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. However, considerable heterogeneity in their structure, mode of formation and functional properties has emerged, suggesting the existence of distinct subclasses. Open-ended tunneling nanotubes allow for the trafficking of cytoplasmic material, e.g. endocytic vesicles, or the transmission of calcium signals. 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. Recent live cell imaging suggested that membrane nanotubes between T cells could present a novel route for HIV-1 transmission. Here, we describe detailed protocols for observing membrane nanotubes and HIV-1 trafficking by live cell fluorescence microscopy.
Sowinski
Stefanie
S
Gladstone Institute of Virology and Immunology, San Francisco, CA 94158-2216, USA. stefanie.sowinski@gladstone.ucsf.edu
Alakoskela
Juha-Matti
JM
Jolly
Clare
C
Davis
Daniel M
DM
D018929:Cell Culture Techniques; D021961:Cell Membrane Structures / Q000648:ultrastructure*; D058948:Cell Tracking; D005353:Fibronectins / Q000378:metabolism; D005404:Fixatives; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D054992:Immunological Synapses / Q000648:ultrastructure; D019169:Jurkat Cells; D018613:Microscopy, Confocal; D011554:Pseudopodia / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D013601:T-Lymphocytes / Q000648:ultrastructure* / Q000821:virology; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000096:biosynthesis / Q000235:genetics
D005353:Fibronectins; D005404:Fixatives; D011993:Recombinant Fusion Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; D049452:Green Fluorescent Proteins