Calpain 6 Is Involved in Microtubule Stabilization and Cytoskeletal Organization

GFP-Capn6 overexpression impairs cytokinesis and causes the formation of multinucleated cells. (A and B) Multinucleation of HeLa cells by GFP-Capn6 overexpression. Many GFP-Capn6-transfected cells become binucleated (arrowheads) 48 h after transfection (A), whereas most control GFP-transfected cells are mononucleated (B). The scale bars indicate 100 μm. (C) Comparison of multinucleated-cell numbers between control and GFP-Capn6-transfected cells 48 h after transfection. The data represent the mean and standard deviation of four independent experiments. (D to F) Representative images from time-lapse recordings of HeLa cells transiently expressing GFP (D) or GFP-Capn6 (E and F). (D) In control GFP-expressing cells, the cleavage furrow (arrows) started to ingress within 5 min and daughter cells flattened out within 30 min after the onset of anaphase. The cytoplasmic bridge (arrowhead) disappeared within 90 min. (E) In many GFP-Capn6-expressing cells, furrow formation started normally, but its progression was retarded. (F) In some cases, GFP-Capn6 overexpression caused regression of the cleavage furrow to yield binucleated cells. Time is in h/min after anaphase onset (0:00 time point). The scale bars indicate 20 μm.

Subcellular localization of GFP-Capn6 in HeLa cells at various stages of the cell cycle and its stabilizing effect on microtubules. (A to G) Cells were transfected with expression plasmids containing GFP (A and F) or GFP-Capn6 (B to E and G). After 24 h, the cells were stained with the anti-GFP antibody and anti-α-tubulin (A to E) or anti-acetylated α-tubulin (F and G) antibody. Notably, perinuclear microtubule bundling was observed in GFP-Capn6-transfected cells at interphase (B). During mitosis, GFP-Capn6 was distributed in association with the mitotic spindle (C). At telophase, GFP-Capn6 colocalized to the central spindle (D). At the late stage of cytokinesis, GFP-Capn6 colocalized to the midbody, whereas a large portion of the GFP signals were distributed throughout the cytoplasm (E). Three-dimensional Z-stack images show that the acetylated α-tubulin contents (Ac-tubulin), especially in the perinuclear region, were increased in GFP-Capn6-transfected cells (F and G). The scale bars indicate 20 μm. (H) HeLa cells were transfected with GFP (lane 1) or GFP-Capn6 (lane 2), lysed 16 h after transfection, and blotted with anti-acetylated α-tubulin, anti-α-tubulin, and anti-β-actin antibodies. HeLa cells were treated with DMSO (lane 3), 500 nM paclitaxel (lane 4), or 5 μM nocodazole (lane 5) for 1 h and blotted similarly to serve as controls for changes in acetylated α-tubulin levels. Acetylated-α-tubulin levels were increased in GFP-Capn6-transfected cells compared with GFP-transfected cells, whereas total α-tubulin levels were not increased. Blotting for β-actin served as an internal control. Similar results were obtained in three independent experiments. Representative data are shown.

GFP-Capn6 colocalizes to the microtubule network and induces microtubule bundling. NIH 3T3 cells were transfected with expression plasmids containing GFP (A and B) or GFP-Capn6 (C and D). After 16 h, the cells were treated with DMSO (vehicle) (A and C) or 500 nM nocodazole (B and D) for 15 min and stained with anti-GFP and anti-α-tubulin antibodies. GFP-Capn6 induced thick microtubule bundling (C), which was resistant to the destabilizing effect of nocodazole (D). The scale bars indicate 20 μm.

Detection of Capn6 by rabbit polyclonal antibody. (A) Whole-cell lysates of untrasfected (lane 1) and Capn6-transfected (lane 2) NIH 3T3 cells were immunoblotted. The Capn6 antibody recognizes a band of ∼74 kDa in untransfected cell lysates and more strongly in Capn6-transfected cell lysates. (B) NIH 3T3 cell lysates were separated into 0.1% NP-40-insoluble (lane I) and -soluble (lane S) fractions and immunoblotted with the indicated antibodies. The ∼74-kDa band was found in the insoluble fraction with acetylated α-tubulin (Ac-tubulin) and MAP4. (C to F) Endogenous Capn6 colocalizes to the microtubule network. NIH 3T3 cells were treated with DMSO (vehicle) (C and F), 500 nM paclitaxel (D), or 500 nM nocodazole (E) for 30 min, and double stained with the anti-Capn6 (C to E) or anti-Capn1 (F) and anti-α-tubulin antibodies. The scale bars indicate 20 μm.

Capn6 biochemically interacts with microtubules. (A and B) Lysates of untransfected (A) or GFP-Capn6-transfected (B) NIH 3T3 cells were subjected to microtubule cosedimentation assay. (A) Endogenous Capn6 was detected in the microtubule-containing pellet in the presence of paclitaxel, although a large quantity of Capn6 remained in the supernatants (Sup). MAP4 also coprecipitated with microtubules in the pellet, whereas Capn1, detected as autolytic products, remained in the supernatants regardless of the presence of paclitaxel. (B) Overexpressed GFP-Capn6 was recovered in the pellet in the presence of paclitaxel. In contrast, control GFP did not correlate with the amount of precipitated α-tubulin in the pellet. Arrow, GFP-Capn6; arrowhead, GFP. (C) Unfused GST (lane 1), GST fused to full-length Capn6 (lane 2), and purified MAP2 (lane 3) were subjected to a microtubule binding assay on filter paper. GST-Capn6 and MAP2 (positive control), but not unfused GST, bound to microtubules as detected by anti-α-tubulin antibody. The filter was sequentially reblotted with anti-GST, anti-Capn6, and anti-MAP2 antibodies to confirm the presence of intact proteins.

Mapping of Capn6 domains interacting with microtubules. (A) Structures of GFP-tagged full-length Capn6 and its deletion mutants. (B) Localization of GFP-Capn6 mutants. NIH 3T3 cells were transfected with expression plasmids encoding GFP-Capn6 mutants, treated with 500 nM paclitaxel for 1 h, and stained with anti-GFP and anti-α-tubulin antibodies. Note that only GFP-Capn6 mutants containing domain III (amino acids 327 to 503) colocalized to microtubule bundles. The scale bars indicate 20 μm. (C) GST pull-down assay for microtubule binding. Microtubules were detected by anti-α-tubulin and anti-acetylated tubulin antibodies. α-Tubulin was pulled down from NIH 3T3 cell lysates by GST-full-length Capn6, GST-Capn6(327-503) (domain III), and GST-Capn6(504-641) (domain T), but not by GST-Capn6(1-56) (domain I), GST-Capn6(57-326) (domain II), or GST alone (right). Blotting with anti-acetylated tubulin showed that stable microtubules were more likely to bind to domain III than to domain T. The amounts of GFP fusion proteins were grossly estimated by Ponceau staining (left). The arrows indicate GST fusion proteins.

RNAi-mediated inactivation of Capn6 destabilizes microtubules. NIH 3T3 cells were transfected with Capn6-targeted or control siRNAs. Forty-eight hours after transfection, the effects of siRNAs were evaluated. (A and B) RT-PCR for Capn6 mRNA (A) and Western blotting for Capn6 protein (B). Stealth siRNAs targeting two different regions of the Capn6 transcript (1298 and 1715 for nucleotides 1298 to 1322 and 1715 to 1739, respectively) downregulated Capn6 mRNA and protein levels. (C) Immunostaining with anti-Capn6 antibody. Signals superimposed on microtubules were evident in paclitaxel-treated control siRNA-transfected cells (a), but not in paclitaxel-treated cells transfected with 1298 (b) or 1715 (c) siRNA, although background cytosolic staining was comparable in the three groups. (D) NIH 3T3 cells were transfected with control (a), 1298 (b), or 1715 (c) siRNA and immunostained for acetylated α-tubulin (Ac-tubulin). The levels of acetylated α-tubulin decreased in cells transfected with Capn6-targeted siRNA. (E) NIH 3T3 cells stably expressing GFP-tubulin were transfected with control (a), 1298 (b), or 1715 (c) siRNA and immunostained for GFP. a′, b′, and c′ are magnified images of the boxed areas in a, b, and c, respectively. Microtubule network structures were largely disrupted in cells transfected with Capn6-targeted siRNA. The scale bars indicate 20 μm (C, D, and a to c in panel E) and 5 μm (a′ to c′ in panel E). (F) Western blotting of control and Capn6 siRNA-transfected NIH 3T3 cell extracts with anti-acetylated α-tubulin, anti-α-tubulin, and anti-β-actin antibodies. Acetylated α-tubulin levels decreased in Capn6 siRNA (1298 and 1715)-transfected cells compared with control siRNA-transfected cells, whereas total α-tubulin levels did not decrease. Blotting for β-actin served as an internal control. Similar results were obtained in three independent experiments. Representative data are shown.

RNAi-mediated inactivation of Capn6 affects cytoskeletal reorganization during cytokinesis. (A and B) Representative images from time-lapse recordings of control (A) and Capn6 (B) siRNA-transfected NIH 3T3 cells. Cleavage furrows were similarly formed after anaphase onset in control and Capn6 siRNA (1715)-transfected cells. At subsequent stages, Capn6 siRNA-transfected cells tended to form lamellipodial protrusions with ruffling and to become flat much earlier than control cells. Times are in h/min after anaphase onset (0:00 time point). Arrowheads, first appearance of lamellipodial protrusion; arrows, completion of cell abscission. The scale bars indicate 20 μm. (C) Comparison of the times from anaphase onset to the first appearance of lamellipodial protrusions. The time was significantly shorter in Capn6-downregulated cells (17.9 [mean] ± 4.5 [standard deviation] min; n = 30) than in control cells (34.4 ± 12.4 min; n = 30). *, P < 0.0001; Mann-Whitney nonparametric test. (D) Comparison of the times from anaphase onset to the completion of cell abscission. No significant difference was observed between control cells (118.8 ± 35.1 min; n = 25) and Capn6-downregulated cells (108.6 ± 27.6 min; n = 24).