The cDNA and deduced amino acid sequence of the gamma subunit of the L-type calcium channel from rabbit skeletal muscle

Complementary DNAs for the gamma subunit of the calcium channel of rabbit skeletal muscle were isolated on the basis of peptide sequences derived from the purified protein. The deduced primary structure is without homology to other known protein sequences and is consistent with the gamma subunit being an integral membrane protein.     

The Ndc80p complex from Saccharomyces cerevisiae contains conserved centromere components and has a function in chromosome segregation

We have purified a complex from Saccharomyces cerevisiae containing the spindle components Ndc80p, Nuf2p, Spc25p, and Spc24p. Temperature-sensitive mutants in NDC80, SPC25, and SPC24 show defects in chromosome segregation. In spc24-1 cells, green fluorescence protein (GFP)-labeled centromeres fail to split during spindle elongation, and in addition some centromeres may detach from the spindle. Chromatin immunoprecipitation assays show an association of all four components of the complex with the yeast centromere. Homologues of Ndc80p, Nuf2p, and Spc24p were found in Schizosaccharomyces pombe and GFP tagging showed they were located at the centromere. A human homologue of Nuf2p was identified in the expressed sequence tag database. Immunofluorescent staining with anti-human Nuf2p and with anti-HEC, the human homologue of Ndc80p, showed that both proteins are at the centromeres of mitotic HeLa cells. Thus the Ndc80p complex contains centromere-associated components conserved between yeasts and vertebrates.     

Importance of the pleckstrin homology domain of dynamin in clathrin-mediated endocytosis

The GTPase dynamin plays an essential role in clathrin-mediated endocytosis [1] [2] [3]. Substantial evidence suggests that dynamin oligomerisation around the necks of endocytosing vesicles and subsequent dynamin-catalysed GTP hydrolysis is responsible for membrane fission [4] [5]. The pleckstrin homology (PH) domain of dynamin has previously been shown to interact with phosphoinositides, but it has not been determined whether this interaction is essential for dynamin's function in endocytosis [6] [7] [8] [9]. In this study, we address the in vivo function of the PH domain of dynamin by assaying the effects of deletions and point mutations in this region on transferrin uptake in COS-7 fibroblasts. Overexpression of a dynamin construct lacking its entire PH domain potently blocked transferrin uptake, as did overexpression of a dynamin construct containing a mutation in the first variable loop of the PH domain. Structural modelling of this latter mutant suggested that the lysine residue at position 535 (Lys535) may be critical in the coordination of phosphoinositides, and indeed, the purified mutant no longer interacted with lipid nanotubes. Interestingly, the inhibitory phenotype of cells expressing this dynamin mutant was partially relieved by a second mutation in the carboxy-terminal proline-rich domain (PRD), one that prevents dynamin from binding to the Src homology 3 (SH3) domain of amphiphysin. These data demonstrate that dynamin's interaction with phosphoinositides through its PH domain is essential for endocytosis. These findings also support our hypothesis that PRD-SH3 domain interactions are important in the recruitment of dynamin to sites of endocytosis.     

Localization of a gene for syndactyly type 1 to chromosome 2q34-q36

Syndactyly type 1 (SD1) is an autosomal dominant limb malformation characterized in its classical form by complete or partial webbing between the third and fourth fingers and/or the second and third toes. After exclusion of a candidate region previously identified for syndactyly type 2 (synpolydactyly), we performed a genomewide linkage analysis in a large German pedigree. We found evidence for linkage of SD1 to polymorphic markers on chromosome 2q34-q36, with a maximum LOD score of 12.40 for marker D2S301. Key recombination events in affected individuals defined a 9.4-cM region between markers D2S2319 and D2S344. The identification of the responsible gene will give further insights into the molecular basis of limb development.     

Cdc42 and phosphoinositide 3-kinase drive Rac-mediated actin polymerization downstream of c-Met in distinct and common pathways

Activation of c-Met, the hepatocyte growth factor (HGF)/scatter factor receptor induces reorganization of the actin cytoskeleton, which drives epithelial cell scattering and motility and is exploited by pathogenic Listeria monocytogenes to invade nonepithelial cells. However, the precise contributions of distinct Rho-GTPases, the phosphatidylinositol 3-kinases, and actin assembly regulators to c-Met-mediated actin reorganization are still elusive. Here we report that HGF-induced membrane ruffling and Listeria invasion mediated by the bacterial c-Met ligand internalin B (InlB) were significantly impaired but not abrogated upon genetic removal of either Cdc42 or pharmacological inhibition of phosphoinositide 3-kinase (PI3-kinase). While loss of Cdc42 or PI3-kinase function correlated with reduced HGF- and InlB-triggered Rac activation, complete abolishment of actin reorganization and Rac activation required the simultaneous inactivation of both Cdc42 and PI3-kinase signaling. Moreover, Cdc42 activation was fully independent of PI3-kinase activity, whereas the latter partly depended on Cdc42. Finally, Cdc42 function did not require its interaction with the actin nucleation-promoting factor N-WASP. Instead, actin polymerization was driven by Arp2/3 complex activation through the WAVE complex downstream of Rac. Together, our data establish an intricate signaling network comprising as key molecules Cdc42 and PI3-kinase, which converge on Rac-mediated actin reorganization essential for Listeria invasion and membrane ruffling downstream of c-Met.     

Chloroplast Signaling Gates Thermotolerance in Arabidopsis

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Methane emissions from rice microcosms: The balance of production, accumulation and oxidation

In rice microcosms (Oryza sativa, var. Roma, type japonica),CH₄ emission, CH₄ production, CH₄oxidation and CH₄ accumulation were measured over an entirevegetation period. Diffusive CH₄ emission was measured inclosed chambers, CH₄ production was measured in soil samples,CH₄ oxidation was determined from the difference between oxicand anoxic emissions, and CH₄ accumulation was measured byanalysis of porewater and gas bubbles. The sum of diffusiveCH₄ emission, CH₄ oxidation, andCH₄ accumulation was only 60% of the cumulativeCH₄ production. The two values diverged during the first 50days (vegetative phase) and then again during the last 50 days (latereproductive phase and senescence) of the 150 day vegetation period. Duringthe period of day 50–100 (early reproductive phase/flowering), theprocesses were balanced. Most likely, gas bubbles and diffusion limitationare responsible for the divergence in the early and late phases. The effectof rice on CH₄ production rates and CH₄concentrations was studied by measuring these processes also in unplantedmicrocosms. Presence of rice plants lowered the CH₄concentrations, but had no net effect on the CH₄ productionrates.     

Influence of Photorespiration on ATP/ADP Ratios in the Chloroplasts, Mitochondria, and Cytosol, Studied by Rapid Fractionation of Barley (Hordeum vulgare) Protoplasts

Using the principle described by R McC Lilley, M Stitt, G Mader, HW Heldt (1982 Plant Physiol 70: 965-970), an apparatus for rapid fractionation of barley leaf (Hordeum vulgare) protoplasts by membrane filtration was built. From studies of ATP/ADP ratios, it is concluded that the quenching of metabolic reactions is very fast, making it possible to use the method for studies on metabolic interactions between different compartments in plant cells. The fractionation method was used to study the influence of photorespiration on ATP/ADP ratios in the chloroplasts, mitochondria, and cytosol of barley leaf protoplasts. The cytosolic ATP/ADP ratio was higher under photorespiratory conditions than under nonphotorespiratory conditions. Aminoacetonitrile, an inhibitor of the photorespiratory conversion of glycine to serine, had a very small effect on the ATP/ADP ratios in the different subcellular compartments during photosynthesis in nonphotorespiratory conditions (saturating CO₂). In photorespiratory conditions (limiting CO₂), on the other hand, aminoacetonitrile increased the ATP/ADP ratio in the chloroplasts and decreased the ATP/ADP ratios in the mitochondria and the cytosol. These results are consistent with the hypothesis, that during photorespiration glycine oxidation is coupled to oxidative phosphorylation to provide ATP to the cytosol.