CrkL directs ASAP1 to peripheral focal adhesions

Searching for proteins in platelets that can interact with the N-terminal SH3 domain of CrkL (using a combination of a pull-down assay followed by mass spectrometry), we have found that human platelets express an ADP-ribosylation factor (Arf)-specific GTPase-activating protein (GAP), ASAP1, as a CrkL-binding protein. In spreading platelets, most endogenous ASAP1 is localized at peripheral focal adhesions. To determine the physiologic significance of the CrkL-ASAP1 association, we overexpressed CrkL, ASAP1, or both in combination in COS7 cells. Unlike endogenous ASAP1 in platelets, overexpressed ASAP1 showed diffuse cytoplasmic distribution. However, when co-expressed with wild-type CrkL, both endogenous and expressed ASAP1 accumulated at CrkL-induced focal adhesions. An SH2-mutated CrkL, which cannot localize at focal adhesions, failed to recruit ASAP1 into focal adhesions. Thus, CrkL appears to be a lynchpin between ASAP1 and peripheral focal adhesions.     

Comparison of the macromolecular MR contrast agents with ethylenediamine-core versus ammonia-core generation-6 polyamidoamine dendrimer

Two novel macromolecular MRI contrast agents based upon generation-6 polyamidoamine dendrimers (G6) of presumed similar molecular size, but of different molecular weight, were compared in terms of their blood retention, tissue distribution, and renal excretion. Two G6s with either ammonia core (G6A) or with ethylenediamine core (G6E), which possessed 192 and 256 exterior primary amino groups, respectively, were used. These dendrimers were reacted with 2-(p-isothiocyanatobenzyl)-6-methyl-diethylenetriaminepentaacetic acid (1B4M). The G6--1B4M conjugates were reacted with (153)Gd for studying biodistribution and blood clearance or Gd(III) for the MRI study. 3D-micro-MR angiography of the mice were taken with injection of 0.033 mmol of Gd/kg of G6A--(1B4M-Gd)(192) or G6E--(1B4M-Gd)(256) using a 1.5-T superconductive MRI unit. Although numerous fine vessels of approximately 100 microm diameter were visualized on subtracted 3D-MR-angiography with both G6A--(1B4M-Gd)(192) and G6E--(1B4M-Gd)(256), (153)Gd-labeled saturated G6E-(1B4M)(256) remained in the blood significantly more than (153)Gd-labeled saturated G6A--(1B4M)(192) at later than 15 min postinjection (p < 0.01). In addition, G6E--(1B4M-Gd)(256) visualized these finer vessels longer than G6A--(1B4M-Gd)(192). The G6A--(1B4M-Gd)(192) showed higher signal intensity in the kidney on the dynamic MR images and brighter kidney images than G6E--(1B4M-Gd)(256). In conclusion, the G6A--(1B4M-Gd)(192) was observed to go through glomerular filtration more efficiently than G6E--(1B4M-Gd)(256) resulting faster clearance from the blood and higher renal accumulation, even though both of G6--1B4M conjugates have almost similar molecular size and same chemical structure. In terms of the ability of intravascular contrast agents, G6E--(1B4M-Gd)(256) was better due to more Gd(III) atoms per molecule and longer retention in the circulation than G6A--(1B4M-Gd)(192).     

Role of Rab3 GDP/GTP exchange protein in synaptic vesicle trafficking at the mouse neuromuscular junction

The Rab3 small G protein family consists of four members, Rab3A, -3B, -3C, and -3D. Of these members, Rab3A regulates Ca(2+)-dependent neurotransmitter release. These small G proteins are activated by Rab3 GDP/GTP exchange protein (Rab3 GEP). To determine the function of Rab3 GEP during neurotransmitter release, we have knocked out Rab3 GEP in mice. Rab3 GEP-/- mice developed normally but died immediately after birth. Embryos at E18.5 showed no evoked action potentials of the diaphragm and gastrocnemius muscles in response to electrical stimulation of the phrenic and sciatic nerves, respectively. In contrast, axonal conduction of the spinal cord and the phrenic nerve was not impaired. Total numbers of synaptic vesicles, especially those docked at the presynaptic plasma membrane, were reduced at the neuromuscular junction approximately 10-fold compared with controls, whereas postsynaptic structures and functions appeared normal. Thus, Rab3 GEP is essential for neurotransmitter release and probably for formation and trafficking of the synaptic vesicles.     

Multiple roles of Arf1 GTPase in the yeast exocytic and endocytic pathways

ADP-ribosylation factors, a family of small GTPases, are believed to be key regulators of intracellular membrane traffic. However, many biochemical in vitro experiments have led to different models for their involvement in various steps of vesicular transport, and their precise role in living cells is still unclear. We have taken advantage of the powerful yeast genetic system and screened for temperature-sensitive (ts) mutants of the ARF1 gene from Saccharomyces cerevisiae. By random mutagenesis of the whole open reading frame of ARF1 by error-prone PCR, we isolated eight mutants and examined their phenotypes. arf1 ts mutants showed a variety of transport defects and morphological alterations in an allele-specific manner. Furthermore, intragenic complementation was observed between certain pairs of mutant alleles, both for cell growth and intracellular transport. These results demonstrate that the single Arf1 protein is indeed involved in many different steps of intracellular transport in vivo and that its multiple roles may be dissected by the mutant alleles we constructed.     

Effect of gravity change on thrombopoiesis in mice

It is reported that the stay in the space develops anemia, thrombocytopenia, and altered function and structure of red blood cell. The mechanism of these abnormalities was not clarified yet. Therefore, it is necessary to elucidate the mechanism of the effect of the gravity change on the thrombocytopoiesis, which plays the important role for the hemostasis, using animal models. The cloning of thrombopoietin (TPO), followed by the analysis of TPO and c-mpl (its cellular receptor) knockout mice confirmed its role as the primary regulator of thrombopoiesis. TPO has been shown to stimulate both megakaryocyte colony growth from marrow progenitor cells and the maturation of immature megakaryocyte to form functional platelet. This process includes the massive cytoskeletal rearrangement, such as proplatelet formation and fragmentation of proplatelet. In this study we have focused on the thrombopoiesis in mice those were exposed to gravity change by parabolic flight (PF).     

Analysis of Mn(2+)/Ca(2+) influx and release during Ca(2+) oscillations in mouse eggs injected with sperm extract

Repetitive Ca(2+) release from the endoplasmic reticulum (ER) is necessary for activation of mammalian eggs. Influx and release of Mn(2+) and Ca(2+) during Ca(2+) oscillations induced by injection of sperm extract (SE) into mouse eggs were investigated by Mn(2+)-quenching of intracellular Fura-2 after adding Mn(2+) to external medium. Mn(2+)/Ca(2+) influx was detected at the resting state. A marked Mn(2+)/Ca(2+) influx occurred during the first Ca(2+) release upon SE injection, and persistently facilitated Mn(2+)/Ca(2+) influx was observed during steady Ca(2+) oscillations. As intracellular Mn(2+) concentration ([Mn(2+)](i)) increased progressively, periodic [Mn(2+)](i) rises appeared, corresponding to each Ca(2+)transient but taking a slower time course. A numerical simulation based on continuous Mn(2+)/Ca(2+) influx-extrusion across the plasma membrane and release-uptake across the ER membrane in a competitive manner mimicked well the Mn(2+) oscillations calculated from experimental data, strongly suggesting that repetitive Mn(2+) release develops after Mn(2+) entry and uptake into the ER. In other experiments, a marked Mn(2+) influx occurred upon Mn(2+) addition to Ca(2+)-free medium after depletion of the ER using an ER Ca(2+) pump inhibitor plus repeated injection of inositol 1,4,5-trisphosphate (InsP(3)). No significant increase in Mn(2+) influx was induced by injection of SE, InsP(3), or Ca(2+), when Ca(2+) release was prevented by pre-injection of an antibody against the InsP(3) receptor. We concluded that Ca(2+) influx is activated during the initial large Ca(2+)release possibly by a capacitative mechanism and kept facilitated during steady Ca(2+) oscillations. The finding that repetitive Mn(2+) release is caused by continuous Mn(2+) entry suggests that continuous Ca(2+) influx may play a critical role in refilling the ER and, thereby, maintaining Ca(2+)oscillations in mammalian fertilization.     

Crystal structures of substrate free and complex forms of reactivated BphC, an extradiol type ring-cleavage dioxygenase

BphC derived from Pseudomonas sp. strain KKS102, an extradiol type catecholic dioxygenase, is a non-heam iron-containing enzyme, playing an important role in the degradation of biphenyl/PCB (Poly Chlorinated Biphenyls) in the microbe. Although we had earlier solved the crystal structure of KKS102 BphC, it was the inactive form with Fe(III) in the active site. In order to determine the active form structure, BphC was re-activated by anaerobic incubation with Fe(II) and ascorbate, and crystallized anaerobically. The crystal structures of activated BphC and its substrate complex (E x S complex) were determined at 2.0 A resolution under cryogenic condition. In addition, crystal structures of unactivated BphC in substrate free and complex forms were also re-determined. Comparison of activated and unactivated E x S complexes reveals that the orientation of the bound substrate in the active site is significantly different between the two. The structural comparison of the substrate free and complex forms of activated BphC show certain small conformational shifts around the active site upon substrate binding. As a result of the conformational shifts, His194, which has been suggested as the catalytic base, takes part in a weak hydrogen bond with hydroxyl group of the substrate.