Studies on targeting of vesicle transport using virally medicated expression system

Abstract

Neurons are composed of two morphologically and molecularly distinct domains, axons and dendrites. The accurate localization of proteins to these domains is critical for the neuronal functions. The proteins destined to localize into different cellular domains are probably packaged into different populations of carrier vesicles in the trans‐Golgi network. The objectives of this study are to examine the mechanisms to regulate targeting of transferrin receptor (TfR), a dendritic protein, neuron‐glia cell adhesion molecule (NgCAM), an axonal protein, and amyloid precursor protein (APP) and synaptophysin (p38), synaptic proteins, using live cell imaging system. Previous studies were complicated by overexpression artifacts. Our approach to this study was to apply an adenovirus mediated regulatable expression system for TfR, NgCAM, APP and p38 in NT‐2 cells, a neuronal precursor cell line, and BHK cells. Live cell imaging was performed with confocal microscopy. There are two specific features of our adenovirus systems: (1) proteins are tagged with green fluorescent protein (GFP) or its variants (YFP and CFP) for live cell imaging; (2) protein expression is under the control of tetracycline responsive element (Tet‐On), for tight control of both timing and levels of expression. Our results showed that APP, p38 and TfR vesicle movements in both BHK and NT‐2 cells were bi‐directional. NgCAM in BHK cells showed plasma membrane localization and minimal vesicle movement whereas expression of NgCAM in NT‐2 cells showed both plasma membrane and vesicle localization with bidirectional vesicle movements. In conclusion, our adenovirus system appears well suited for live cell imaging and could be adapted with other proteins of interest for studies of protein transport and sorting during neuronal development.