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First published online 16 September 2003
doi: 10.1242/jcs.00749

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The formation of vault-tubes: a dynamic interaction between vaults and vault PARP

¹ Department of Hematology, Erasmus Medical Center, PO Box 1738, 3000 DR Rotterdam, The Netherlands
² Department of Pathology, Erasmus Medical Center/Josephine Nefkens Institute, PO Box 1738, 3000 DR Rotterdam, The Netherlands
³ Department of Pathology, Free University Medical Center, PO Box 7057, 1007 MB Amsterdam, The Netherlands
⁴ Department of Medical Oncology, Erasmus Medical Center/Josephine Nefkens Institute, PO Box 1738, 3000 DR Rotterdam, The Netherlands

View larger version (74K): [in a new window]   Fig. 1. Incorporation of GFP-tagged MVP molecules in vaults. (A) The SW1573 transfectant expressing MVP-GFP (a) was examined by fluorescence microscopy. The cell line SW1573 (b) was processed for indirect immunofluorescence and stained with anti-MVP (LRP-56). Bar, 10 µm. (B) Vault particles were pelleted from a total cell lysate (TL) of SW1573, SW1573/MVP-GFP transfectants and SW1573 transfectants expressing a GFP-tagged truncated MVP (SW1573/MVP706-GFP) by a 100,000 g centrifugation step. Immunoblotting using rabbit polyclonal anti-MVP was performed to determine the presence of MVP and the GFP-fusion proteins in the resulting pellet (P) and supernatant (S) fractions. Note that SW1573/MVP-GFP and SW1573/MVP706-GFP show two bands, representing endogenous (lower band) and GFP-tagged MVP or truncated MVP (upper band). (C) Immunoblot analysis showing the presence of the minor vault protein VPARP in immunoprecipitates obtained from MVP-GFP transfectants using anti-GFP (lane 2). Lane 1 contains control immunoprecipitates in which a polyclonal rabbit pre-immune serum was used.

View larger version (13K): [in a new window]   Fig. 2. Dynamics of the vault complex measured by FRAP. (A) The graph shows the mobility of fluorescent MVP-GFP (red line) compared with free GFP (green line) and a GFP-tagged truncated MVP that is not incorporated into vaults (MVP706-GFP, blue line), each measured in 12 cells. Fluorescence intensity before the bleach pulse was set at 1. Note that the recovery of fluorescence is never 100% because of removal of fluorescence by the bleach pulse. This effect differs between proteins depending on their residence time in the strip during bleaching. (B) The redistribution of fluorescent MVP-GFP within a bleached strip was monitored at 37°C (red line) and at 28°C (blue line) with intervals of 100 milliseconds. The graph depicts the data of an experiment in which 21 cells were measured.

View larger version (75K): [in a new window]   Fig. 3. Formation of vault-tubes. (A) SW1573/MVP-GFP cells were incubated at 37°C (a-c), at 21°C for 60 minutes (d-f) and at 37°C for 30 minutes after the treatment at 21°C (g-i). Shown is the MVP-GFP fluorescent signal (a,d,g), the indirect immunofluorescence staining for VPARP (b,e,h) and the merged images (c,f,i). Incubation at 21°C resulted in the formation of large cytoplasmic, tube-like structures (vault-tubes, d). Under the same conditions VPARP staining reveals a dramatic decrease in the number of non-vault-associated VPARP-rods. The VPARP signal almost completely colocalizes with the tube structures (e and f). Both the MVP and VPARP distribution returns to normal when the cells cultured at 21°C were placed at 37°C for 30 minutes (g-i). Bar, 10 µm. (B) Partial colocalization of GFP-tagged MVP (a and d) and the VPARP-rods (b and e) in the merged picture (c and f) after incubation of SW1573/MVP-GFP cells at 21°C for 10 minutes. The arrowheads point to sites where MVP-GFP clusters have formed, which are in close proximity to the VPARP-rods. Bars, 5 µm.

View larger version (29K): [in a new window]   Fig. 4. Cytoplasmic VPARP-rods and their relation to MVP expression levels. (A) Anti-VPARP staining of untransfected SW1573 (a) and HeLa (b) cells showed a normal VPARP distribution, including the VPARP-rods. Background staining was absent as revealed by the staining of SW1573 cells with an isotype antibody (c). Bar, 10 µm. (B) The left-hand graph depicts the average length of the VPARP-rods in untransfected SW1573 cells, in its vault-overexpressing drug-resistant derivative SW1573/2R120 and in the SW1573/MVP-GFP transfectant. The numbers in the bars indicate the number of VPARP-rods measured. The data were analyzed by the Student's t-test. The P-value was in all cases below 0.05, indicating statistically significant differences. The right-hand graph shows the quantification of the MVP (black bars) and VPARP (gray bars) levels as determined by immunoblotting (see inlay; lane 1, SW1573/MVP-GFP; lane 2, SW1573/2R120; and lane 3, SW1573, with equal amounts of total protein loaded). The levels of VPARP and MVP in the SW1573 cells are arbitrarily set at 1 and corrected for loading using the ß-tubulin signal as a reference. Note that the two protein bands visible in the MVP panel in lane 1 represent the endogenous MVP and the MVP-GFP fusion product.

View larger version (42K): [in a new window]   Fig. 5. Coiled-coil domain of MVP essential for incorporation into vault-tubes. (A) Vault-tube formation, by a 60 minute incubation at 21°C, could be observed in nontransfected SW1573 (a) and SW1573/2R120 (b) cells immunodetected with anti-MVP (LRP-56 mAb). Bar, 10 µm. (B) Stably transfected SW1573 cells expressing either full-length MVP (a and c) or the amino acids 1-706 of MVP (b and d) fused to GFP were incubated at 21°C for 60 minutes. The GFP-tagged proteins in which a part of the coiled-coil domain of MVP is deleted (MVP706-GFP) are not incorporated into vault-tubes. Bar, 10 µm.

View larger version (39K): [in a new window]   Fig. 6. Dimensions and dynamics of the vault-tubes. (A) Images obtained by confocal laser scan microscopy on living SW1573/MVP-GFP cells cultured at 21°C clearly show that vault-tubes are hollow cylinders. Depicted are transverse and longitudinal optical sections of the vault-tubes. Bar, 5 µm. (B) Dynamics of the vault-tubes were studied with FRAP. Shown are images from a representative FRAP experiment of a vault-tube before the bleach pulse (pb), directly after bleaching (0 s) and 50, 100 and 200 seconds after the bleach pulse. The rapid recovery of fluorescent staining in the bleached area indicated that vault-tubes are dynamic structures. Bar, 5 µm.

View larger version (62K): [in a new window]   Fig. 7. Vault-tube formation and the stability of microtubules. SW1573/MVP-GFP cells were cultured at 37°C for 60 minutes in the absence (a and d) or presence of 30 µM nocodazole (b and e) or 20 µM taxol (c and f). Subsequently, the cells were placed at 21°C for 60 minutes and monitored for vault-tube formation by fluorescence microscopy. Bar, 10 µm.