BACKGROUND INFORMATION: Cells, especially those of the immune system, can form long and thin connections termed tunnelling nanotubes (TNTs). These structures can reach >100 µm in length and, in T-cells, contain actin but no tubulin and are not open ended. T-cell TNTs were found to form following cell contact and to enable the transfer of HIV-1 from an infected- to a connected-T-cell. TNTs are poorly characterised at molecular level. RESULTS: We found Rab11 and tetraspanins, especially CD81, all along T-cells TNTs, whereas Rab4 and Rab35 were absent from these structures. Regarding actin cytoskeleton regulators, Exo70, N-WASP and especially ezrin accumulated at the level of the TNT tip that contacts the connected cell. Phosphoinositides such as PI(4,5)P2 were also concentrated at this level together with HIV-1 Gag. Gag spots on cells and TNTs were essentially immobile, and likely correspond to area of Gag multimerisation for budding to form virus-like particles. Mobility of PHPLCδ , a specific probe for PI(4,5)P2 , was reduced > threefold at the level of TNT basis or tip compared with the cell body. CONCLUSION: Our study identified the TNT tip as an active zone of actin cytoskeleton reorganisation with the presence of ezrin, Exo70, N-WASP and PI(4,5)P2 . The latter is also known to enable HIV-1 Gag recruitment for viral budding, and the presence of Gag at this level, contacting the connected cell, indicates that the TNT tip is also a favourite place for HIV-1 assembly and budding.
BACKGROUND INFORMATION: Cells, especially those of the immune system, can form long and thin connections termed tunnelling nanotubes (TNTs). These structures can reach >100 µm in length and, in T-cells, contain actin but no tubulin and are not open ended. T-cell TNTs were found to form following cell contact and to enable the transfer of HIV-1 from an infected- to a connected-T-cell. TNTs are poorly characterised at molecular level. RESULTS: We found Rab11 and tetraspanins, especially CD81, all along T-cells TNTs, whereas Rab4 and Rab35 were absent from these structures. Regarding actin cytoskeleton regulators, Exo70, N-WASP and especially ezrin accumulated at the level of the TNT tip that contacts the connected cell. Phosphoinositides such as PI(4,5)P2 were also concentrated at this level together with HIV-1Gag. Gag spots on cells and TNTs were essentially immobile, and likely correspond to area of Gag multimerisation for budding to form virus-like particles. Mobility of PHPLCδ , a specific probe for PI(4,5)P2 , was reduced > threefold at the level of TNT basis or tip compared with the cell body. CONCLUSION: Our study identified the TNT tip as an active zone of actin cytoskeleton reorganisation with the presence of ezrin, Exo70, N-WASP and PI(4,5)P2 . The latter is also known to enable HIV-1Gag recruitment for viral budding, and the presence of Gag at this level, contacting the connected cell, indicates that the TNT tip is also a favourite place for HIV-1 assembly and budding.