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First published online November 24, 2004
doi: 10.1242/10.1242/jcs.01550

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Dynamic targeting of microtubules by TPPP/p25 affects cell survival

Atilla Lehotzky^1,*, László Tirián^2,*, Natália Tökési¹, Péter Lénárt³, Bálint Szabó⁴, János Kovács⁵ and Judit Ovádi^1,

¹ Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, 1113 Budapest, Hungary
² Department of Biology, Faculty of Medicine, University of Szeged, 6720 Szeged, Hungary
³ European Molecular Biology Laboratory, 69117 Heidelberg, Germany
⁴ Biological Physics Research Group, Hungarian Academy of Sciences, 1117 Budapest, Hungary
⁵ Department of General Zoology, Eötvös University of Sciences, 1117 Budapest, Hungary

View larger version (98K): [in a new window]   Fig. 1. Phase-contrast and fluorescent images of HeLa cells transfected with pEGFP-TPPP/p25 from time-lapse recordings. (A) Transfected cultures of HeLa cells were maintained and imaged on a microscope stage by phase-contrast and epifluorescence. A pair of cells is shown with increasing fluorescence over time. (B) Phase-contrast frames were selected from a time-lapse recording with fluorescent insets. Cells marked 1 and 2 did not show detectable fluorescence before division. Later, they became fluorescent, as shown in the image captured at 42.5 hours. Cell 2 died at 46 hours; cell 1 divided into cells 3 and 4 at 60 hours. Cells 3 and 4 were still alive after 91 hours. Time elapsed after transfection with pEGFP-TPPP/p25 is indicated in the lower left corners. Bar, 100 µm.

View larger version (91K): [in a new window]   Fig. 2. Overview of HeLa cells transfected with pEGFP-TPPP/p25. (A) Cells were fixed in cold-buffered ethanol for immunocytochemistry with anti-tubulin antibody. The representative image shows three TPPP/p25-transfected cells with distinct morphologies in the same microscopic field. Note the colocalization (orange) of EGFP-TPPP/p25 (green) with the microtubular network (red) at low expression levels (cell marked 1). The other two cells with bright fluorescence show an aberrant microtubular network and morphological changes with colocalization (marked 2 and 3). Comparing the fluorescence of these three transfected cells suggests low (cell 1) and high (cells 2 and 3) levels of expressed protein. (B) TMRE uptake in transfected HeLa cells. TMRE (red) was used to investigate mitochondrial membrane state in live cells. At low levels of expression, the cells show a polarization state similar to that of untransfected cells. (C) HeLa cells transfected with pEGFP-C1 plasmid. Note the strong nuclear presence of the EGFP protein. (D-F) HeLa cells transfected with EGFP-TPPP/p25 plasmid. Representative examples of the different expression `classes' are presented: (D) low-level expressing; (E) aggresome-like; (F) nuclear cage. Bars, 10 µm.

View larger version (81K): [in a new window]   Fig. 3. HeLa cells transfected with pEGFP-TPPP/p25 and their filamentous networks. (A-C) Cells were transfected and incubated for 20 hours, fixed with cold ethanol for immunocytochemistry to detect the alignment of TPPP/p25 (green) with tubulin (A), actin (B) and vimentin (C) (all red). (A) The microtubular network is not affected in the transfected cell, however, accumulation of fluorescent material started at the perinuclear position. Virtually complete colocalization with microtubules is observed. (B) Actin filaments show complete dislocation with the green EGFP-TPPP/p25 signal. Red and green images were taken at different positions on the z-axis to get focused signals. (C) Intermediate filaments (vimentin) do not show colocalization with EGFP-TPPP/p25. Bars, 10 µm.

View larger version (110K): [in a new window]   Fig. 4. EGFP-TPPP/p25 localization in transfected NRK cells at different stages of the cell cycle. Tubulin, TPPP/p25 and DNA are red, green and blue in the merged images, respectively. (A-D) At interphase TPPP/p25 localizes over the microtubules throughout the cell, but accumulates at a higher levels in the vicinity of the centrosome (green dominates in the merged image D). (F and J) From prophase (not shown) to late anaphase, free TPPP/p25 level is considerably higher in the cytoplasm. In metaphase (E-H) and anaphase (I-L) TPPP/p25 accumulation is detectable only over the spindle microtubules and the centrosomes. (M-P) The level of free TPPP/p25 decreases again at the telophasecytokinesis transition and TPPP/p25 becomes preferentially enriched over the microtubules spanning the cytokinetic cleavage furrow.

View larger version (97K): [in a new window]   Fig. 5. Rapid exchange of EGFP-TPPP/p25 on microtubules demonstrated by FRAP. (A-C) NRK cells were transfected with pEGFP-TPPP/p25. The experimental region (white square shown in B) containing a thick microtubule bundle was bleached with a high-intensity laser beam. Fluorescent intensities were measured over the blue rectangles (rectangle 1 resides within the bleached area; rectangle 2 is in a control region; values from rectangle 3 were used for background correction). Insets from B and C are shown at 1 and 19 seconds, respectively. Fluorescence of the bleached region reappeared nearly completely during the observation period of 20 seconds indicating the very rapid exchange of TPPP/p25 on the microtubule bundle (compare the intensities of the enlarged insets for rectangle 1 below A and C). (D) The intensities of rectangle 1 compared to the bleached area (), and rectangle 2 compared to the control area () over the course of the experiment.

View larger version (90K): [in a new window]   Fig. 6. Transfected HeLa cells in the high expression state. HeLa cells were transfected with pEGFP-TPPP/p25 (A and B) or pcDNA3-TPPP/p25 (C). After 20 hours, cells were fixed with ice-cold buffered ethanol. Tubulin was stained with anti--tubulin antibody (red) (A and C). (B) Nuclei were stained with ethidium bromide (red). Protein construct was detected as green EGFP signal in A and B. Expressed TPPP/p25 was detected by FITC-conjugated secondary antibody in C. Bars, 10 µm.

View larger version (178K): [in a new window]   Fig. 7. Electron microscopy of the aggresome in a HeLa cell expressing TPPP/p25. (A) Aggresome-like body (marked by arrows) located in the cavity of a crescent-shaped nucleus. (B) High-power magnification of the area boxed in A showing the heavy accumulation of microtubules and intermediate filaments in this region. Golgi cisternae (G) and mitochondria (M) are located around the body. Bar, 600 nm (A); 300 nm (B). N, nucleus.

View larger version (221K): [in a new window]   Fig. 8. Electron microscopy of the perinuclear cage in a HeLa cell expressing TPPP/p25. (A) Low power view of the perinuclear cage. (B) Enlarged view of the area framed in A showing that the cage consists of microtubules (arrows) organized into roughly parallel bundles running at different angles in close proximity to the nuclear membrane. N, nucleus. Bar, 300 nm (A); 150 nm (B).

View larger version (11K): [in a new window]   Fig. 9. Quantitative analysis of fluorescence intensity of transfected HeLa cells. Digital images were acquired through a 10x objective and analyzed with ImageJ software. Individual cells were identified and measured manually. The overall fluorescence of a cell was determined as the product of the measured area and average fluorescence intensity of the measured area (with average of background subtracted). Statistical comparison was done using the Student's t-test. The number of detected (transfected) cells within a microscopic field was 40±6 and 65±7 for the control and MG132-treated samples, respectively (P 0.001).

View larger version (53K): [in a new window]   Fig. 10. Stability of TPPP/p25 bundled microtubules. (A) For the in vivo assay, transfected cells were treated with 100 nM vinblastin in the final 2 hours of the incubation period. Tubulin was detected by immunocytochemistry (red). Green signal originated from EGFP-TPPP/p25. In the presence of vinblastin, in non-transfected cells (red only), the microtubular network is completely dissolved. Fine fibers in the cytoplasm are not detected after vinblastin treatment, but bundled, tubulin-positive fibers were preserved. Note the colocalization pattern of red and green signal in merged image in A. (B-D) Effect of low temperature (4°C; B), plus 0.75 mM Ca2+ (C) or plus 5 µM vinblastin (D) on the taxol-stabilized microtubules treated () and untreated () with TPPP/p25. 15 µM tubulin was polymerized by addition of 20 µM taxol with and without 3 µM TPPP/p25 at 37°C for 15 minutes in PEM buffer. Ca2+ and vinblastin were added and the depolymerization of microtubules was followed by turbidimetry for 60 minutes at 4°C. Bar, 10 µm.

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