Optimized methods for imaging membrane nanotubes between T cells and trafficking of HIV-1

A wide variety of cell types, including immune cells, have been observed to frequently interact via transient, long-distance membrane connections [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17]. However, considerable heterogeneity in their structure, mode of formation and functional properties has emerged, suggesting the existence of distinct subclasses [18], [19], [20], [21]. Open-ended tunneling nanotubes allow for the trafficking of cytoplasmic material, e.g. endocytic vesicles, or the transmission of calcium signals [1], [8]. Closed-ended membrane nanotubes do not seamlessly connect the cytoplasm between two interacting cells and a junction exists within the nanotube or where the nanotube meets a cell body [4], [5], [7]. Recent live cell imaging suggested that membrane nanotubes between T cells could present a novel route for HIV-1 transmission [7], [22]. Here, we describe detailed protocols for observing membrane nanotubes and HIV-1 trafficking by live cell fluorescence microscopy.