Inter-cellular transport of ran GTPase.

Ran, a member of the Ras-GTPase superfamily, has a well-established role in regulating the transport of macromolecules across the nuclear envelope (NE). Ran has also been implicated in mitosis, cell cycle progression, and NE formation. Over-expression of Ran is associated with various cancers, although the molecular mechanism underlying this phenomenon is unclear. Serendipitously, we found that Ran possesses the ability to move from cell-to-cell when transiently expressed in mammalian cells. Moreover, we show that the inter-cellular transport of Ran is GTP-dependent. Importantly, Ran displays a similar distribution pattern in the recipient cells as that in the donor cell and co-localizes with the Ran binding protein Nup358 (also called RanBP2). Interestingly, leptomycin B, an inhibitor of CRM1-mediated export, or siRNA mediated depletion of CRM1, significantly impaired the inter-cellular transport of Ran, suggesting a function for CRM1 in this process. These novel findings indicate a possible role for Ran beyond nucleo-cytoplasmic transport, with potential implications in inter-cellular communication and cancers

Inter-cellular transfer of Ran.

<p>HeLa cells were transfected with indicated constructs for 9 h and were then co-cultured with untransfected NIH3T3 cells for 18 h. Cells were stained with GFP antibody (green) and the DNA dye Hoechst 33342 (pseudocoloured in red). Arrows indicate NIH3T3 cells as detected by the characteristic punctate staining of the nucleus. Scale bar, 25 μm.</p

Ectopic expression of Ran GTPase.

<p>HeLa cells were transfected with indicated constructs for 24 h and fixed using methanol (A) or paraformaldehyde (B) and were processed for fluorescence microscopy. GFP is detected with a specific polyclonal antibody (green) and DNA was stained with Hoechst 33342 (blue). (C) COS-7 cells were transfected with indicated constructs for 24 h and the unfixed cells were directly visualized under fluorescence microscope. In all the above experiments, the adjacent respective graph represents quantitative data indicating the percentage of cells showing the GFP proteins and was derived from three independent experiments (in each experiment at least 100 cells were counted). Data are expressed as mean ± SD. Scale bar, 20 μm. (D) HeLa cells transfected with indicated constructs were lysed, separated on 10% SDS-PAGE and analysed by western blotting (WB) with GFP and Ran antibodies. α-tubulin was used as loading control. Molecular weights (in kDa) are shown in numbers.</p

Transient transfection assay for inter-cellular transport of Ran.

<p>HeLa cells were co-transfected with mCherry-α-tubulin (transfection marker, red) and indicated GFP constructs (green) for 9 h. Cells were fixed and analysed for the presence of mCherry and GFP. DNA was stained in blue. Scale bar, 20 μm. Quantitative data showing the number of recipient cells displaying GFP staining surrounding the mCherry-α-tubulin positive donor cell. Cells were counted from 15 individual fields randomly across three independent experiments. Data are expressed as mean ± SD.</p