AGAP1, an endosome-associated,phosphoinositide-dependent ADP-ribosylation factor GTPase-activating protein that affects actin cytoskeleton

We have identified three members of the AGAP subfamily of ASAP family ADP-ribosylation factor GTPase-activating proteins (Arf GAPs). In addition to the Arf GAP domain, these proteins contain GTP-binding protein-like, ankyrin repeat and pleckstrin homology domains. Here, we have characterized the ubiquitously expressed AGAP1/KIAA1099. AGAP1 had Arf GAP activity toward Arf1>Arf5>Arf6. Phosphatidylinositol 4,5-bisphosphate and phosphatidic acid synergistically stimulated GAP activity. As found for other ASAP family Arf GAPs, the pleckstrin homology domain was necessary for activity. Deletion of the GTP-binding protein-like domain affected lipid dependence of Arf GAP activity. In vivo effects of AGAP1 were distinct from other ASAP family Arf GAPs. Overexpressed AGAP1 induced the formation of and was associated with punctate structures containing the endocytic markers transferrin and Rab4. AP1 was redistributed from the trans-Golgi to the punctate structures. Like other ASAP family members, AGAP1 overexpression inhibited the formation of PDGF-induced ruffles. However, distinct from other ASAP family members, AGAP1 also induced the loss of actin stress fibers. Thus, AGAP1 is a phosphoinositide-dependent Arf GAP that impacts both the endocytic compartment and actin.     

The face of TSR revealed: an extracellular signaling domain is exposed

In this issue, Tan et al. (2002) report the first high resolution (1.9 A) structural data for thrombospondin (TSP)-1, a large multifunctional protein that regulates cell adhesion, angiogenesis, cell proliferation and survival, TGFbeta activation, and protease function (for review see Chen et al., 2000). Because TSP-1 has multiple binding partners and many functions, precise structural information is crucial to understanding its biology. The structure now reported, derived from crystals of the second and third type I repeats of TSP-1 is of particular interest because of the specific functions attributed to these repeats and because domains homologous to the repeats appear in many other proteins in nature. The novel layered fold motif described brings great insight into how the complicated functions of TSP-1 and related molecules are affected.     

Genomic evidence for the absence of a functional cholesteryl ester transfer protein gene in mice and rats

Mice and rats are naturally deficient in cholesteryl ester transfer protein (CETP) activity, although the reason behind the deficiency in activity is unknown. A search of mouse genome databases revealed sequences resembling 7 of the 16 human exons. However, these sequences could not code for a functional CETP. Analysis of the rat genome using Southern blotting revealed sequences complementary to human CETP cDNA, but RNase protection assays were unable to detect any Cetp gene expression in liver, adipose, or muscle. A search of rat whole-genome shotgun databases revealed exon-like sequences that would be unable to code for a functional CETP. An Ap3s1 pseudogene lay immediately upstream of the CETP-like sequences in mouse, but was nearly identical to the functional gene and unlikely to have been inserted prior to mouse-rat divergence. In contrast, a deletion leading to a nonsense codon was found in the exon 11-like sequences of both rat and mouse and not in any other species. Thus, the lack of CETP activity in both the mouse and the rat is most likely due to an evolutionary event that occurred before these species diverged and not to altered regulation of the gene or function of the gene product.     

Cellular lipid dynamics

During the past years, the notion of microdomains at the surface of cellular membranes has been developed. These are constituted by lipid rafts which involve sphingoglycolipids and cholesterol. To these rafts are associated proteins which have a lipid anchor or are transmembrane proteins. These lipid rafts target specific proteins at the plasma membrane surface and can remain associated with them. They are present in surface receptors and endocytosis occurs upon binding of the specific ligands. Thus these rafts participate to major aspects of cellular dynamics. These rafts are complex structures, insoluble in non-ionic detergents. According to the detergent used, many types of rafts can be isolated. Any alteration of cholesterol, sphingoglycolipids, or abnormalities of the proteins themselves, can lead to abnormal targeting at the membrane surface. It is possible that specific sphingoglycolipids are necessary to target specific proteins at the membrane surface. This may explain the complexity of the sphingoglycolipid molecules, both in relation to their oligosaccharide and to their ceramide structures. There is both a cellular and a tissue specificity of these constituents. Complex sphingoglycolipids are involved in cellular differentiation, cellular polarization, and modified in relation to cancer. Virus and bacteria can be linked to the sphingoglycolipids of these microdomains and alter cellular signaling and function. Sphingoglycolipids are involved in autoimmune diseases as antibody targets and in neurolipidoses which are genetic diseases involving their catabolism. The dynamics of the lipid rafts, in relation to cholesterol, can be altered in Niemann-Pick's disease type C and in Alzheimer's disease. Thus these microdomains are involved in many aspects related to normal and pathological cellular dynamics.     

Putative roles of the CNF2 and CDTIII toxins in experimental infections with necrotoxigenic Escherichia coli type 2 (NTEC2) strains in calves

Newborn colostrum-restricted calves were orally inoculated with an Escherichia coli strain, identified originally as non-pathogenic, and into which the plasmid pVir was conjugally transferred. This resulted in diarrhea, intestinal lesions and extra-intestinal invasion, suggesting that factors affecting these pathogenic properties are located on pVir. In order to analyze the respective roles of the toxins CNF2 and CDTIII in the pathogenesis, colostrum-restricted calves were inoculated with isogenic mutants in the cnf2 and the cdt-III genes. The loss of cnf2 is associated with a reduction in the pathogenicity, since diarrhea does not occur in calves challenged, in spite of successful colonization of the intestine. Nevertheless, the mutant strain remains able to invade the bloodstream and to localize in the internal organs. Conversely, the calves inoculated with mutant in the cdt-III gene evolved in the same way as wild-type strain-inoculated calves with regard to clinical signs and macroscopic or microscopic lesions.     

Starfish: Fault-Tolerant Dynamic MPI Programs on Clusters of Workstations

This paper reports on the architecture and design of Starfish, an environment for executing dynamic (and static) MPI-2 programs on a cluster of workstations. Starfish is unique in being efficient, fault-tolerant, highly available, and dynamic as a system internally, and in supporting fault-tolerance and dynamicity for its application programs as well. Starfish achieves these goals by combining group communication technology with checkpoint/restart, and uses a novel architecture that is both flexible and portable and keeps group communication outside the critical data path, for maximum performance.     

Genetics of schizophrenia and bipolar disorder: recent success of linkage studies with evidence of specific and shared susceptibility loci

Results claiming linkage on two chromosomes for schizophrenia (SZ) and bipolar affective disorder (BP) were prematurely published in Nature at the end of the '80s. This ended up into disappointment. The knowledge accumulated from the first generation of unsuccessful molecular genetics studies of SZ and BP provided a stronger basis for the following generation of linkage studies that are now yielding encouraging converging results. Hence, we report several genomics susceptibility loci for SZ and BP, some of them being probably shared by the two major psychiatric illnesses whereas others could be specific to each.     

Role of the promyelocytic leukemia body in the dynamic interaction between the androgen receptor and steroid receptor coactivator-1 in living cells

The dynamic interaction between the androgen receptor (AR) and steroid receptor coactivator-1 (SRC-1) was explored in living cells expressing chimeric forms of the receptor and the coactivator containing two spectral variants of jellyfish fluorescent protein. Laser scanning confocal imaging of transfected cells expressing fluorescently labeled SRC-1 revealed that in an unsynchronized cell population, the coactivator is distributed in approximately 40% cells as nuclear bodies of 0.2-1.0 microm in diameter. Immunostaining of cyan fluorescent protein-labeled SRC-1 (CFP-SRC1)-expressing cells with antibody to promyelocytic leukemia (PML) protein showed significant overlap of the CFP fluorescence with the antibody stain. Cotransfection of cells with a plasmid expressing the CFP conjugate of Sp100 (another marker protein for the PML nuclear body) also showed colocalization of the yellow fluorescent protein (YFP)-SRC1 containing nuclear foci with the PML bodies in living cells. Analysis of the three-dimensional structure revealed that the PML bodies are round to elliptical in shape with multiple satellite bodies on their surface. Some of these satellite bodies contain the SRC-1. Activation and nuclear import of CFP-AR by the agonistic ligand 5alpha-dihydrotestosterone, but not by the antagonist casodex, transferred YFP-SRC1 from the PML bodies to an interlacing filamentous structure. In a single living cell, agonist-activated AR caused a time-dependent movement of YFP-SRC1 from the PML bodies to this filamentous structure. Additionally, coexpression of a constitutively active mutant of AR (AR-deltaligand binding domain) also displaced YFP-SRC1 from the PML bodies to this intranuclear filamentous structure. The fluorescence recovery after photobleaching approach was used to examine changes in the kinetics of movement of YFP-SRC1 during its mobilization from the PML bodies to the intranuclear filamentous structure by the agonist-activated AR. Results of the relative half-times (t(1/2)) of replacement of YFP-SRC1 within the photobleached region of a single PML body from its surrounding nuclear space supported the conclusion that SRC-1 is actively transported from the PML bodies to the intranuclear filamentous structure by the ligand-activated AR. This observation also suggests an interaction between AR and SRC-1 before its binding to the target gene. The PML bodies have been implicated as a cross-road for multiple regulatory pathways that control cell proliferation, cellular senescence, and apoptosis. Our present results along with other recent reports expand the role of this subnuclear structure to include the regulation of steroid hormone action.