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

First published online 27 July 2004
doi: 10.1242/jcs.01242

This Article Summary Full Text Full Text (PDF) Movies 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 Beardmore, V. A. Articles by Kallio, M. J. Search for Related Content PubMed PubMed Citation Articles by Beardmore, V. A. Articles by Kallio, M. J.

Survivin dynamics increases at centromeres during G2/M phase transition and is regulated by microtubule-attachment and Aurora B kinase activity

Victoria A. Beardmore^1,*,, Leena J. Ahonen^2,*, Gary J. Gorbsky² and Marko J. Kallio^1,3,

¹ University of Oklahoma Health Sciences Center, Department of Cell Biology, 940 Stanton L. Young Boulevard, Oklahoma City, OK 73104, USA
² Oklahoma Medical Research Foundation, Molecular, Cell and Developmental Biology Research Program, 825 N.E. 13th Street, Oklahoma City, OK 73104, USA
³ VTT-Medical Biotechnology and University of Turku, P.O. Box 106, 20521 Turku, Finland

View larger version (72K): [in a new window]   Fig. 1. Survivin concentrates near centromeres in G2 and forms an extendable bipartite structure between the sister kinetochores in mitosis. (A) Accumulation of survivin-GFP adjacent to centromeres of early G2 cells. See text for details. The arrow and arrowheads denote the midbody and the spindle poles, respectively. The merge shows overlay of survivin-GFP (green) and autoimmune anti-centromere marker (red). The insets show higher magnification of selected areas. (B) Aurora B does not co-localize with survivin-GFP foci (thin arrows) until M phase (short arrows). (C) Survivin (red) undergoes extensive stretching between the sister kinetochores (anti-hCdc20, green) in a manner dependent on microtubule attachment and chromosome bipolarity. The arrows indicate a gap separating the two survivin accumulations on chromosomes that are under robust tension. (a) prophase, (b) unattached prometaphase, (c) attached prometaphase, (d) metaphase, (e) metaphaseanaphase transition. The numbers show the average length of the survivin segments under each condition (n=10-20 chromosomes per each category). The three linescan graphs show the average gray values for the above prometaphase (f), metaphase (g), and metaphase-anaphase transition (h) chromosomes (red survivin; green kinetochores). (D) Aurora B kinase associates with survivin-GFP in mitotic HeLa cells. Immunoprecipitations were performed with rabbit anti-GFP antibody utilizing HeLa cell populations expressing either survivin-GFP or control GFP alone. The arrow denotes a 42 kDa band detected with anti-hAurora B antibody and the asterisk the antibody heavy chain at 55 kDa. IP, immunoprecipitate; S, supernatant. Bars = 10 µm (panels A,B) and 0.5 µm (panel C).

View larger version (91K): [in a new window]   Fig. 2. Indirect immunolocalization of endogenous survivin in LLCPK cells. (A) Survivin accumulates near the centromeres at G2 phase (arrows). (B) In late prophase cells, survivin is between the sister kinetochores (arrows). (C) During prometaphase, survivin changes its morphology as microtubules attach to the kinetochores and chromosomes become bipolarly oriented (the short arrow shows a `relaxed' chromosome and the long arrow denotes a chromosome under tension from both spindle poles). (D) Metaphase. (E) Early anaphase. (F) Anaphase B. The arrowheads denote survivin strands at the central spindle. (G) In telophase cells, survivin concentrates at the cleavage furrow (arrowheads) before final accumulation at the midbody (arrow). Merge images of survivin (red, detected with the monoclonal anti-survivin antibody), kinetochores (green, detected with anti-hCdc20 antibodies or Crest anti-centromere serum), and DNA (blue, stained with DAPI) are shown. (H) The specificity of monoclonal anti-survivin antibody. A single band corresponding to a 16 kDa protein was detected in interphase (I) and mitotic (M, 12 h taxol treatment) HeLa and LLC-PK cells. Bars, 5 µm.

View larger version (85K): [in a new window]   Fig. 3. Redistribution of survivin-GFP during cell division. (A) Time-lapse sequence of an LLC-PK cell expressing survivin-GFP during prophase, nuclear envelope breakdown (NEB) and prometaphase. The amount of survivin-GFP at the inner centromeres increases substantially around NEB (time point 0 minutes) and shows further accumulation during prometaphase. The white circle denotes a centromere that remained in focus throughout the observation period. In contrast to inner centromere labeling, the intensity of chromosome arm labeling diminishes during prometaphase after peaking at NEB. A video corresponding to the still images is available as supplementary information (see Movie 1, http://jcs.biologists.org/supplemental/). (B) Changes in the average integrated fluorescence of chromosome arm (, n=5) and inner centromere localized survivin-GFP (, n=8) from late prophase to onset of anaphase. (C) Co-localization of survivin-GFP with the spindle microtubules in anaphase cells is lost by early telophase when survivin-GFP forms separate filamentous accumulations (arrows) that run between the microtubules of the central spindle. The area inside the white boxes is shown in higher magnification. mts, microtubules; surv, survivin-GFP. The merge shows an overlay of DNA (blue), survivin-GFP (green), and spindle microtubules (red). Bars = 10 µm (panel A) and 3 µm (panel C).

View larger version (132K): [in a new window]   Fig. 4. The turnover rate of survivin peaks at early M phase and is partially dependent on microtubule-kinetochore attachment. (A-G) Target areas (indicated with white circles) in HeLa cells (panel A) or LLC-PK cells (panels B-G) were photo-bleached and followed by fluorescence time-lapse microscopy. Pre-bleach and post-bleach images representing partial and maximal recovery are shown. The insets show higher magnification views of the target area. At the end of each row are corresponding graphs of survivin-GFP recovery (arrows indicate the pre-bleach fluorescence level of the target area). Proportions of fluorescence recovery (recf) and half time of recovery (t1/2) are shown for each graph, as percentages. Supplemental Movies 2-6 corresponding to the still images of panels A, B, C, E and F, respectively, are available online (http://jcs.biologists.org/supplemental/).

View larger version (40K): [in a new window]   Fig. 5. Aurora B regulates survivin turnover at inner centromeres. (Top) FRAP analysis in an LLC-PK cell shows that survivin-GFP turnover at metaphase centromeres occurs at its normally rapid rate in a cell treated with the proteaseome inhibitor MG132. (Bottom) In a cell pretreated for 30 minutes with MG132 then treated for 10 minutes with ZM447439 in the continued presence of MG132, survivin-GFP at the inner centromeres shows slow and limited turnover. Pre-bleach and post-bleach images representing partial and maximal recovery are shown. The white circles denote the target area. The graphs show survivin-GFP recovery (arrows indicates the pre-bleach fluorescence level of the target area). Proportion of fluorescence recovery (recf) and half time of recovery (t1/2) are shown as percentages. Movie 7 corresponding to the still images of panel B is available online (http://jcs.biologists.org/supplemental/).