QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online April 3, 2008
doi: 10.1242/10.1242/jcs.027417

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mukai, A. Articles by Komada, M. Search for Related Content PubMed PubMed Citation Articles by Mukai, A. Articles by Komada, M. Social Bookmarking                         What's this?

Dynamic regulation of ubiquitylation and deubiquitylation at the central spindle during cytokinesis

¹ Department of Biological Sciences, Tokyo Institute of Technology, 4259-B-16 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan
² Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan

View larger version (52K): [in this window] [in a new window]   Fig. 1. UBPY and AMSH localize to the central spindle and midbody during cytokinesis. (A) Classification of the four stages (S1S4) of cytokinesis used in this study. HeLa cells were double-stained for microtubules (red) and DNAs (blue) with anti-tubulin antibody and TO-PRO-3, respectively. See text for details. (B-B'') HeLa cells at stage 4 of cytokinesis were triple-stained with anti-UBPY antibody (B), anti-tubulin antibody (B', red) and TO-PRO-3 (B', blue). (C-C'') HeLa cells were transfected with FLAG-tagged UBPY, and triple-stained with anti-FLAG antibody (C), anti-tubulin antibody (C', red) and TO-PRO-3 (C', blue) at stage 4 of cytokinesis. (D-G'') HeLa cells at stages 1 (D-D''), 2 (E-E'',F-F'') and 4 (G-G'') of cytokinesis were triple-stained with anti-AMSH antibody (D-G), anti-tubulin antibody (D'-G', red) and TO-PRO-3 (D'-G', blue). B''-G'' are merged images. Asterisks in E,E'',F,F'' indicate the midbody remnants in neighboring interphase cells. Insets show high-magnification images of the central spindle/midbody regions. Scale bars: 10 µm.

View larger version (48K): [in this window] [in a new window]   Fig. 2. Ubiquitylated proteins localize to the central spindle during cytokinesis. (A-D'') HeLa cells at stages 1 (A-A''), 2 (B-B''), 3 (C-C'') and 4 (D-D'') of cytokinesis were triple-stained with FK2 (A-D), anti-tubulin antibody (A'-D', red) and TO-PRO-3 (A'-D', blue). (E-F'') HeLa cells were transfected with HA-tagged Ub, and triple-stained with anti-HA antibody (E,F), FK2 (E', green) or anti-AMSH antibody (F', green) and TO-PRO-3 (E',F', blue). A''-F'' are merged images. Insets show high-magnification images of the central spindle/midbody regions. Scale bars: 10 µm.

View larger version (53K): [in this window] [in a new window]   Fig. 3. Marker analysis of the cleavage regions positive for UBPY, AMSH and ubiquitylated proteins. (A-A'') HeLa cells were transfected with HA-Ub, and triple-stained with anti-HA antibody (A), anti-Aurora B antibody (A', red) and TO-PRO-3 (A', blue) at stage 2 of cytokinesis. (B-B'') HeLa cells at stage 2 of cytokinesis were triple-stained with FK2 (B), anti-MKLP1 antibody (B', red) and TO-PRO-3 (B', blue). (C-C'') HeLa cells at stage 4 of cytokinesis were triple-stained with anti-UBPY antibody (C), anti-Aurora B antibody (C', red) and TO-PRO-3 (C', blue). (D-D'') HeLa cells at stage 2 of cytokinesis were triple-stained with anti-AMSH antibody (D), anti-MKLP1 antibody (D', red) and TO-PRO-3 (D', blue). (E-E'') HeLa cells at stage 4 of cytokinesis were double-stained with anti-Hrs (E) and anti-tubulin (E') antibodies. Cytoplasmic punctate staining in green indicates endosomes. A''-E'' are merged images. Insets show high-magnification images of the central spindle/midbody regions. Scale bars: 10 µm.

View larger version (27K): [in this window] [in a new window]   Fig. 4. Depletion of cellular UBPY and AMSH causes cytokinesis defects. (A) Immunoblot analysis of the lysates of HeLa cells transfected with the mock, two UBPY siRNA or two AMSH siRNA expression vectors with anti-UBPY (top), anti-AMSH (middle) and anti-tubulin (bottom) antibodies. (B) HeLa cells were transfected with the mock, UBPY siRNA-1 or AMSH siRNA-1 expression vector, and double-stained with anti-tubulin antibody (red) and SYTOX green (green). Scale bars: 50 µm. (C) Percentage of multi-nucleated cells in randomly chosen 400500 HeLa cells transfected with the mock, two UBPY siRNA or two AMSH siRNA expression vectors. Mean±s.d. of five independent experiments is shown (*P<0.01, t-test). (D) HeLa cells transfected with AMSH siRNA-1 were double-stained with anti-tubulin antibody (red) and FK2 (green) at stage 4 of cytokinesis. Double-headed arrow indicates an atypically long central spindle between two daughter cells (asterisks). Arrowhead indicates the midbody ring. Inset shows a high-magnification image of the midbody region. Scale bar: 10 µm. (E) HeLa cells were transfected with the mock, two UBPY siRNA or two AMSH siRNA expression vectors, and stained with anti-tubulin antibody. Cells with the long central spindle (>20 µm; e.g. arrow in D) were counted in a 15 mm diameter cover glass, and the numbers are shown as mean±s.d. of three independent experiments (*P<0.01, t-test).

View larger version (38K): [in this window] [in a new window]   Fig. 5. VAMP8 undergoes ubiquitylation. (A-B'') HeLa cells were transfected without (A-A'') or with (B-B'') FLAG-VAMP8, and stained with anti-VAMP8 (A) or anti-FLAG (B) antibody, together with FK2 (A',B', red) and TO-PRO-3 (A', blue), at stage 2 or 3 of cytokinesis. A'' and B'' are merged images. Insets show high magnification images of the central spindle region. Scale bar: 10 µm. (C) Schematic structure of mouse VAMP8 and its mutants used in this study. Positions of the SNARE motif, transmembrane domain and the seven Lys residues are indicated. (D) FLAG-tagged VAMP8 and its mutants were transfected into HeLa cells, and immunoprecipitated with anti-FLAG antibody. The immunoprecipitates were immunoblotted with anti-FLAG antibody (left) or FK2 (right). Black arrowheads indicate the positions of non-ubiquitylated FLAG-VAMP8 (band 0, 14 kDa) and FLAG-VAMP8 proteins conjugated with 1 (band 1, 22 kDa), 2 (band 2, 30 kDa) and 3 (band 3, 38 kDa) Ub molecules. Asterisks indicate the IgG light (L) chain used for immunoprecipitation. The identity of the bands indicated by a white arrowhead is unknown. Ubiquitylated VAMP8 was often detected as a doublet band. The reason for this is unknown.

View larger version (48K): [in this window] [in a new window]   Fig. 6. UBPY and AMSH deubiquitylate VAMP8. (A) HeLa cells were transfected with FLAG-VAMP8, together with HA-tagged UBPY, UBPYC748A, AMSH or AMSHD348A. FLAG-VAMP8 was immunoprecipitated from their lysates with anti-FLAG antibody, and immunoblotted with FK2 (top), anti-HA (second from top) and anti-FLAG (third from top) antibodies. The expression levels of HA-tagged DUB constructs were assessed by immunoblotting of the total lysates with anti-HA antibody (bottom). (B) HeLa cells were transfected with FLAG-VAMP8, together with the mock, two UBPY siRNA or two AMSH siRNA expression vectors. FLAG-VAMP8 was immunoprecipitated from their lysates with anti-FLAG antibody, and immunoblotted with FK2 (top) and anti-FLAG (second from top) antibodies. The expression levels of UBPY and AMSH were assessed by immunoblotting of the total lysates with corresponding antibodies (third and fourth from top). As a loading control, the lysates were also blotted with anti-tubulin antibody (bottom).

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter