Technical Reference #1675

1675.

Gamma-protocadherin Homophilic Interactions and Intracellular Trafficking is Controlled by the Cytoplasmic Domain in Neurons Monica Fernández-Monreal; Semie Kang; Greg R. Phillips, Mount Sinai School of Medicine, Molecular and Cellular Neuroscience, 40(1675), (2009)

Abstract:
Gamma-protocadherins (Pcdh-γs) are good candidates to mediate specificity in synaptogenesis but their role in cell-cell interactions is a matter of debate. We proposed that Pcdh-γs modify preformed synapses via trafficking of Pcdh-γs-containing organelles insertion into synaptic membranes and homophilic transcellular interaction. Here we provide evidence in support of this model. We show for the first time that Pcdh-γs have homophilic properties and that they accumulate at dendro-dendritic and axo-dendritic interfaces during neuronal development. Pcdh-γs are maintained in a substantial mobile intracellular pool in dendrites and cytoplasmic deletion shifts the molecule to the surface and reduces the number and velocity of the mobile packets.We monitored Pcdh-γ temporal and spatial dynamics in transport organelles. Pcdh-γ organelles bud and fuse with stationary clusters near synapses. These results suggest that Pcdh-γ-mediated cell-cell interactions in synapse development or maintenance are tightly regulated by control of intracellular trafficking via the cytoplasmic domain.

Keywords:
adhesion; sunaptogenesis; cadherin; live cell imaging; FRAP; trafficking

Materials & Methods:
Neuronal cultures Hippocampi were dissected from E18 rat embryos and cells dissociated with trypsin as described (Banker and Goslin 1991). Neurons were plated at 0.9–1.0×104 cells/cm2 onto poly-L-lysinecoated coverslips or 35 mm glass bottomed Petri dishes (MatTek Corporation) and cultured in Neurobasal medium including B27 supplements (Invitrogen) for periods ranging from 2 days in vitro (DIV) to 21 DIV. Cells were maintained at 37 °C and 5% CO2.

Microscopic Technique
Confocal Microscopy, Time-Lapse Microscopy, Fluorescence Microscopy

Cell Type(s)
Neurons - Hippocampal, E18 rat embryos