Elimination of host cell PtdIns(4,5)P(2) by bacterial SigD promotes membrane fission during invasion by Salmonella

Salmonella invades mammalian cells by inducing membrane ruffling and macropinocytosis through actin remodelling. Because phosphoinositides are central to actin assembly, we have studied the dynamics of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)) in HeLa cells during invasion by Salmonella typhimurium. Here we show that the outermost parts of the ruffles induced by invasion show a modest enrichment in PtdIns(4,5)P(2), but that PtdIns(4,5)P(2) is virtually absent from the invaginating regions. Rapid disappearance of PtdIns(4,5)P(2) requires the expression of the Salmonella phosphatase SigD (also known as SopB). Deletion of SigD markedly delays fission of the invaginating membranes, indicating that elimination of PtdIns(4,5)P(2) may be required for rapid formation of Salmonella-containing vacuoles. Heterologous expression of SigD is sufficient to promote the disappearance of PtdIns(4,5)P(2), to reduce the rigidity of the membrane skeleton, and to induce plasmalemmal invagination and fission. Hydrolysis of PtdIns(4,5)P(2) may be a common and essential feature of membrane fission during several internalization processes including invasion, phagocytosis and possibly endocytosis.     

Effects of digestive status on the reptilian gut

Reptiles, including the Burmese python, Python molurus bivittatus, that feed at infrequent intervals show a prominent increase in gastrointestinal mass, metabolism and brush border transport rates after feeding. Current knowledge and theories around these phenomena, as well as studies on the innervation of the reptilian gut, are summarised in this review. Little is known about the putative changes in the nervous and humoral control systems of the gut, and it is not known whether feeding affects innervation and motility of the stomach and intestine. Using immunohistochemistry, we have investigated possible up/down regulation of several neurotransmitters in specimens that had been fasted for a minimum of 3 weeks and specimens that had ingested a large meal 2 days before the experiments were conducted. There were no major changes in the innervation by nerves containing calcitonin gene-related peptide (CGRP), galanin, nitric oxide synthase (NOS), pituitary adenylate cyclase-activating polypeptide (PACAP), somatostatin (SOM), substance P/neurokinin A (SP/NKA), or vasoactive intestinal polypeptide (VIP)-like immunoreactivity. Nor did we find any differences in the effect of substance P (stomach and intestine), galanin (intestine), or bradykinin (intestine) on motility in strip preparations from the gut wall. A significant increase in dry weight of the intestine was obtained 48 h after feeding. We conclude that although there are considerable changes in gut thickness and absorptive properties after feeding, the smooth muscle and its control appear little affected.     

Lateral diffusion in substrate-supported lipid monolayers as a function of ambient relative humidity

We analyzed the influence of water activity on the lateral self-diffusion of supported phospholipid monolayers. Lipid monolayer membranes were supported by polysaccharide cushions (chitosan and agarose), or glass. A simple diffusion model was derived, based on activated diffusion with an activation energy, E(a), which depends on the hydration state of the lipid headgroup. A crucial assumption of the derived model is that E(a) can be calculated assuming an exponential decay of the humidity-dependent disjoining pressure in the monolayer/substrate interface with respect to the equilibrium separation distance. A plot of ln(D) against ln(p(0)/p), where D is the measured diffusion coefficient and p(0) and p are the partial water pressures at saturation and at a particular relative humidity, respectively, was observed to be linear in all cases (i.e., for differing lipids, lateral monolayer pressures, temperatures, and substrates), in accordance with the above-mentioned diffusion model. No indications for humidity-induced first-order phase transitions in the supported phospholipid monolayers were found. Many biological processes such as vesicle fusion and recognition processes involve dehydration/hydration cycles, and it can be expected that the water activity significantly affects the kinetics of these processes in a manner similar to that examined in the present work.     

Mitochondrial respiratory rates and activities of respiratory chain complexes correlate linearly with heteroplasmy of deleted mtDNA without threshold and independently of deletion size

To clarify the importance of deleted protein and tRNA genes on the impairment of mitochondrial function, we performed a quantitative analysis of biochemical, genetic and morphological findings in skeletal muscles of 16 patients with single deletions and 5 patients with multiple deletions of mtDNA. Clinically, all patients showed chronic progressive external ophthalmoplegia (CPEO). The size of deletions varied between 2.5 and 9 kb, and heteroplasmy between 31% and 94%. In patients with single deletions, the citrate synthase (CS) activity was nearly doubled. Decreased ratios of pyruvate- and succinate-dependent respiration were detected in fibers of all patients in comparison to controls. Inverse and linear correlations without thresholds were established between heteroplasmy and (i) CS referenced activities of the complexes of respiratory chain, (ii) CS referenced maximal respiratory rates, (iii) and cytochrome-c-oxidase (COX) negative fibers. In patients with single and multiple deletions, all respiratory chain complexes as well as the respiratory rates were decreased to a similar extent. All changes detected in patients with single deletions were independent of deletion size. In one patient, only genes of ND5, ND4L as well as tRNA(Leu(CUN)), tRNA(Ser(AGY)), and tRNA(His) were deleted. The pronounced decrease in COX activity in this patient points to the high pathological impact of these missing tRNA genes. The activity of nuclear encoded SDH was also significantly decreased in patients, but to a lesser extent. This is an indication of secondary disturbances of mitochondria at CPEO.In conclusion, we have shown that different deletions cause mitochondrial impairments of the same phenotype correlating with heteroplasmy. The missing threshold at the level of mitochondrial function seems to be characteristic for large-scale deletions were tRNA and protein genes are deleted.     

The cytoplasmic filaments of the nuclear pore complex are dispensable for selective nuclear protein import

Oxysterol binding proteins (OSBPs) comprise a large conserved family of proteins in eukaryotes. Their ubiquity notwithstanding, the functional activities of these proteins remain unknown. Kes1p, one of seven members of the yeast OSBP family, negatively regulates Golgi complex secretory functions that are dependent on the action of the major yeast phosphatidylinositol/phosphatidylcholine Sec14p. We now demonstrate that Kes1p is a peripheral membrane protein of the yeast Golgi complex, that localization to the Golgi complex is required for Kes1p function in vivo, and that targeting of Kes1p to the Golgi complex requires binding to a phosphoinositide pool generated via the action of the Pik1p, but not the Stt4p, PtdIns 4-kinase. Localization of Kes1p to yeast Golgi region also requires function of a conserved motif found in all members of the OSBP family. Finally, we present evidence to suggest that Kes1p may regulate adenosine diphosphate-ribosylation factor (ARF) function in yeast, and that it may be through altered regulation of ARF that Kes1p interfaces with Sec14p in controlling Golgi region secretory function.     

NAD+-dependent DNA ligases of Mycobacterium tuberculosis and Streptomyces coelicolor

Sequencing of the genomes of Mycobacterium tuberculosis H37Rv and Streptomyces coelicolor A3(2) identified putative genes for an NAD(+)-dependent DNA ligase. We have cloned both open reading frames and overexpressed the protein products in Escherichia coli. In vitro biochemical assays confirm that each of these proteins encodes a functional DNA ligase that uses NAD(+) as its cofactor. Expression of either protein is able to complement E. coli GR501, which carries a temperature-sensitive mutation in ligA. Thus, in vitro and in vivo analyses confirm predictions that ligA genes from M. tuberculosis and S. coelicolor are NAD(+)-dependent DNA ligases.     

Aluminum rapidly depolymerizes cortical microtubules and depolarizes the plasma membrane: evidence that these responses are mediated by a glutamate receptor

Efforts to understand how plants respond to aluminum have focused on describing the symptoms of toxicity and elucidating mechanisms of tolerance; however, little is known about the signal transduction steps that initiate the plant's response. Here, we image cortical microtubules and quantify plasma-membrane potential in living, root cells of intact Arabidopsis seedlings. We show that aluminum depolymerizes microtubules and depolarizes the membrane, and that these responses are prevented by calcium channel blockade. Calcium influx might involve glutamate receptors, which in animals are ligand-gated cation channels and are present in the Arabidopsis genome. We show that glutamate depolymerizes microtubules and depolarizes the plasma membrane. These responses, and also the inhibition of root elongation, occur within the first few min of treatment, but are evoked more rapidly by glutamate than by aluminum. Microtubule depolymerization and membrane depolarization, induced by either glutamate or aluminum, are blocked by a specific antagonist of ionotropic glutamate receptors, 2-amino-5-phosphonopentanoate; whereas an antagonist of an aluminum-gated anion channel blocks the two responses to aluminum but not to glutamate. For growth, microtubule integrity, and membrane potential, responses to combined glutamate and aluminum were not greater than to glutamate alone. We propose that signaling in response to aluminum is initiated by efflux of a glutamate-like ligand through an anion channel and the binding of this ligand to a glutamate receptor.     

Switch-of-function mutants based on morphology classification of Ras superfamily small GTPases

Signaling proteins from the same family can have markedly different roles in a given cellular context. Here, we show that expression of one hundred constitutively active human small GTPases induced cell morphologies that fell into nine distinct classes. We developed an algorithm for pairs of classes that predicted amino acid positions that can be exchanged to create mutants with switched functionality. The algorithm was validated by creating switch-of-function mutants for Rac1, CDC42, H-Ras, RalA, Rap2B, and R-Ras3. Contrary to expectations, the relevant residues were mostly outside known interaction surfaces and were structurally far apart from each other. Our study shows that specificity in protein families can be explored by combining genome-wide experimental functional classification with the creation of switch-of-function mutants.