡CPh)]. The crystal belongs to the monoclinic system, space group P21/c, with eight formula units in a cell with dimensions of a=17.844(3), b=9.476(3), c=36.061(4) A, β=98.18(1)°. Two independent molecules have similar structures except for the conformation of the phenylethynyl groups and one of the triethylphosphine groups. The 1,2-bis(methoxycarbonyl)vinyl and phenylethynyl groups are σ-bonded to the palladium atom. No significant difference is observed between the Pd–C(sp)[2.04(2) and 2.03(2) A] and Pd–C(sp2)[2.05(2) and 2.06(2) A] bond lengths. The environment of the palladium atom is approximately square-planar: Pd–P=2.321(5), 2.312(5), and 2.305(5), 2.311(5) A.