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 [1–17]. However, considerable heterogeneity in their structure, mode of formation and functional properties has emerged, suggesting the existence of distinct subclasses [18–21]. 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.