Shigella enters epithlial cells via internalization into a vacuole. Subsequent vacuolar membrane rupture allows bacterial escape into the cytosol for replication and cell-to-cell spread. Bacterial ...effectors such as IpgD, a PI(4,5)P2 phosphatase that generates PI(5)P and alters host actin, facilitate this internalization. Here, we identify host proteins involved in Shigella uptake and vacuolar membrane rupture by high-content siRNA screening and subsequently focus on Rab11, a constituent of the recycling compartment. Rab11-positive vesicles are recruited to the invasion site before vacuolar rupture, and Rab11 knockdown dramatically decreases vacuolar membrane rupture. Additionally, Rab11 recruitment is absent and vacuolar rupture is delayed in the ipgD mutant that does not dephosphorylate PI(4,5)P2 into PI(5)P. Ultrastructural analyses of Rab11-positive vesicles further reveal that ipgD mutant-containing vacuoles become confined in actin structures that likely contribute to delayed vacular rupture. These findings provide insight into the underlying molecular mechanism of vacuole progression and rupture during Shigella invasion.
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•High-content screen reveals host proteins involved in Shigella vacuolar rupture•Rab11-positive vesicles are targeted by Shigella to induce vacuolar rupture•The bacterial inositol phosphatase IpgD controls Rab11 recruitment before rupture•Correlative microscopy analyses provides ultrastructural details of the rupture process
Shigella enters cells via vacuolar internalization followed by vacuole rupture and bacterial release into the cytosol. Mellouk et al. show that Shigella subverts the host cell recycling compartment via Rab11 to achieve vacuolar rupture. Rab11 is targeted by the enzymatic phosphatase activity of the injected bacterial effector IpgD.
mRNA localization is an evolutionary conserved mechanism that underlies the establishment of cellular polarity and specialized cell functions. To identify mRNAs localized in subcellular compartments ...of developing neurons, we took an original approach that combines compartmentalized cultures of rat sympathetic neurons and sequential analysis of gene expression (SAGE). Unexpectedly, the most abundant transcript in axons was mRNA for myo-inositol monophosphatase-1 (Impa1), a key enzyme that regulates the inositol cycle and the main target of lithium in neurons. A novel localization element within the 3' untranslated region of Impa1 mRNA specifically targeted Impa1 transcript to sympathetic neuron axons and regulated local IMPA1 translation in response to nerve growth factor (NGF). Selective silencing of IMPA1 synthesis in axons decreased nuclear CREB activation and induced axonal degeneration. These results provide insights into mRNA transport in axons and reveal a new NGF-responsive localization element that directs the targeting and local translation of an axonal transcript.
Fructose-2,6-bisphosphate is responsible for mediating glucagon-stimulated gluconeogenesis in the liver. This discovery has led to the realization that this compound plays a significant role in ...directing carbohydrate fluxes in all eukaryotes. Biophysical studies of the enzyme that both synthesizes and degrades this biofactor have yielded insight into its molecular enzymology. Moreover, the metabolic role of fructose-2,6-bisphosphate has great potential in the treatment of diabetes.
Embryonic stem cells and induced pluripotent stem cells represent potentially important therapeutic agents in regenerative medicine. Complex interlinked transcriptional and signaling networks control ...the fate of these cells towards maintenance of pluripotency or differentiation. In this study we have focused on how mouse embryonic stem cells begin to differentiate and lose pluripotency and, in particular, the role that the ERK MAP kinase and GSK3 signaling pathways play in this process. Through a genome-wide siRNA screen we have identified more than 400 genes involved in loss of pluripotency and promoting the onset of differentiation. These genes were functionally associated with the ERK and/or GSK3 pathways, providing an important resource for studying the roles of these pathways in controlling escape from the pluripotent ground state. More detailed analysis identified MAP kinase phosphatases as a focal point of regulation and demonstrated an important role for these enzymes in controlling ERK activation kinetics and subsequently determining early embryonic stem cell fate decisions.
In Arabidopsis root tips cultured in medium containing sufficient nutrients and the membrane-permeable protease inhibitor E-64d, parts of the cytoplasm accumulated in the vacuoles of the cells from ...the meristematic zone to the elongation zone. Also in barley root tips treated with E-64, parts of the cytoplasm accumulated in autolysosomes and pre-existing central vacuoles. These results suggest that vacuolar and/or lysosomal autophagy occurs constitutively in these regions of cells. 3-Methyladenine, an inhibitor of autophagy, inhibited the accumulation of such inclusions in Arabidopsis root tip cells. Such inclusions were also not observed in root tips prepared from Arabidopsis T-DNA mutants in which AtATG2 or AtATG5, an Arabidopsis homolog of yeast ATG genes essential for autophagy, is disrupted. In contrast, an atatg9 mutant, in which another homolog of ATG is disrupted, accumulated a significant number of vacuolar inclusions in the presence of E-64d. These results suggest that both AtAtg2 and AtAtg5 proteins are essential for autophagy whereas AtAtg9 protein contributes to, but is not essential for, autophagy in Arabidopsis root tip cells. Autophagy that is sensitive to 3-methyladenine and dependent on Atg proteins constitutively occurs in the root tip cells of Arabidopsis.
We recently characterized gene expression patterns in gastrointestinal stromal tumors (GISTs) using cDNA microarrays, and found that the gene
FLJ10261
(
DOG1
, discovered on GIST-1), encoding a ...hypothetical protein, was specifically expressed in GISTs. The immunoreactivity of a rabbit antiserum to synthetic DOG1 peptides was assessed on two soft tissue tumor microarrays. The tissue microarrays included 587 soft tissue tumors, with 149 GISTs, including 127 GIST cases for which the
KIT
and
PDGFRA
mutation status was known. Immunoreactivity for DOG1 was found in 136 of 139 (97.8%) of scorable GISTs. All seven GIST cases with a
PDGFRA
mutation were DOG1-positive, while most of these failed to react for KIT. The immunohistochemical findings were confirmed with
in situ
hybridization probes for
DOG1
,
KIT
, and
PDGFRA
. Other neoplasms in the differential diagnosis of GIST, including desmoid fibromatosis (0 of 17) and Schwannoma (0 of 3), were immunonegative for DOG1. Only 4 of 438 non-GIST cases were immunoreactive for DOG1. DOG1, a protein of unknown function, is expressed strongly on the cell surface of GISTs and is rarely expressed in other soft tissue tumors. Reactivity for DOG1 may aid in the diagnosis of GISTs, including
PDGFRA
mutants that fail to express KIT antigen, and lead to appropriate treatment with imatinib mesylate, an inhibitor of the KIT tyrosine kinase.
The enzyme MTH1 cleanses the cellular nucleotide pool of oxidatively damaged 8-oxo-dGTP, preventing mutagenesis by this nucleotide. The enzyme is considered a promising therapeutic target; however, ...methods to measure its activity are indirect and laborious and have low sensitivity. Here we describe a novel ATP-linked chimeric nucleotide (ARGO) that enables luminescence signaling of the enzymatic reaction, greatly simplifying the measurement of MTH1 activity. We show that the reporting system can be used to identify inhibitors of MTH1, and we use it to quantify enzyme activity in eight cell lines and in colorectal tumor tissue. The ARGO reporter is likely to have considerable utility in the study of the biology of MTH1 and potentially in analyzing patient samples during clinical testing.
The imbalance between the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) in DNA and the efficiency of cellular systems of DNA protection and repair is considered an important factor in ...the age-dependent development of cancer. This study investigated the relationship between oxidatively damaged DNA and the activity of the DNA repair system and 8-oxo-7,8-dihydro-2′-deoxyguanosine 5′-triphosphate pyrophosphohydrolase (8-oxodGTPase) activity in liver and lung tissue from mice at 10–100 weeks of age. The level of 8-oxodG increased with age, whereas the level of formamidopyrimidine DNA glycosylase sites was unaltered. The enzyme activity toward single oxygen-induced DNA damage and mRNA expression levels of
Ercc1,
Neil1, and
Ogg1 remained unaltered with age. However, the 8-oxodGTPase activity in the liver was 18% (95% CI: 0.2–37%) lower in mice at 25 and 50 weeks than in 10-week-old mice. The 10- and 100-week-old mice had similar 8-oxodGTPase activity. In contrast, the mRNA expression of
Nudt1 was statistically unaltered that likely resulted from higher variation of measurements. The accumulation of 8-oxodG with age is not a direct consequence of decreased enzyme activity toward singlet oxygen-induced substrate DNA. An age-related higher level of 8-oxodG even occurs concomitantly with high 8-oxodGTPase activity.
Cellular signaling is initially confined to the plasma membrane, where the cytoplasmic tails of surface receptors and other membrane-anchored proteins are phosphorylated in response to ligand ...binding. These proteins often contain multiple phosphorylation sites that are regulated by membrane-confined enzymes. Phosphorylation of these proteins is thought to be tightly regulated, because they initiate and regulate signaling cascades leading to cellular activation, yet how their phosphorylation is regulated is poorly understood. Ultrasensitive or switchlike responses in their phosphorylation state are not expected because the modifying enzymes are in excess. Here, we describe a novel mechanism of ultrasensitivity exhibited by multisite membrane-anchored proteins, but not cytosolic proteins, even when enzymes are in excess. The mechanism underlying this concentration-independent ultrasensitivity is the local saturation of a single enzyme by multiple sites on the substrate. Local saturation is a passive process arising from slow membrane diffusion, steric hindrances, and multiple sites, and therefore may be widely applicable. Critical to this ultrasensitivity is the brief enzymatic inactivation that follows substrate modification. Computations are presented using ordinary differential equations and stochastic spatial simulations. We propose a new role, to our knowledge, for multisite membrane-anchored proteins, discuss experiments that can be used to probe the model, and relate our findings to previous theoretical work.
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