Molecular mechanisms specifying epithelial and neuronal polarity : segregation of membrane domains and polarized trafficking
Tesi di Dottorato
Data di Pubblicazione:
2009
Citazione:
Molecular mechanisms specifying epithelial and neuronal polarity : segregation of membrane domains and polarized trafficking / V. Padovano ; coordinatore: F. Guidobono Cavalchini ; tutor: G. Pietrini. DIPARTIMENTO DI FARMACOLOGIA, CHEMIOTERAPIA E TOSSICOLOGIA MEDICA, 2009. 22. ciclo, Anno Accademico 2008/2009.
Abstract:
Cell polarity is involved in the processes of differentiation, proliferation and
morphogenesis in both unicellular and multicellular organisms. In polarized cells,
such as epithelial cells and neurons, it is possible to distinguish a junctional
domain (cell-cell and cell-matrix) and an extra-junctional domain (apical domain
in epithelial cells and extra-synaptic domain in neurons). In epithelial cells,
assembly of tight junctions (TJs) leads to the formation of apical and basolateral
plasma membrane domains, while the functional analogous of these junctions in
neurons is still unknown.
By organizing protein complexes through protein-protein interaction domains, scaffold proteins play an important role in the assembly of TJs and in the organization of the post-synaptic density (PSD) at post-synaptic sites, and here we have investigated the role of the L27 and PDZ domains for proteinprotein interaction of the small scaffold protein LIN7 in Madin-Darby canine kidney (MDCK) cells and in the motor neuronal NSC-34 cell line.
In MDCK cells, we have found that the stable expression of a LIN7 mutantlacking the L27 domain (delL27 mutant) acts as a dominant interfering protein by inhibiting TJ localization of endogenous LIN7. The loss of LIN7 did not alter the localization of the PALS1 partner of the L27 domain, but prevented TJlocalization of the insulin receptor substrate p53 (IRSp53), a partner of the PDZ domain of LIN7. The function of both L27 and PDZ domain of LIN7 in IRSp53
localization to TJs has been further demonstrated by reducing the expression of
LIN7 (LIN7shRNA experiments), and by expression of IRSp53 deleted of its motif
for PDZ interaction (IRSp53del5) or fused to the L27 domain of LIN7 (L27-IRSp53del5). Cell lines with decreased localization of LIN7 and IRSp53 to TJs showed defects during assembly of TJs and cyst polarization, and failed to activate Rac1, a member of the Rho GTPase family crucially involved in actin
organization and orientation of apico-basal polarity. These data indicate that LIN7-IRSp53 association plays a role during assembly of functional TJs and surface polarization in epithelial cells.
In NSC-34 cells, a motor neuron-like hybrid cell line, we examined the role of LIN7 in the localization and function of the IRSp53 partner of its PDZ domain.
To this purpose we analyzed the effects of transient expression of IRSp53 and
LIN7 constructs (expressed individually or together) in NSC-34 cells. We found that overexpression of IRSp53 induced the formation of an exceeding number of filopodia and pseudo-fiopodia (without detectable filamentous actin), while a
number of filopodia comparable to untransfected cells and absence of pseudofilopodia were observed when IRSp53 was coexpressed together with LIN7 and
when the L27-IRSp53del5 chimera was expressed. Our data indicate that LIN7-
IRSp53 association is also involved in the polarization of neuronal cells by
regulating the formation of the precursors of dendritic spines.
In order to maintain cell polarity, epithelial cells have also to ensure proper
delivery and/or retention of apical and basolateral cargo to their respective target
location. Moreover, the recycling of proteins from and to different plasma
membrane domains must be finely regulated. In this study, we have used glutamate and GABA transporters differentially located to apical or lateral surface as molecular tool to study the sorting mechanisms regulating the polarized distribution of plasma membrane proteins.
In MDCK cells, we have investigated the PKC mediated regulation of the apically located EAAC1 glutamate transporter. We found that stimulation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) treatment induces a time-dependent decrease in glutamate trans
Tipologia IRIS:
13 - Tesi di dottorato discussa entro ottobre 2010
Elenco autori:
V. Padovano
Link alla scheda completa: