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The insulin receptor substrate protein of 53 kDa (IRSp53) is crucially involved in the formation of filopodia and neurites through mechanisms that have only partially been clarified. We have investigated the role of the small scaffold protein LIN7, which interacts with IRSp53. We found that formation of actin-filled protrusions in neuronal NSC34 cells and neurites in neuroblastoma N2A cells depends on motifs mediating the LIN7:IRSp53 association, as both the coexpression of LIN7 with IRSp53 or the expression of the L27-IRSp53 chimera (a fusion protein between IRSp53 and the LIN7L27 domain for plasma membrane protein complexes association) prevented actin-deficient protrusions induced by overexpressed IRSp53, and enhanced the formation of actin-filled protrusions. The regulatory role of LIN7 in IRSp53-mediated extension of filopodia in neuronal N2A cells was demonstrated by live-cell imaging experiments. Moreover, LIN7 silencing prevented the extension of filopodia and neurites, induced by ectopic expression of IRSp53 or serum starvation, respectively, in undifferentiated and differentiated N2A cells. The expression of full-length IRSp53 or the LIN7ΔPDZ mutant lacking the domain for association with IRSp53 was unable to restore neuritogenesis in LIN7-silenced cells. Conversely, defective neuritogenesis could be rescued by the expression of RNAi-resistant full-length LIN7 or chimeric L27-IRSp53. Finally, LIN7 silencing prevented the recruitment of IRSp53 in Triton X-100-insoluble complexes, otherwise occurring in differentiated cells. Collectively these data indicate that LIN7 is a novel regulator of IRSp53, and that the association of these proteins is required to promote the formation of actin-dependent filopodia and neurites.

POF1B is a candidate gene for premature ovarian failure (POF); it is mainly expressed in polarised epithelial tissues, but its function in these tissues and the relationship with the disorder are unknown. Here we show colocalisation of POF1B with markers of both adherens and tight junctions in human jejunum. The tight junction localisation was maintained by the human POF1B stably expressed in the MDCK polarised epithelial cell line, whereas it was lost by the POF1B R329Q variant associated with POF. Localisation of apico-basal polarity markers and ultrastructure of the tight junctions were maintained in cells expressing the mutant. However, tight junction assembly was altered, cells were dysmorphic and the monolayer organisation was also altered in three-dimensional culture systems. Moreover, cells expressing the POF1B R329Q variant showed defects in ciliogenesis and cystogenesis as a result of misorientation of primary cilia and mitotic division. All of these defects were explained by interference of the mutant with the content and organisation of F-actin at the junctions. A role for POF1B in the regulation of the actin cytoskeleton was further verified by shRNA silencing of the endogenous protein in human intestinal Caco-2 cells. Taken together, these data indicate that localisation of POF1B to tight junctions has a key role in the organisation of epithelial monolayers by regulating the actin cytoskeleton., (@ 2011. Published by The Company of Biologists Ltd)

It has been suggested that glutamate-induced excitotoxicity plays a central role in the development of motor neuron diseases such as amyotrophic lateral sclerosis (ALS). The GLT-1 isoform of the glutamate transporter gene family is the most important transporter involved in keeping extracellular glutamate concentration below neurotoxic levels. Its loss and an increase in extracellular glutamate has been documented in cases of sporadic and familial ALS, as well as in animal models expressing ALS-linked Cu2+-Zn2+ superoxide dismutase (SOD1) mutations, but the underlying molecular mechanisms are still unclear. We developed and characterised a cell model consisting of polarised epithelial Madin-Darby Canine Kidney (MDCK) cell lines stably expressing wild-type SOD1 or the ALS-linked SOD1 G93A mutant, and analysed the expression of glutamate transporters after transient transfection of the corresponding cDNAs. Like ALS patients and animal models of ALS, the G93A-expressing MDCK cell system showed reduced total glial GLT-1 expression, with no change in the expression of the neuronal EAAC1 glutamate transporter isoform. Morphological analysis revealed the intracellular redistribution of GLT-1 to acidic compartments, whereas the surface distribution of other glutamate transporters (neuronal EAAC1 and glial GLAST) was not affected. Moreover, mutant SOD1 affected the cytosolic tail of GLT-1 because reduced protein expression of EAAC-GLT but not GLT-EAAC chimeras was found in G93A-expressing cell lines. GLT-1 downregulation was greatly induced by inhibition of protein synthesis, and prevented by treatment with chloroquine aimed at inhibiting the activity of acidic degradative compartments. Negligible effect on the protein level or distribution of GLT-1 was observed in cells overexpressing wild-type SOD1. The specific decrease in the GLT-1 isoform of glutamate transporters is therefore recapitulated in G93A-expressing MDCK cell lines, thus suggesting an autonomous cell mechanism underlying the loss of GLT-1 in ALS. Our data indicate that the continuous expression of mutant SOD1 causes the downregulation of GLT-1 by increasing the internalisation and degradation of the surface transporter, and suggest that the cytosolic tail of GLT-1 is required to target the transporter to degradation.

Stardust (Sdt) and Discs-Large (Dlg) are membrane-associated guanylate kinases (MAGUKs) involved in the organization of supramolecular protein complexes at distinct epithelial membrane compartments in Drosophila. Loss of either Sdt or Dlg affects epithelial development with severe effects on apico-basal polarity. Moreover, Dlg is required for the structural and functional integrity of synaptic junctions. Recent biochemical and cell culture studies have revealed that various mammalian MAGUKs can interact with mLin-7/Veli/MALS, a small PDZ-domain protein. To substantiate these findings for their in vivo significance with regard to Sdt- and Dlg-based protein complexes, we analyzed the subcellular distribution of Drosophila Lin-7 (DLin-7) and performed genetic and biochemical assays to characterize its interaction with either of the two MAGUKs. In epithelia, Sdt mediates the recruitment of DLin-7 to the subapical region, while at larval neuromuscular junctions, a particular isoform of Dlg, Dlg-S97, is required for postsynaptic localization of DLin-7. Ectopic expression of Dlg-S97 in epithelia, however, was not sufficient to induce a redistribution of DLin-7. These results imply that the recruitment of DLin-7 to MAGUK-based protein complexes is defined by cell-type specific mechanisms and that DLin-7 acts downstream of Sdt in epithelia and downstream of Dlg at synapses.

As little is known about the role of cadherin-mediated cell-cell adhesion in astrocytes and its alteration in migrating and invasive glioblastomas, we investigated its molecular composition and organisation in primary cultured astrocytes and the T98G and U373MG glioblastoma cell lines. Biochemical and morphological analysis indicated that all three cell types express all of the structural components of the adhesion system, including the LIN-7 PDZ protein, a novel component involved in the organisation of the junctional domain in epithelia and neurons. However, only the astrocytes and T98G cells generated and maintained mature adhesive junctional domains to which LIN-7 was recruited. Alterations in the junctional domain of U373MG cells were associated with higher motility in a poly-L-lysine migration assay. When the T98G cells were cultured on Matrigel matrix, they acquired invasive properties but, despite unchanged cadherin adhesion system protein levels, the invasive T98G cell-cell contacts failed to accumulate LIN-7 and failed to mature. These results identify the LIN-7 PDZ protein as a marker of cell adhesion maturity and cell invasion and indicate that instability and disorganisation of cadherin-mediated junctions rather than reduced expression of cadherin-catenin system components are required to promote migration and invasiveness in glioblastoma cell lines.