Publisher Correction: Retrograde trafficking of ß-dystroglycan from the plasma membrane to the nucleus.

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Giardipain-1, a protease secreted by Giardia duodenalis trophozoites, causes junctional, barrier and apoptotic damage in epithelial cell monolayers.

The adhesion of Giardia duodenalis trophozoites to intestinal epithelial cells allows the onset and maintenance of giardiasis. During these interactions, epithelial cells can be committed to apoptosis by enzymes secreted by the parasites, including cysteine proteases that are increasingly identified as virulence factors in parasitic protozoa. In this work, a monoclonal antibody (mAb1G3) raised against G. duodenalis surface components was found to react with a 25 kDa protein expressed in the cell surface and flagella of G. duodenalis trophozoites. When trophozoites expressing this protein were cultured with IEC-6 intestinal epithelial cell monolayers, a dynamic release of this protein was observed with mAbIG3. Proteomic analysis identified the protein as a mature cathepsin B-like (gCatB) enzyme, whose proteolytic activity, detected in zymograms, was eliminated by CatB inhibitor E-64. This protein was named giardipain-1 due to its functional papain-like features and was purified by affinity chromatography using mAbIG3. Upon exposure to the purified, mature and secreted forms of giardipain-1, IEC-6 epithelial cell monolayers displayed membrane blebbing and phosphatidylserine exposure on the outer cell surface, indicating an apoptotic process. In Madin Darby Canine Kidney (MDCK) cell monolayers, giardipain-1 leads to the appearance of pore-like regions and of gaps along cell-cell junctions, to decreased transepithelial electrical resistance (TER), caspase-3 activation and poly-ADP-ribose polymerase (PARP) fragmentation. At early times during exposure, giardipain-1 co-localized at cell-cell junctions, associated with occludin and induced the delocalization and degradation of tight junction proteins occludin and claudin-1. The damage caused to epithelial monolayers by giardipain-1 was blocked by pre-incubation with the CatB B Inhibitor E-64. Furthermore, silencing the giardipain-1 gene in trophozoites lowered the proteolytic activity of giardipain-1 and reduced the damage in IEC-6 monolayers. The damage observed appears to be specific to giardipain activity since almost no damage was observed when IEC-6 monolayers were incubated with papain, a non-related cysteine protease. Hence this study suggests that giardipain-1 triggers, in epithelial cells, degradation of cell-cell junctional components and apoptotic damage, supporting the notion of giardiapain-1 as a virulence factor of Giardia.

Control of nuclear ß-dystroglycan content is crucial for the maintenance of nuclear envelope integrity and function.

ß-Dystroglycan (ß-DG) is a plasma membrane protein that has ability to target to the nuclear envelope (NE) to maintain nuclear architecture. Nevertheless, mechanisms controlling ß-DG nuclear localization and the physiological consequences of a failure of trafficking are largely unknown. We show that ß-DG has a nuclear export pathway in myoblasts that depends on the recognition of a nuclear export signal located in its transmembrane domain, by CRM1. Remarkably, NES mutations forced ß-DG nuclear accumulation resulting in mislocalization and decreased levels of emerin and lamin B1 and disruption of various nuclear processes in which emerin (centrosome-nucleus linkage and ß-catenin transcriptional activity) and lamin B1 (cell cycle progression and nucleoli structure) are critically involved. In addition to nuclear export, the lifespan of nuclear ß-DG is restricted by its nuclear proteasomal degradation. Collectively our data show that control of nuclear ß-DG content by the combination of CRM1 nuclear export and nuclear proteasome pathways is physiologically relevant to preserve proper NE structure and activity.

Retrograde trafficking of ß-dystroglycan from the plasma membrane to the nucleus.

ß-Dystroglycan (ß-DG) is a transmembrane protein with critical roles in cell adhesion, cytoskeleton remodeling and nuclear architecture. This functional diversity is attributed to the ability of ß-DG to target to, and conform specific protein assemblies at the plasma membrane (PM) and nuclear envelope (NE). Although a classical NLS and importin α/ß mediated nuclear import pathway has already been described for ß-DG, the intracellular trafficking route by which ß-DG reaches the nucleus is unknown. In this study, we demonstrated that ß-DG undergoes retrograde intracellular trafficking from the PM to the nucleus via the endosome-ER network. Furthermore, we provided evidence indicating that the translocon complex Sec61 mediates the release of ß-DG from the ER membrane, making it accessible for importins and nuclear import. Finally, we show that phosphorylation of ß-DG at Tyr890 is a key stimulus for ß-DG nuclear translocation. Collectively our data describe the retrograde intracellular trafficking route that ß-DG follows from PM to the nucleus. This dual role for a cell adhesion receptor permits the cell to functionally connect the PM with the nucleus and represents to our knowledge the first example of a cell adhesion receptor exhibiting retrograde nuclear trafficking and having dual roles in PM and NE.