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Inter-cellular transport of ran GTPase.

Khuperkar D, Helen M, Magre I, Joseph J - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: National Centre for Cell Science, Ganeshkhind, Pune, India.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Leptomycin B treatment interferes with inter-cellular transport of Ran.(A) COS-7 cells were co-transfected with indicated GFP-constructs and mCherry-α-tubulin (transfection marker), and after 6h, were untreated (-) or treated (+) with leptomycin B (LMB, 5 ng/ml) and continued till the indicated time points (hours post-transfection). The inter-cellular transport of GFP-proteins was monitored by visualizing the unfixed cells directly under fluorescence microscope and calculating the fold increase in the number of GFP positive cells over the number of cells expressing the transfection marker mCherry-α-tubulin. Quantitative representation is shown. (B) LMB treatment in donor cells impairs inter-cellular transport of Ran. HeLa cells were co-transfected with HA-GAPDH and 2xGFP control or 2xGFP-Ran constructs as indicated. After 12 h, LMB treatment (5 ng/mL) was given for 12 h. Then the cells were washed, trypsinized and co-cultured with untreated HeLa cells (1:4 ratio of transfected to untransfected cells) and stained after 24 h for HA-GAPDH (transfection marker; blue) and endogenous Nup358 (red). GFP (green) was directly visualized by epifluorescence. Scale bar, 20 μm. Lower Panel: Quantitative representation of inter-cellular transport of Ran when untreated (-) and LMB treated (+) transfected (donor) cells were co-cultured with untransfected cells. The quantitation was performed as mentioned for Fig 3, except that cells were counted from 30 different fields across three independent experiments. Data are expressed as mean ± SD.
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pone.0125506.g005: Leptomycin B treatment interferes with inter-cellular transport of Ran.(A) COS-7 cells were co-transfected with indicated GFP-constructs and mCherry-α-tubulin (transfection marker), and after 6h, were untreated (-) or treated (+) with leptomycin B (LMB, 5 ng/ml) and continued till the indicated time points (hours post-transfection). The inter-cellular transport of GFP-proteins was monitored by visualizing the unfixed cells directly under fluorescence microscope and calculating the fold increase in the number of GFP positive cells over the number of cells expressing the transfection marker mCherry-α-tubulin. Quantitative representation is shown. (B) LMB treatment in donor cells impairs inter-cellular transport of Ran. HeLa cells were co-transfected with HA-GAPDH and 2xGFP control or 2xGFP-Ran constructs as indicated. After 12 h, LMB treatment (5 ng/mL) was given for 12 h. Then the cells were washed, trypsinized and co-cultured with untreated HeLa cells (1:4 ratio of transfected to untransfected cells) and stained after 24 h for HA-GAPDH (transfection marker; blue) and endogenous Nup358 (red). GFP (green) was directly visualized by epifluorescence. Scale bar, 20 μm. Lower Panel: Quantitative representation of inter-cellular transport of Ran when untreated (-) and LMB treated (+) transfected (donor) cells were co-cultured with untransfected cells. The quantitation was performed as mentioned for Fig 3, except that cells were counted from 30 different fields across three independent experiments. Data are expressed as mean ± SD.

Mentions: CRM1, an important RanGTP binding protein, is involved in the export of cargos from the nucleus to the cytoplasm. We were interested to test if leptomycin B (LMB), a known inhibitor of the export receptor CRM1 that affects its interaction with RanGTP [48], impaired cell-to-cell transfer of Ran. We co-transfected COS-7 cells with GFP-control, GFP-Ran-Q69L, GFP-Ran-T24N or GFP-Ran-WT and mCherry-α-tubulin (transfection marker) and monitored the cell-to-cell transfer using live microscopy at different time intervals in the absence or presence of LMB. We found that LMB significantly interfered with cell-to-cell transfer of Ran-Q69L (Fig 5A). Moreover, siRNA mediated depletion of CRM1 in HeLa cells significantly impaired the inter-cellular transfer of Ran (S4 Fig).


Inter-cellular transport of ran GTPase.

Khuperkar D, Helen M, Magre I, Joseph J - PLoS ONE (2015)

Leptomycin B treatment interferes with inter-cellular transport of Ran.(A) COS-7 cells were co-transfected with indicated GFP-constructs and mCherry-α-tubulin (transfection marker), and after 6h, were untreated (-) or treated (+) with leptomycin B (LMB, 5 ng/ml) and continued till the indicated time points (hours post-transfection). The inter-cellular transport of GFP-proteins was monitored by visualizing the unfixed cells directly under fluorescence microscope and calculating the fold increase in the number of GFP positive cells over the number of cells expressing the transfection marker mCherry-α-tubulin. Quantitative representation is shown. (B) LMB treatment in donor cells impairs inter-cellular transport of Ran. HeLa cells were co-transfected with HA-GAPDH and 2xGFP control or 2xGFP-Ran constructs as indicated. After 12 h, LMB treatment (5 ng/mL) was given for 12 h. Then the cells were washed, trypsinized and co-cultured with untreated HeLa cells (1:4 ratio of transfected to untransfected cells) and stained after 24 h for HA-GAPDH (transfection marker; blue) and endogenous Nup358 (red). GFP (green) was directly visualized by epifluorescence. Scale bar, 20 μm. Lower Panel: Quantitative representation of inter-cellular transport of Ran when untreated (-) and LMB treated (+) transfected (donor) cells were co-cultured with untransfected cells. The quantitation was performed as mentioned for Fig 3, except that cells were counted from 30 different fields across three independent experiments. Data are expressed as mean ± SD.
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Related In: Results  -  Collection

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pone.0125506.g005: Leptomycin B treatment interferes with inter-cellular transport of Ran.(A) COS-7 cells were co-transfected with indicated GFP-constructs and mCherry-α-tubulin (transfection marker), and after 6h, were untreated (-) or treated (+) with leptomycin B (LMB, 5 ng/ml) and continued till the indicated time points (hours post-transfection). The inter-cellular transport of GFP-proteins was monitored by visualizing the unfixed cells directly under fluorescence microscope and calculating the fold increase in the number of GFP positive cells over the number of cells expressing the transfection marker mCherry-α-tubulin. Quantitative representation is shown. (B) LMB treatment in donor cells impairs inter-cellular transport of Ran. HeLa cells were co-transfected with HA-GAPDH and 2xGFP control or 2xGFP-Ran constructs as indicated. After 12 h, LMB treatment (5 ng/mL) was given for 12 h. Then the cells were washed, trypsinized and co-cultured with untreated HeLa cells (1:4 ratio of transfected to untransfected cells) and stained after 24 h for HA-GAPDH (transfection marker; blue) and endogenous Nup358 (red). GFP (green) was directly visualized by epifluorescence. Scale bar, 20 μm. Lower Panel: Quantitative representation of inter-cellular transport of Ran when untreated (-) and LMB treated (+) transfected (donor) cells were co-cultured with untransfected cells. The quantitation was performed as mentioned for Fig 3, except that cells were counted from 30 different fields across three independent experiments. Data are expressed as mean ± SD.
Mentions: CRM1, an important RanGTP binding protein, is involved in the export of cargos from the nucleus to the cytoplasm. We were interested to test if leptomycin B (LMB), a known inhibitor of the export receptor CRM1 that affects its interaction with RanGTP [48], impaired cell-to-cell transfer of Ran. We co-transfected COS-7 cells with GFP-control, GFP-Ran-Q69L, GFP-Ran-T24N or GFP-Ran-WT and mCherry-α-tubulin (transfection marker) and monitored the cell-to-cell transfer using live microscopy at different time intervals in the absence or presence of LMB. We found that LMB significantly interfered with cell-to-cell transfer of Ran-Q69L (Fig 5A). Moreover, siRNA mediated depletion of CRM1 in HeLa cells significantly impaired the inter-cellular transfer of Ran (S4 Fig).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: National Centre for Cell Science, Ganeshkhind, Pune, India.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus