IIBIO   27936
INSTITUTO DE INVESTIGACIONES BIOTECNOLOGICAS
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Outgrowth of filopodia is associated with intracellular trafficking of Gpm6a
Autor/es:
FRASCH, A. C.; FUCHSOVA, B.; ROSAS, N. M.
Lugar:
Montreal
Reunión:
Congreso; 2019 ISN Biennal Meeting; 2019
Institución organizadora:
The International Society for Neurochemistry (ISN) and the American Society for Neurochemistry (ASN)
Resumen:
Outgrowth of filopodia is associated withintracellular trafficking of Gpm6aRosas N.M.,Frasch A.C. and Fuchsova B.Instituto de InvestigacionesBiotecnol├│gicas (IIB-INTECH, UNSAM, CONICET),San Martin, Buenos Aires, Argentina Gpm6a is aneuronal membraneglycoprotein that functions in the processes of neuronal development and itsoverexpression leads to the extensive formation offilopodia. Neuropsychiatric disorders and chronic stressexposure have been linked to the alterations in Gpm6a expression levels or sequence. However, the mechanism of action of Gpm6a is notclearly understood.Previously, we identifiedK250, K255, and E258 in the C-terminal of Gpm6aas key functional residues for the formation of filopodia. Subsequent bioinformaticanalysis revealed that K250, K255, andE258 are predicted as part of sorting signals of transmembraneproteins. Colocalization assay showed that deletion of the C-terminus diminishes the association of Gpm6awith clathrin in hippocampal neurons implying involvement of clathrin-mediatedtrafficking events. Moreover, using flow cytometry we found that subtitionof K250, K255, and E258 with alanine dimishes the amount of Gpm6a on cellsurface and in case of K255 and E258 also leads to the lower amount of total expressedprotein.Here usingconfocal microscopy we analyze the subcellular localization of the mutant formsof Gpm6a that fail to induce filopodia formation. K250A and E258A displayincreased intracellular accumulation of the protein and a preferentiallocalization to Lamp1-positive structures. To determine if K250, K255 and E258 substitionsleads to an increased protein degradation we use flow cytometry to quantify theGpm6a expression levels upon treatment with different protease inhibitors. Thelocalization of Gpm6a mutants to LC3- and Calnexin- positive structures wasalso evaluated.<!-- /* Font Definitions */@font-face{font-family:"MS 明朝";panose-1:0 0 0 0 0 0 0 0 0 0;mso-font-charset:128;mso-generic-font-family:roman;mso-font-format:other;mso-font-pitch:fixed;mso-font-signature:1 134676480 16 0 131072 0;}@font-face{font-family:"MS 明朝";panose-1:0 0 0 0 0 0 0 0 0 0;mso-font-charset:128;mso-generic-font-family:roman;mso-font-format:other;mso-font-pitch:fixed;mso-font-signature:1 134676480 16 0 131072 0;}@font-face{font-family:Calibri;panose-1:2 15 5 2 2 2 4 3 2 4;mso-font-charset:0;mso-generic-font-family:auto;mso-font-pitch:variable;mso-font-signature:3 0 0 0 1 0;}@font-face{font-family:ArialMT;panose-1:0 0 0 0 0 0 0 0 0 0;mso-font-alt:Arial;mso-font-charset:77;mso-generic-font-family:swiss;mso-font-format:other;mso-font-pitch:auto;mso-font-signature:3 0 0 0 1 0;} /* Style Definitions */p.MsoNormal, li.MsoNormal, div.MsoNormal{mso-style-unhide:no;mso-style-qformat:yes;mso-style-parent:"";margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:115%;mso-pagination:widow-orphan;font-size:11.0pt;font-family:Calibri;mso-ascii-font-family:Calibri;mso-ascii-theme-font:minor-latin;mso-fareast-font-family:Calibri;mso-fareast-theme-font:minor-latin;mso-hansi-font-family:Calibri;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:minor-bidi;mso-ansi-language:ES;}.MsoChpDefault{mso-style-type:export-only;mso-default-props:yes;font-size:11.0pt;mso-ansi-font-size:11.0pt;mso-bidi-font-size:11.0pt;font-family:Calibri;mso-ascii-font-family:Calibri;mso-ascii-theme-font:minor-latin;mso-fareast-font-family:Calibri;mso-fareast-theme-font:minor-latin;mso-hansi-font-family:Calibri;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:minor-bidi;mso-ansi-language:ES;}.MsoPapDefault{mso-style-type:export-only;margin-bottom:10.0pt;line-height:115%;}@page WordSection1{size:8.5in 11.0in;margin:1.0in 1.25in 1.0in 1.25in;mso-header-margin:.5in;mso-footer-margin:.5in;mso-paper-source:0;}div.WordSection1{page:WordSection1;}-->