Bazile F et al. (2009), Complex relationship between TCTP, microtubules...

XB-ART-39137

Carcinogenesis 2009 Apr 01;304:555-65. doi: 10.1093/carcin/bgp022.

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Complex relationship between TCTP, microtubules and actin microfilaments regulates cell shape in normal and cancer cells.

Bazile F , Pascal A , Arnal I , Le Clainche C , Chesnel F , Kubiak JZ .

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Translationally controlled tumor-associated protein (TCTP) is a ubiquitous and highly conserved protein implicated in cancers. Here, we demonstrate that interactions of TCTP with microtubules (MTs) are functionally important but indirect, and we reveal novel interaction of TCTP with the actin cytoskeleton. Firstly, immunofluorescence in Xenopus XL2 cells revealed cytoplasmic fibers stained with TCTP but not with tubulin antibodies, as well as MTs free of TCTP. Furthermore, TCTP localized to a subset of actin-rich fibers in migrating cells. Secondly, Xenopus laevis TCTP did not affect in vitro assembly/disassembly of MTs and lacked MT-binding affinity both in pull-down assays and in cell-free extracts. Although TCTP also failed to bind to purified filamentous actin (F-actin), it was associated with microfilaments in cell-free extracts. Thirdly, TCTP concentrated in mitotic spindle did not colocalize with MTs and was easily dissociated from these structures except at the poles. Finally, RNA interference knockdown of TCTP in XL2 and HeLa cells provoked drastic, MT-dependent shape change. These data show that although TCTP interacts with MTs, it does not behave as classic MT-associated protein. Our evidence for an association of TCTP with F-actin structures, and for an involvement in cell shape regulation, implicates this protein in integrating cytoskeletal interactions both in interphase and mitosis providing a new avenue to fully understand the role of TCTP.

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Species referenced: Xenopus laevis
Genes referenced: actl6a plk1 tpt1

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	Fig. 1. TCTP and MTs form similar, but not identical, networks in XL2 cells. ( A ) Western blotting showing specificity of the serum used to detect Xenopus laevis TCTP (a). Lanes: 1 and 1’ recombinant His-TCTP; 2 and 2’ XL2 cells extract; 3 and 3’ CSF extract made of Xenopus ovulated MII oocytes. Preimmune serum (b) used in 1:5000 concentration, the same as the immune serum shown in (a) delivers virtually no background. Therefore, we show here a western blot with more concentrated (1:1000) preimmune serum (b) showing only traces of non-specific bands. Immunofluorescence localization of endogenous TCTP (c and d in the top panel) and Myc-TCTP localization (f and g in the bottom panel) following expression in XL2 cells. In mitotic cells, TCTP localizes to the division spindle as well as it forms a granular network in the cytoplasm (c; a single confocal section through metaphase cell). In interphase cells, TCTP forms dense cytoplasmic network similar to MTs (d). Midbody stained with anti-tubulin (white arrow in the inset) is, however, free of TCTP (white arrow in d). Control staining with preimmune serum (e) shows the specificity of the immunofluorescence staining with the serum. Preimmune serum gives a faint background with no fibrous staining. Myc-TCTP was expressed and localized by immunofluorescence using anti-Myc antibody in XL2 cells (f and g; bottom panel). In mitotic cells, Myc-TCTP accumulates in the spindle (f; here in metaphase). In interphase cells (g), Myc-TCTP forms a fibrous MT-like network. Non-transfected cells were entirely negative for anti-Myc antibody as shown in Figure 2C (negative cells around the central positive one). Bars = 20 μm. ( B ) Endogenous TCTP was visualized by immunofluorescence (a) in parallel with MTs (b) using monoclonal α-tubulin. The merged image (c) shows differences between TCTP and MTs fibers localization in the most flattened cell parts. Red arrows point out MTs that do not correspond to TCTP fibers and appear red. Bars in a–c = 20 μm. Higher magnifications of cells illustrate distinct TCTP (green) and MT (red) fibers (d–f). Green arrows show examples of TCTP fibers not overlapping with MTs and red arrows point out MTs free of TCTP. In colchicine-treated cells (e and f), MTs become less dense and the lack of colocalization between MTs and TCTP fibers is visible even in the cell body and not only in their most flattened parts. Note that most green TCTP fibers have no equivalent in MTs (green arrows in e and f). Inversely, most, if not all, MTs are free of TCTP (red arrows in e and f). Bars: d = 10 μm, e = 20 μm, f = 15 μm. All cells in (A) and (B) were fixed with methanol–formaldehyde.
	Fig. 2. TCTP is abundant in the mitotic spindle, but it is not associated with spindle MTs. ( A ) Mitotic spindles were assembled in the CSF extract following addition of sperm heads and rhodamine tubulin. Samples were frozen in liquid nitrogen, post-fixed in cold methanol (crude spindles) and stained for DNA (a), TCTP (b) and MTs (c; merged image in d). Spindles show high, but not fibrous, TCTP staining and a granular network of TCTP in the adjacent cytoplasm that does not colocalize to MTs (especially visible in corners of b–d). Two examples of MT fibers (black arrows in c) that do not correspond to any TCTP fibers (white arrows in b) are shown. In parallel, spindles assembled in the extract were purified on glycerol cushion, fixed (purified spindles) and processed for DNA staining (e), TCTP immunofluorescence (f) and MTs labeling with rhodamine tubulin (g; merged image in h). The majority of TCTP staining disappeared from purified spindles and discrete fluorescent dots of TCTP remained on the spindle poles (white arrows in f). Merged image of DNA, TCTP and rhodamine tubulin (h) shows clear red staining of spindle MTs with yellow dots on its poles indicating that only in these areas the colocalization of TCTP and tubulin is detected. Inset in h shows details of a merged image of another spindle pole with TCTP remnants in higher magnification. Bars = 20 μm. ( B ) Either 6xHis- Xl TCTP or 6xHis- Xl TCTP ΔMAP1B does not modify assembly/disassembly of MTs in vitro . Tubulin alone (control) or mixed with two concentrations of recombinant wild-type TCTP (TCTP 2 and 8 μM) or ΔMAP1B deletion mutant (ΔMAP1B 2 and 8 μM) was incubated at 37°C (0–35 min) or 4°C (35–60 min) and optical density of samples was measured at 350 nm light wave to follow assembly (0–35 min) and disassembly (35–60 min) of MTs. All curves are very similar indicating that neither 6xHis- Xl TCTP nor 6xHis- Xl TCTP ΔMAP1B modifies MTs dynamics. ( C ) MAP1 domain does not influence localization of TCTP. Myc-TCTP ΔMAP1B localizes to mitotic spindles (b; here in metaphase) after expression in XL2 cells and forms a network in the cytoplasm of both mitotic (b) and interphasic cells (a). XL2 cells were transfected with an appropriate vector and were fixed and processed for anti-Myc immunofluorescence. Single confocal sections of DNA (blue) and anti-Myc immunofluorescence (red) in an interphase (a) and metaphase (b) cells are shown. Note that peripheral cells that do not express Myc-TCTP are negative, while stained with anti-Myc antibody providing the control of the specificity of anti-Myc detection for the exogenous protein. Bars = 20 μm.
	Fig. 2. TCTP is abundant in the mitotic spindle, but it is not associated with spindle MTs. ( A ) Mitotic spindles were assembled in the CSF extract following addition of sperm heads and rhodamine tubulin. Samples were frozen in liquid nitrogen, post-fixed in cold methanol (crude spindles) and stained for DNA (a), TCTP (b) and MTs (c; merged image in d). Spindles show high, but not fibrous, TCTP staining and a granular network of TCTP in the adjacent cytoplasm that does not colocalize to MTs (especially visible in corners of b–d). Two examples of MT fibers (black arrows in c) that do not correspond to any TCTP fibers (white arrows in b) are shown. In parallel, spindles assembled in the extract were purified on glycerol cushion, fixed (purified spindles) and processed for DNA staining (e), TCTP immunofluorescence (f) and MTs labeling with rhodamine tubulin (g; merged image in h). The majority of TCTP staining disappeared from purified spindles and discrete fluorescent dots of TCTP remained on the spindle poles (white arrows in f). Merged image of DNA, TCTP and rhodamine tubulin (h) shows clear red staining of spindle MTs with yellow dots on its poles indicating that only in these areas the colocalization of TCTP and tubulin is detected. Inset in h shows details of a merged image of another spindle pole with TCTP remnants in higher magnification. Bars = 20 μm. ( B ) Either 6xHis- Xl TCTP or 6xHis- Xl TCTP ΔMAP1B does not modify assembly/disassembly of MTs in vitro . Tubulin alone (control) or mixed with two concentrations of recombinant wild-type TCTP (TCTP 2 and 8 μM) or ΔMAP1B deletion mutant (ΔMAP1B 2 and 8 μM) was incubated at 37°C (0–35 min) or 4°C (35–60 min) and optical density of samples was measured at 350 nm light wave to follow assembly (0–35 min) and disassembly (35–60 min) of MTs. All curves are very similar indicating that neither 6xHis- Xl TCTP nor 6xHis- Xl TCTP ΔMAP1B modifies MTs dynamics. ( C ) MAP1 domain does not influence localization of TCTP. Myc-TCTP ΔMAP1B localizes to mitotic spindles (b; here in metaphase) after expression in XL2 cells and forms a network in the cytoplasm of both mitotic (b) and interphasic cells (a). XL2 cells were transfected with an appropriate vector and were fixed and processed for anti-Myc immunofluorescence. Single confocal sections of DNA (blue) and anti-Myc immunofluorescence (red) in an interphase (a) and metaphase (b) cells are shown. Note that peripheral cells that do not express Myc-TCTP are negative, while stained with anti-Myc antibody providing the control of the specificity of anti-Myc detection for the exogenous protein. Bars = 20 μm.
	Fig. 4. TCTP colocalizes with F-actin in XL2 cells and has an affinity for F-actin fibers in a cosedimentation assay with cell-free extracts. ( A ) TCTP forms curly fibers at the cell border in flattened and migrating XL2 cells (fixed with paraformaldehyde). Endogenous (left) and Myc-TCTP (right) shows distinct curly fibers at the cell border (arrows). ( B ) TCTP partially colocalizes with F-actin. Double staining for TCTP (left) and F-actin (right) in control (upper row) and cytochalasin D-treated (bottom row) cells fixed with paraformaldehyde. Note clear colocalization of TCTP and F-actin on the cell's border in control cells. Cytochalasin D induces actin-rich foci (bottom, right) in which TCTP accumulates preferentially (bottom, left). TCTP (left) was immunolocalized, whereas F-actin was stained with phalloidin rhodamin (right). Peripheral fibers in control cells in which TCTP and actin colocalize disappear after cytochalasin treatment. Bars = 20 μm. ( C ) Recombinant 6xHis- Xl TCTP is not enriched in F-actin-containing pellet in vitro . Increasing concentrations (0.5, 1, 2.5 μM) of recombinant TCTP alone (−) were incubated in the buffer without F-actin and centrifuged (top row). Supernatants contain growing amounts of TCTP, while a similar amount of TCTP is found in the pellet. When F-actin is added (+) to the mixture, it is found exclusively in the pellets (middle row), whereas TCTP remains in unchanged proportions in supernatants and pellets in the same samples (+; bottom row) as in the absence of F-actin (top row). ( D ) Endogenous Xl TCTP is enriched in the pellet containing F-actin incubated both with CSF and interphase extracts similarly as endogenous tropomyosin known to associate with F-actin. F-actin was not added (−; left) or added (+; right) to CSF and interphase extracts. Endogenous actin is detected in upper (US) and lower (LS) supernatants (both in − and +). Exogenous F-actin is detected in the pellet (P) in F-actin-supplemented extracts (top row; rightmost). TCTP is almost absent in the pellet (P) in the absence of exogenous F-actin (second row, − P), but it appears in the pellet (P) in the presence of exogenous F-actin (second row, + P). Tubulin western blot was used as a negative control (third row). Tubulin is detected largely in the LS both in the absence and presence of exogenous F-actin (third row). Tropomyosin, used here as a positive control (bottom row), accumulates in the pellet (P) only in the presence of exogenous F-actin (bottom row, + P, rightmost). Note that non-specific bands recognized by anti-tropomyosin antibody on the western blot are not present in the pellet confirming the specificity of the assay (compare + P with + and − LS and US lanes).
	Fig. 5. Knockdown of Xl TCTP by siRNA induces drastic change in cell shape. ( A ) TCTP siRNA diminishes significantly the quantity of TCTP in XL2 cells (western blot; left) and induces changes in their shape (Nomarski differential interference contrast; right). Western blot: untreated cells (−), HiPerfect-treated control cells (HiPerfect), siRNA-treated cells (RNAi TCTP). XL2 cells grow in islands of well-attached cells (control). siRNA TCTP-treated cells are highly elongated and attached with each other only through very limited areas (RNAi TCTP). Bar = 40 μm. ( B ) Knockdown of Hs TCTP in HeLa cells by siRNA using either of three different oligonucleotides diminishes significantly the levels of TCTP protein (western blot) and induces cells elongation and MT bundling (color images). Immunofluorescence: DNA, TCTP and MTs staining followed by merge of control cells (top row); the oligonucleotide A RNAi (second row) shows the mildest phenotype (TCTP signal diminishes in the cytoplasm and in mitotic spindles as well as some cells become elongated and form protrusions); oligonucleotide B (third row) gives the strongest phenotype (cells become long and fusiform, their proliferation slows down and longitudinal cables of MTs are formed in the cytoplasm) and oligo C (bottom row) gives an intermediate phenotype (cells elongate, form protrusions and their proliferation is slowed down).
	Fig. 6. Phenotype of HeLa cells upon TCTP knockdown. ( A ) Upon RNAi of TCTP, the staining of TCTP disappears from the mitotic spindle and long protrusions formed during interphase may persist upon mitosis (DNA, TCTP, merge—from left to right). Top row—control. Second, third and bottom row—rounded mitotic cells treated with oligos A, B and C, respectively, in which spindle TCTP staining disappeared or diminished significantly (bottom). Fourth row—metaphase cell treated with oligo B with long protrusion oriented along the spindle axis. ( B ) Actin localization in control and TCTP siRNA-treated cells. Flattened cells with abundant cortical actin and stress fibers (control) transform into highly elongated ones (RNAi TCTP). Actin staining remains cortical with numerous fibers oriented perpendicularly to the cell axis. Bars = 20 μm. ( C ) Elongation of HeLa cells upon TCTP knockdown is MT dependent. Control −: control cells. Control +: nocodazole treated for 24 h. Nocodazole increases the number of mitotic cells (rounded up) in comparison with the untreated control (−). Nocodazole treatment does not influence the shape of interphase cells that remain flattened (control +). In oligo B-treated cells (RNAi TCTP −), highly elongated interphase cells are present in the absence of nocodazole (−). The drug treatment (RNAi TCTP +) induces massive flattening of interphase cells, which become morphologically undistinguishable from the controls; compare interphase cells in control (−) with RNAi TCTP (+). Bar = 200 μm. ( D ) Cell protrusions induced by TCTP RNAi participate in unequal division of daughter cells during cytokinesis. The long protrusions (arrows) formed during interphase remain oriented along the spindle axis during mitosis. They participate in unequal mitotic division (compare the size of daughter cells upon cytokinesis of two dividing cells at 90 min time point). Arrows at 150 min time point show daughter cells that contain the protrusion. Photographs of time-lapse video microscopy of two dividing cells filmed during 150 min. Bars = 20μm.
	Supplementary Figure 1

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