correlative-light-electron-microscopy-clem-as-tool-to-visualize

Correlative Light and Electron Microscopy (CLEM) as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets


The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity ...

University of Texas Southwestern Medical Center 

Bacterial toxins and effector proteins hijack eukaryotic enzymes that are spatially localized and display rapid signaling kinetics. However, the molecular mechanisms by which virulence factors engage highly dynamic substrates in the host cell environment are poorly understood. Here, we demonstrate that the enteropathogenic Escherichia coli (EPEC) type III effector protein EspF nucleates a multiprotein signaling complex composed of eukaryotic sorting nexin 9 (SNX9) and neuronal Wiskott-Aldrich syndrome protein (N-WASP). We demonstrate that a specific and high affinity association between EspF and SNX9 induces membrane remodeling in host cells. These membrane-remodeling events are directly coupled to N-WASP/Arp2/3-mediated actin nucleation. In addition to providing a biochemical mechanism of EspF function, we find that EspF dynamically localizes to membrane-trafficking organelles in a spatiotemporal pattern that correlates with SNX9 and N-WASP activity in living cells. Thus, our findings suggest that the EspF-dependent assembly of SNX9 and N-WASP represents a novel form of signaling mimicry used to promote EPEC pathogenesis and gastrointestinal disease.

The type III effector EspF coordinates membrane trafficking by the spatiotemporal activation of two eukaryotic signaling pathways.

Enteropathogenic Escherichia coli (EPEC) have been previously shown to alter sodium hydrogen exchanger 3 (NHE3) activity in human intestinal epithelial cells. To further characterize these observations, PS120 fibroblasts transfected with NHE3 were studied. EPEC E2348/69 infection decreased NHE3 activity in PS120 fibroblasts. The effect on NHE3 was enhanced when PS120 cells were co-transfected with the scaffolding/regulatory proteins NHERF1 or NHERF2 or EBP50 and E3KARP respectively. The decrease in NHE3 activity was dependent on an intact type III secretion system, although intimate attachment mediated by translocated intimin receptor was not required. Despite its ability to bind to NHERF proteins, the EPEC effector Map had no impact on the regulation of NHE activity. Instead, EspF was found to be responsible for decreased NHE3 activity. However, neither EspF-induced apoptosis nor the interaction of EspF with sorting nexin-9, an endocytic protein, were involved.

The enteropathogenic Escherichia coli effector protein EspF decreases sodium hydrogen exchanger 3 activity.

Polarity in mammalian cells emerges from the assembly of signaling molecules into extensive biochemical interaction networks. Despite their complexity, bacterial pathogens have evolved parsimonious mechanisms to hijack these systems. Here, we develop a tractable experimental and theoretical model to uncover fundamental operating principles, in both mammalian cell polarity and bacterial pathogenesis. Using synthetic derivatives of the enteropathogenic Escherichia coli guanine-nucleotide exchange factor (GEF) Map, we discover that Cdc42 GTPase signal transduction is controlled by the interaction between Map and F-actin. Mathematical modeling reveals how actin dynamics coupled to a Map-dependent positive feedback loop spontaneously polarizes Cdc42 on the plasma membrane. By rewiring the pathogenic signaling circuit to operate through β-integrin stimulation, we further show how Cdc42 is polarized in response to an extracellular spatial cue. Thus, a molecular pathway of polarity is proposed, centered on the interaction between GEFs and F-actin, which is likely to function in diverse biological systems.

Article	Year
The Journal of cell biology\| PubMed ID: 12186851	2002
Journal of vascular surgery : official publication, the Society for Vascular Surgery [and] International Society for Cardiovascular Surgery, North American Chapter\| PubMed ID: 12514595	2003
Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 12672969	2003
Cell biochemistry and biophysics\| PubMed ID: 15289655	2004
Current opinion in microbiology\| PubMed ID: 15694853	2005
Cell\| PubMed ID: 16413487	2006
The type III effector EspF coordinates membrane trafficking by the spatiotemporal activation of two eukaryotic signaling pathways. The Journal of cell biology\| PubMed ID: 17893247	2007
Cellular microbiology\| PubMed ID: 18190559	2008
Developmental cell\| PubMed ID: 18194648	2008
Methods in enzymology\| PubMed ID: 18374161	2008
The enteropathogenic Escherichia coli effector protein EspF decreases sodium hydrogen exchanger 3 activity. Cellular microbiology\| PubMed ID: 18433466	2008
Cell host & microbe\| PubMed ID: 18996344	2008
Annals of the New York Academy of Sciences\| PubMed ID: 19538303	2009
Nature structural & molecular biology\| PubMed ID: 19620963	2009
Cellular microbiology\| PubMed ID: 20088948	2010
Cell host & microbe\| PubMed ID: 20542244	2010
The Journal of biological chemistry\| PubMed ID: 20670942	2010
Nature\| PubMed ID: 21170023	2011
Cellular microbiology\| PubMed ID: 21951829	2012
Small GTPases\| PubMed ID: 22145094	2011
Identification of F-actin as the dynamic hub in a microbial-induced GTPase polarity circuit. Cell\| PubMed ID: 22341450	2012

Neal M. Alto

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