Mutations in the spin gene are characterized by an extraordinarily strong rejection behavior of female flies in response to male courtship. They are also accompanied by decreases in the viability, adult life span, and oviposition rate of the flies. In spin mutants, some oocytes and adult neural cells undergo degeneration, which is preceded by reductions in programmed cell death of nurse cells in ovaries and of neurons in the pupal nervous system, respectively. The central nervous system (CNS) of spin mutant flies accumulates autofluorescent lipopigments with characteristics similar to those of lipofuscin. The spin locus generates at least five different transcripts, with only two of these being able to rescue the spin behavioral phenotype; each encodes a protein with multiple membrane-spanning domains that are expressed in both the surface glial cells in the CNS and the follicle cells in the ovaries. Orthologs of the spin gene have also been identified in a number of species from nematodes to humans. Analysis of the spin mutant will give us new insights into neurodegenerative diseases and aging.

Key Points• Clot retraction of sphingomyelin-rich raftdepleted platelets from sphingomyelin synthase knockout mouse is delayed.• Translocation of fibrin to sphingomyelin-rich rafts in platelet membrane is induced by thrombin in the presence of FXIII crosslinking activity.Membrane rafts are spatially and functionally heterogenous in the cell membrane. We observed that lysenin-positive sphingomyelin (SM)-rich rafts are identified histochemically in the central region of adhered platelets where fibrin and myosin are colocalized on activation by thrombin. The clot retraction of SM-depleted platelets from SM synthase knockout mouse was delayed significantly, suggesting that platelet SM-rich rafts are involved in clot retraction. We found that fibrin converted by thrombin translocated immediately in platelet detergent-resistant membrane (DRM) rafts but that from Glanzmann's thrombasthenic platelets failed. The fibrinogen g-chain C-terminal (residues 144-411) fusion protein translocated to platelet DRM rafts on thrombin activation, but its mutant that was replaced by A398A399 at factor XIII crosslinking sites (Q398Q399) was inhibited. Furthermore, fibrin translocation to DRM rafts was impaired in factor XIII A subunitdeficient mouse platelets, which show impaired clot retraction. In the cytoplasm, myosin translocated concomitantly with fibrin translocation into the DRM raft of thrombin-stimulated platelets. Furthermore, the disruption of SM-rich rafts by methyl-b-cyclodextrin impaired myosin activation and clot retraction. Thus, we propose that clot retraction takes place in SM-rich rafts where a fibrin-aIIbb3-myosin complex is formed as a primary axis to promote platelet contraction. (Blood. 2013;122(19):3340-3348) IntroductionMembrane rafts are dynamic assemblies of sphingolipids, cholesterol, and proteins that can be stabilized into platforms involved in the regulation of a number of vital cellular processes. 1 The important role of rafts at the cell surface may be their function in signal transduction. A number of studies provide considerable evidence that rafts are integral to the regulation of immune and neuronal signaling. Membrane rafts are also involved in hemostasis and thrombosis. Among blood cells, platelets are critical for maintaining the integrity of the blood coagulation system. Platelet rafts are critical membrane domains in physiological responses such as adhesion and aggregation. 2 The localization of the adhesion receptor glycoprotein (GP)Ib-IX-V complex to membrane rafts is required for platelet adhesion to the vessel wall by binding the von Willebrand factor. 3 Membrane rafts are also required for platelet aggregation via the collagen receptor GPVI, 4 the adenosine 59-diphosphate (ADP) receptor P2Y12, 5 the Fcg receptor FcgRIIa, 6 and the C-type lectinlike receptor CLEC-2.7 Detergent-resistant membrane (DRM) rafts of platelets show round vesicles of heterogeneous sizes ranging from 20 to 500 nm, which are enriched in CD36 (GPIV). 8,9 Recent reports have demonstrated that membrane rafts are ...

Factor XIII (FXIII) is a plasma transglutaminase that cross-links fibrin monomers, ␣ 2 -plasmin inhibitor, and so forth. Congenital FXIII deficiency causes lifelong bleeding symptoms. To understand the molecular pathology of FXIII deficiency in vivo, its knockout mice have been functionally analyzed. Because prolonged bleeding times, a sign of defective/abnormal primary hemostasis, were commonly observed in 2 separate lines of FXIII A subunit (FXIII-A) knockout mice, a possible role or roles of FXIII in platelet-related function was investigated in the present study. Although platelet aggregation induced by adenosine diphosphate or collagen was normal, clot retraction (CR) was lost in the platelet-rich plasma (PRP) of FXIII-A knockout mice. IntroductionCoagulation factor XIII (FXIII) is a pro-enzyme of plasma transglutaminase (TGase) consisting of 2 enzymatic A subunits (FXIII-A) and 2 noncatalytic B subunits, and plays a critical role in the generation of a stable hemostatic plug. 1-3 FXIII catalyzes intermolecular cross-linking reactions between fibrin monomers, ␣ 2 -plasmin inhibitor, fibronectin, etc. These reactions increase the mechanical strength of the fibrin clot and its resistance to proteolytic degradation, and enhance the assembly of the extracellular matrix.Congenital FXIII deficiency is a rare autosomal recessive disorder, the cases of most of which are caused by defects in the FXIII-A gene. 3 A lifelong bleeding tendency, abnormal wound healing, and recurrent spontaneous miscarriage are common symptoms of FXIII deficiency. 1,4 FXIII-A exists extracellularly in plasma as well as intracellularly as a cytosolic protein in megakaryocytes/platelets and monocytes/macrophages, although the function(s) of intracellular FXIII-A remain(s) unknown. 5,6 In particular, platelets cause clot retraction (CR) by retracting extended filopodia along fibrin strands. 7 There have been conflicting reports about the effects of FXIII deficiency on CR; investigators reported that the absence of FXIII abolished, 7-9 did not affect, 10,11 or rather enhanced 12 CR in patients with congenital FXIII deficiency. However, platelet aggregation induced by various agents is uniformly normal in patients with congenital FXIII deficiency. 8,9,13,14 To understand the precise molecular pathology of FXIII deficiency in vivo, FXIII-A knockout (KO) mice have been analyzed. FXIII-A KO mice demonstrated a severe bleeding tendency. 15 We also reported that FXIII-A KO mice developed severe uterine bleeding, resulting in spontaneous miscarriage in females, and male-specific intrathoracic hemorrhage as well as excessive cardiac fibrosis. 16,17 Because bleeding times in FXIII-A KO mice were longer than in wild-type, 15,18 we hypothesized that FXIII-A KO mice might have a defective platelet-related function(s). Accordingly, we have explored the contribution of FXIII to the process of CR in the present study. Methods AnimalWild-type C57BL/6J mice were obtained from Japan SLC Inc. Genetargeted mice of FXIII-A were generated as previously reported, ...

Temperature affects the physiology, behavior, and evolution of organisms. We conducted mutagenesis and screens for mutants with altered temperature preference in Drosophila melanogaster and identified a cryophilic (cold-seeking) mutant, named atsugari (atu). Reduced expression of the Drosophila ortholog of dystroglycan (DmDG) induced tolerance to cold as well as preference for the low temperature. A sustained increase in mitochondrial oxidative metabolism caused by the reduced expression of DmDG accounted for the cryophilic phenotype of the atu mutant. Although most ectothermic animals do not use metabolically produced heat to regulate body temperature, our results indicate that their thermoregulatory behavior is closely linked to rates of mitochondrial oxidative metabolism and that a mutation in a single gene can induce a sustained change in energy homeostasis and the thermal responses.

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Absolute energy distribution of hard x rays produced in the interaction of a kilohertz femtosecond laser with tantalum targets Rev. Sci. Instrum. 77, 093302 (2006); Fast protons are observed by a newly developed online time-of-flight spectrometer, which provides shot-to-shot proton-energy distributions immediately after the irradiation of a laser pulse having an intensity of ϳ10 18 W/cm 2 onto a 5-m-thick copper foil. The maximum proton energy is found to increase when the intensity of a fs prepulse arriving 9 ns before the main pulse increases from 10 14 to 10 15 W/cm 2 . Interferometric measurement indicates that the preformed-plasma expansion at the front surface is smaller than 15 m, which corresponds to the spatial resolution of the diagnostics. This sharp gradient of the plasma has the beneficial effect of increasing the absorption efficiency of the main-pulse energy, resulting in the increase in the proton energy. This is supported by the result that the x-ray intensity from the laser plasma clearly increases with the prepulse intensity.

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We found an autoimmune serum, K199, that strongly suppresses nuclear membrane assembly in a cell-free system involving a Xenopus egg extract. Four different antibodies that suppress nuclear assembly were affinity-purified from the serum using Xenopus egg cytosol proteins. Three proteins recognized by these antibodies were identified by partial amino acid sequencing to be glyceraldehyde-3-phosphate dehydrogenase (GAPDH), fructose-1,6-bisphosphate aldolase, and the regulator of chromatin condensation 1. GAPDH is known to be a fusogenic protein. To verify the participation of GAPDH in nuclear membrane fusion, authentic antibodies against human and rat GAPDH were applied, and strong suppression of nuclear assembly at the nuclear membrane fusion step was observed. The nuclear assembly activity suppressed by antibodies was recovered on the addition of purified chicken GAPDH. A peptide with the sequence of amino acid residues 70 -94 of GAPDH, which inhibits GAPDH-induced phospholipid vesicle fusion, inhibited nuclear assembly at the nuclear membrane fusion step. We propose that GAPDH plays a crucial role in the membrane fusion step in nuclear assembly in a Xenopus egg extract cell-free system. The nuclear envelope (NE)1 of eukaryotes is composed of inner and outer nuclear membranes, nuclear pore complexes, and the nuclear lamina. NE breakdown during the mitotic prophase results in the dispersal of both nuclear membranes into the mitotic endoplasmic reticulum (ER) network. Inner nuclear membrane proteins such as lamin B receptor, laminaassociated polypeptide 2␤, and emerin reconcentrate on the surface of decondensing chromatin during the late anaphase, and then the NE is re-formed (1, 2). These intrinsic membrane proteins and lamins are believed to play a critical role in NE reassembly (3-5). NE assembly can be studied in vitro using extracts of meiotic or mitotic cells (6, 7). The assembly requires cytosolic factors and is inhibited by a non-hydrolyzable GTP analogue, N-ethylmaleimide, and a calcium chelator, BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid) (8). In vitro, NE assembly is initiated by the binding of membrane vesicles to decondensed chromatin in an energy-independent manner (9 -11). Once bound to chromatin, membrane vesicles fuse and flatten. The insertion of nuclear pore complexes and the expansion of the NE require both energy and cytosolic components (12). The fusion of vesicles on chromatin is inhibited by a non-hydrolyzable GTP analogue (8, 13) and requires Ran GTPase and a regulator of chromatin condensation 1, RCC1/Ran GTP exchange factor (14 -16). Remarkably, agarose beads coated with Ran allow the assembly of a nuclear pore complex-containing NE (17, 18). After the formation of a closed NE around chromatin, further NE growth requires nucleocytoplasmic transport and further membrane fusion (8,19). Analysis of membrane fractions of a Xenopus egg extract suggested that the population of vesicles required for NE assembly is not uniform and that different membranes might be required fo...