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Jaglarz MK
,
Bazile F
,
Laskowska K
,
Polanski Z
,
Chesnel F
,
Borsuk E
,
Kloc M
,
Kubiak JZ
.
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Translationally Controlled Tumour Protein (TCTP) associates with microtubules (MT), however, the details of this association are unknown. Here we analyze the relationship of TCTP with MTs and centrosomes in Xenopus laevis and mammalian cells using immunofluorescence, tagged TCTP expression and immunoelectron microscopy. We show that TCTP associates both with MTs and centrosomes at spindle poles when detected by species-specific antibodies and by Myc-XlTCTP expression in Xenopus and mammalian cells. However, when the antibodies against XlTCTP were used in mammalian cells, TCTP was detected exclusively in the centrosomes. These results suggest that a distinct pool of TCTP may be specific for, and associate with, the centrosomes. Double labelling for TCTP and γ-tubulin with immuno-gold electron microscopy in Xenopus laevis oogonia shows localization of TCTP at the periphery of the γ-tubulin-containing pericentriolar material (PCM) enveloping the centriole. TCTP localizes in the close vicinity of, but not directly on the MTs in Xenopus ovary suggesting that this association requires unidentified linker proteins. Thus, we show for the first time: (1) the association of TCTP with centrosomes, (2) peripheral localization of TCTP in relation to the centriole and the γ-tubulin-containing PCM within the centrosome, and (3) the indirect association of TCTP with MTs.
Figure 1. Immunofluorescence localization of TCTP in Xenopus laevis mitotic spindles using XlTCTP antibody. (a) Confocal section of an XL2 cell showing the presence of TCTP in the spindle with higher density at the spindle poles. (b) Isolated spindle formed by sperm-head addition to the CSF extract. Red MTs stained with rhodamine-β-tubulin added to the extract, green TCTP detected by immunofluorescence with XlTCTP antibody. White arrows point to spindle poles with TCTP staining. Blue DNA stained with DAPI. Note the presence of yellow staining of TCTP at the spindle poles and the absence of TCTP in the remaining parts of the spindle. (c) Confocal section of murine metaphase NIH3T3 cell stained with XlTCTP antibody (green) and with DAPI for DNA. Note that XlTCTP stains exclusively two distinct spots corresponding to the centrosomes, at the spindle poles corresponding. (d) Confocal section of human HeLa metaphase cell. Green TCTP detected with XlTCTP antibody, blue DNA. XlTCTP stains two spindle poles; the granular background staining is also visible in the cytoplasm. (e) Human HeLa metaphase cell. Green TCTP detected with homologous HsTCTP antibody, blue DNA. HsTCTP stains the whole spindle. (f) Monkey Cos7 metaphase cell incubated with anti-rat TCTP antibody showing a very distinct staining of spindle poles. Bar is equal to 20âμm.
Figure 2. Expression of Myc-XlTCTP in Xenopus laevis XL2 cells. (a) Confocal section of XL2 cell in anaphase with high concentration of XlTCTP at the spindle poles (white arrows). (b) Confocal section of two dividing XL2 cells with high concentration of XlTCTP at the spindle poles. (c) Control mitotic XL2 cells expressing Myc tag only. (d) Interphase XL2 cell expressing Myc-XlTCTP. XLTCTP is localized in distinct fibers in the cytoplasm. (e) Mitotic murine NIHT3T cell expressing Myc-XlTCTP. High concentration of XlTCTP is present at the spindle poles (white arrows). (f) Interphase murine NIH3T3 cells expressing Myc-XlTCTP. Note that XlTCTP forms MT-like fibers in the cytoplasm. Bar is equal to 20âμm.
Figure 3. Immunofluorescence localization of TCTP with anti-XlTCTP antibody in mouse maturing oocytes. GV: prophase I-arrested oocyte, GVBD: the beginning of maturation, MI and MII: oocytes in MI and MII phase of meiosis, respectively, control PI: control MII oocyte stained with the preimmune serum. XlTCTP antibody stains PCM in all stages of maturing mouse oocytes. DNA (blue) stained with DAPI. Bar is equal to 40âμm.
Figure 4. MTs and TCTP in Xenopus laevis tadpoleovary. Interphase cells. Upper panel, left: β-tubulin, right: TCTP localization. Anti-XlTCTP was used for this localization. Single white arrow points to cellular structures positive both for β-tubulin and TCTP. Double arrows point to β-tubulin-positive and TCTP-negative fibers. Triple arrows point to TCTP-positive and β-tubulin-negative fibers. Bar is equal to 20 μm; Bottom panel: electron microscopy gold immunolabeling of TCTP (black particles in the center) in the vicinity of MTs (black arrows). Bar is equal to 100 nm.
Figure 5. MTs and TCTP in mitotic Xenopus laevis oogonia. Left: β-tubulin, right: TCTP localization in tadpole oogonia. Anti-XlTCTP was used for this localization. Upper panel: metaphase cell. Left: mitotic spindle visualized by β-tubulin staining (white arrow). Right: The whole of the spindle is positive for TCTP (white arrow). Bottom panel: two telophase oogonia. Left: prominent midbides are visualized by anti-β-tubulin immunoflorescence (white arrows). White asterisks show the position of two daughter cells. Note the absence of TCTP in the midbodies. Bar is equal to 20 μm.
Figure 6. Double labeling of γ-tubulin and TCTP in the centrosome of Xenopus laevis oogonium. Centriole labeled with black asterisk; 18ânm gold particles (black arrows) correspond to the presence of TCTP, small, and 10ânm gold particles around the centriole mark the presence of γ-tubulin. Inset in the bottom right corner shows the central area around the centriole where γ-tubulin is present (clear central area), and the TCTP-containing external area of the centrosome (dark grey). Bar is equal to 100ânm.
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