Phalloidin-Eosin Followed by Photo-oxidation

Capani, Francisco; Deerinck, Thomas J.; Ellisman, Mark H.; Bushong, Eric A.; Bobik, Marketta; Martone, Maryann E.

doi:10.1177/002215540104901103

Cited by 51 publications

(24 citation statements)

References 20 publications

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“…When L. monocytogenes is present in the cytosol the bacterial surface protein ActA activates actin polimerization [1], [15]. The detection of this polymerizing actin by phalloidin staining is indicative of phagosomal escape [16], [17]. As expected, wild type (wt) L. monocytogenes escaped into the cytosol of J774 macrophages and was coated in actin (Fig.…”

Section: Resultssupporting

confidence: 55%

Inhibition of Calpain Blocks the Phagosomal Escape of Listeria monocytogenes

et al. 2012

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Listeria monocytogenes is a Gram-positive facultative intracellular bacterium responsible for the food borne infection listeriosis, affecting principally the immunocompromised, the old, neonates and pregnant women. Following invasion L. monocytogenes escapes the phagosome and replicates in the cytoplasm. Phagosome escape is central to L. monocytogenes virulence and is required for initiating innate host-defence responses such as the secretion of the cytokine interleukin-1. Phagosome escape of L. monocytogenes is reported to depend upon host proteins such as γ-interferon-inducible lysosomal thiol reductase and the cystic fibrosis transmembrane conductance regulator. The host cytosolic cysteine protease calpain is required in the life cycle of numerous pathogens, and previous research reports an activation of calpain by L. monocytogenes infection. Thus we sought to determine whether host calpain was required for the virulence of L. monocytogenes. Treatment of macrophages with calpain inhibitors blocked escape of L. monocytogenes from the phagosome and consequently its proliferation within the cytosol. This was independent of any direct effect on the production of bacterial virulence factors or of a bactericidal effect. Furthermore, the secretion of interleukin-1β, a host cytokine whose secretion induced by L. monocytogenes depends upon phagosome escape, was also blocked by calpain inhibition. These data indicate that L. monocytogenes co-opts host calpain to facilitate its escape from the phagosome, and more generally, that calpain may represent a cellular Achilles heel exploited by pathogens.

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Section: Resultssupporting

confidence: 55%

Inhibition of Calpain Blocks the Phagosomal Escape of Listeria monocytogenes

et al. 2012

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“…For cells that were probed with the antibodies, cells were immunolabeled as mentioned in ref. 22. Donkey anti-rabbit FITC and donkey anti-mouse Cy5 were used as secondary antibodies at a dilution of 1:100.…”

Section: Methodsmentioning

confidence: 99%

A-kinase-interacting protein localizes protein kinase A in the nucleus

Sastri

Barraclough

Carmichael

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

111

129

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The genetic variability and covalent modifications associated with the amino terminus of the protein kinase A (PKA) catalytic (C) subunit suggest that it may contribute to protein-protein interactions and͞or localization. By using a yeast two-hybrid screen, we identified a PKA-interacting protein (AKIP1) that binds to the amino terminus (residues 1-39) of the C subunit of PKA. The interaction was localized to the A helix (residues 14 -39) of the C subunit and to the carboxyl terminus of AKIP1. AKIP1 thus defines the amino-terminal A helix of PKA as a protein interaction motif. In normal breast (Hs 578 Bst) and HeLa cells, AKIP1 is present in the nucleus as speckles. A nuclear localization signal (Arg-14 and Arg-15) was identified. Upon stimulation with forskolin, HeLa cells expressing AKIP1 accumulated higher levels of the endogenous C subunit in the nucleus. Deletion of the carboxyl terminus of AKIP1 or overexpression of residues 1-39 of the C subunit abolished nuclear localization of the activated endogenous C subunit. Thus, AKIP1 describes a PKA-interacting protein that can contribute to localization by a mechanism that is distinct from A-kinase anchoring proteins that interact with the regulatory subunits.nuclear retention ͉ speckles

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“…We showed previously that ITPKA-enriched bundles form polarized Y-shaped structures in spines, which radiate between the endoplasmic reticulum and the postsynaptic membrane . Such "bundle bridges" have also been depicted in a number of ultrastructural studies (Wilson et al, 1983;Morales and Fifkova, 1989;Capani et al, 2001;Rostaing et al, 2006), but their relationship to structural plasticity is unknown. We also showed previously that ITPKA-labeled actin filaments in dendritic spines undergo a rapid and reversible reorganization in response to Ca 2ϩ influx .…”

Section: Implications For Spine Structural Plasticitymentioning

confidence: 99%

Neuronal IP₃3-Kinase is an F-actin–bundling Protein: Role in Dendritic Targeting and Regulation of Spine Morphology

Johnson

Schell

2009

MBoC

141

148

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The actin microstructure in dendritic spines is involved in synaptic plasticity. Inositol trisphosphate 3-kinase A (ITPKA) terminates Ins(1,4,5)P(3) signals emanating from spines and also binds filamentous actin (F-actin) through its amino terminal region (amino acids 1-66, N66). Here we investigated how ITPKA, independent of its kinase activity, regulates dendritic spine F-actin microstructure. We show that the N66 region of the protein mediates F-actin bundling. An N66 fusion protein bundled F-actin in vitro, and the bundling involved N66 dimerization. By mutagenesis we identified a point mutation in a predicted helical region that eliminated both F-actin binding and bundling, rendering the enzyme cytosolic. A fusion protein containing a minimal helical region (amino acids 9-52, N9-52) bound F-actin in vitro and in cells, but had lower affinity. In hippocampal neurons, GFP-tagged N66 expression was highly polarized, with targeting of the enzyme predominantly to spines. By contrast, N9-52-GFP expression occurred in actin-rich structures in dendrites and growth cones. Expression of N66-GFP tripled the length of dendritic protrusions, induced longer dendritic spine necks, and induced polarized actin motility in time-lapse assays. These results suggest that, in addition to its ability to regulate intracellular Ca(2+) via Ins(1,4,5)P(3) metabolism, ITPKA regulates structural plasticity.

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Phalloidin-Eosin Followed by Photo-oxidation

Cited by 51 publications

References 20 publications

Inhibition of Calpain Blocks the Phagosomal Escape of Listeria monocytogenes

Inhibition of Calpain Blocks the Phagosomal Escape of Listeria monocytogenes

A-kinase-interacting protein localizes protein kinase A in the nucleus

Neuronal IP₃3-Kinase is an F-actin–bundling Protein: Role in Dendritic Targeting and Regulation of Spine Morphology

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Phalloidin-Eosin Followed by Photo-oxidation

Cited by 51 publications

References 20 publications

Inhibition of Calpain Blocks the Phagosomal Escape of Listeria monocytogenes

Inhibition of Calpain Blocks the Phagosomal Escape of Listeria monocytogenes

A-kinase-interacting protein localizes protein kinase A in the nucleus

Neuronal IP33-Kinase is an F-actin–bundling Protein: Role in Dendritic Targeting and Regulation of Spine Morphology

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Neuronal IP₃3-Kinase is an F-actin–bundling Protein: Role in Dendritic Targeting and Regulation of Spine Morphology