Cellular and Molecular Neurobiology
Author: Candela Belliard | Email: candebelliard@gmail.com
Candela Belliard1°, Nicolás Matías Rosas1°, Beata Fuchsova1°
1° Instituto de Investigaciones Biotecnológicas, Escuela de Bio y Nanotecnologías (EByN), Universidad Nacional de San Martín (UNSAM) – Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), San Martín, Buenos Aires, Argentina
The neuronal membrane glycoprotein M6a (Gpm6a) belongs to the proteolipid protein gene family and alterations in its expression and sequence are linked to neuropsychiatric disorders in humans. The mechanism of its action is not clearly understood. However, there is evidence for the role in the processes of neuronal differentiation and development such as filopodium formation, neurite extension, and synaptogenesis.
Recently, we have identified E258 as a key functional residue in the process of filopodium formation. Moreover, we have found that its cell surface expression is diminished while it displays increased intracellular accumulation with the preferential localization to Lamp1-positive structures. Different types of membrane outgrowth require polarized membrane transport and the incapacity of E258A to induce filopodium formation could be linked to the disrupted trafficking of mutant Gpm6a to the cell surface. Therefore, in the present study we used confocal microscopy and live cell imaging to analyze Gpm6a wt and E258A intracellular trafficking in neuroblastoma cells line N2a. Using Trackmate plugin of ImageJ, the number of intracellular particles of Gpm6a wt and E258A was determined and their localization, speed, distance traveled and displacement was evaluated. We also analyzed their colocalization with endosomal markers such as Rab5 (early endosomes), Rab7 (late endosomes), Rab11 (recycling endosomes), and the Lamp1-positive late endosomal and/or lysosomal structures.