The insulin receptor substrate protein of 53 kDa is a critical factor in determining neuronal polarisation, as it initiates membrane protrusions to form filopodia and neurites by coupling membrane deformation with F-actin polymerization. With its C-terminal tail, IRSp53 can bind to PDZ domain containing proteins including LIN7, a small scaffold protein possessing a single L27 domain, necessary for membrane recruitment. Here, we investigated the role of the IRSp53-LIN7 complex in cellular mechanisms that heavily rely on membrane deformation, such as the formation of filopodia and neurites or mitochondrial fission. Concerning the role of the IRSp53:LIN7 complex in filopodia and neurite induction, our findings indicate that the formation of actin-filled filopodia and neurites depends on motifs mediating IRSp53-LIN7 association and filopodia tip localisation. We further showed that co-expression of LIN7 with IRSp53 enhanced the formation of filopodia protrusions in neuronal NSC34 cells, while also preventing the appearance of actin-deficient protrusions induced by the overexpression of IRSp53 alone. The positive regulatory role of LIN7 in IRSp53-mediated extension of filopodia was further demonstrated by live-cell imaging experiments in neuronal N2A cells. Moreover, LIN7 silencing in N2A cells prevented the extension of filopodia and neurites, induced by either the ectopic expression of IRSp53 or serum starvation. Defective neuritogenesis could be rescued by the expression of RNAi-resistant full length LIN7 or chimeric L27-IRSp53, whereas 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. Finally, LIN7 silencing prevented the recruitment of IRSp53 in Triton X-100 insoluble complexes, otherwise occurring in differentiated cells. Collectively, this first set of data identify in LIN7 a novel regulator of the filopodia- and neurites-promoting activity of IRSp53, whose role is to spatially restrict its activity to the plasma membrane for filopodia and neurite initiation, and to further promote the stabilisation of these actin–rich structures. More recently, we tested the hypothesis of a role for the IRSp53-LIN7 complex in the modification of intracellular membranes. To this regard, we found that endogenous LIN7 and IRSp53 localized in punctuate structures along mitochondria, a fact that prompted us to further investigate the possible effects of modifications in the expression levels of IRSp53/LIN7 on mitochondrial morphology. Eukaryotic cells, in fact, strictly regulate the overall morphology of their mitochondrial network thanks to the existence of protein complexes able to control fission and fusion events. We found that, upon overexpression of LIN7 and/or IRSp53, mitochondria morphology was altered, with a significant increase in the percentage of cells showing a less interconnected mitochondrial network compared to GFP-transfected control cells, a phenotype that was blocked by co-expression of the K38A dominant negative mutant of the fission protein Drp1. Downregulation of endogenous LIN7 and/or IRSp53 by shRNA, on the other hand, increased the amount of cells with highly fused mitochondria. Mitochondria hyperfusion in the downregulated cells was associated with an increased resistance to NaN3-induced mitochondrial fragmentation, and by the appearance of cells with aberrantly shaped and often multi-lobed nuclei, a phenotype that we also found in Drp1 K38A-expressing cells, and that others reported to be caused by defective mitochondrial fragmentation during mitosis. Collectively our data strongly suggest a Drp1-dependent function of LIN7 and IRSp53 on mitochondrial division apparatus. Taken together, our data unravel a role of the IRSp53-LIN7 complex on membrane dynamics that is not restricted to plasma membranes, as previously thought, but may also apply to other cellular mechanisms that heavily rely on membrane deformation, as our data on mitochondrial morphology have shown.
THE ROLE OF THE IRSP53-LIN7 COMPLEX IN CELL MEMBRANE DYNAMICS / I. Ferrari ; coordinatore: M. Locati ; tutor: G. Pietrini. - Milano : Università degli studi di Milano. Università degli Studi di Milano, 2015 Jan 12. ((27. ciclo, Anno Accademico 2014.
|Titolo:||THE ROLE OF THE IRSP53-LIN7 COMPLEX IN CELL MEMBRANE DYNAMICS|
|Supervisori e coordinatori interni:||LOCATI, MASSIMO|
|Data di pubblicazione:||12-gen-2015|
|Parole Chiave:||IRSp53; LIN7; IRSp53-LIN7 complex; N2A cells; NSC34 cells; filopodia; neurites; tip complex; ; mitochondrial morphology; mitochondrial fission; mitochondria pre-constriction sites|
|Settore Scientifico Disciplinare:||Settore BIO/13 - Biologia Applicata|
|Citazione:||THE ROLE OF THE IRSP53-LIN7 COMPLEX IN CELL MEMBRANE DYNAMICS / I. Ferrari ; coordinatore: M. Locati ; tutor: G. Pietrini. - Milano : Università degli studi di Milano. Università degli Studi di Milano, 2015 Jan 12. ((27. ciclo, Anno Accademico 2014.|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.13130/ferrari-ilaria_phd2015-01-12|
|Appare nelle tipologie:||Tesi di dottorato|