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First published online 24 July 2007
doi: 10.1242/jcs.007690

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Rapid effects of acute anoxia on spindle kinetochore interactions activate the mitotic spindle checkpoint

Rahul Pandey^*, Sebastian Heeger and Christian F. Lehner^,¶

Department of Genetics, BZMB, University of Bayreuth, 95440 Bayreuth, Germany

View larger version (53K): [in this window] [in a new window]   Fig. 1. Mitotic spindle organization in anoxia. (A) Early Drosophila embryos during the synchronous syncytial blastoderm cycles were incubated in either normoxic (+O2) or anoxic (–O2) buffer for 2, 5 or 20 minutes (2', 5', 20') before fixation and labeling with anti--tubulin (Tub) and a DNA stain (DNA). Metaphase embryos reveal an increasing reduction of centrosome-proximal spindle fibers during anoxia. Bar, 10 µm. (B) The intercentrosomal distances in metaphase spindles (in µm on y-axis ± s.d.) after incubation (2 minutes) in either normoxic (white bars) or anoxic (black bars) buffer before fixation and immunolabeling (see A) was measured during mitosis 10, 11, 12 and 13. At least 25 different spindles from at least five different embryos were measured and averaged for each bar (P values obtained with t-test for mitoses 10, 11, 12 and 13 were 0.028, 0.2, 0.035 and 0.77). (C) Selected frames are shown after time-lapse in vivo imaging of embryos expressing -tubulin-GFP. Time in minutes after prometaphase onset is indicated in the lower-left corners. In the presence of oxygen (+O2, top row), the last metaphase frame is reached after 3.5 minutes, followed by anaphase (not shown). In the embryo made anoxic at the start of mitosis (–O2, lower row), a metaphase arrest is maintained until after reoxygenation (+O2, lower row). The last frame before reoxygenation at 15 minutes reveals the loss of centrosome-proximal spindle fibers (arrowheads) and a reduced spindle width (broken lines) in comparison with normoxic metaphase spindles. Reoxygenation is followed by a pronounced increase in centrosomal fibers (18 minutes, arrow) and spindle shortening (21 minutes) before an apparently normal metaphase spindle (27 minutes) precedes anaphase (not shown). (D) The intercentrosomal distance in mitotic spindles was determined after in vivo imaging of embryos expressing GFP-D-TACC starting in prophase. Anaphase onset in normoxic conditions (x-x) is indicated by the open arrow. During the metaphase arrest in anoxic conditions (o-o), the intercentrosomal distance extends slightly beyond normoxic metaphase values. Reoxygenation (indicated by arrowhead +O2) is followed by a pronounced shortening and eventual anaphase onset (black arrow). The given distances at each time point are average values of five different spindles observed in representative embryos.

View larger version (70K): [in this window] [in a new window]   Fig. 2. Mitotic spindle function in anoxia. (A) Spindle checkpoint-deficient embryos lacking Mps1 protein kinase function were incubated during the synchronous syncytial blastoderm cycles in either normoxic (+O2) or anoxic (–O2) buffer for 5 minutes before fixation and labeling with anti--tubulin (Tub) and a DNA stain (DNA). The effects of anoxia on metaphase spindles do not depend on spindle checkpoint function (compare with Fig. 1A). Bar, 10 µm. (B) Progression through mitosis was analyzed by in vivo imaging of Mps1 mutant embryos expressing a green fluorescent Cenp-A/Cid centromere protein (Cenp-A) and a red fluorescent histone H2A variant (Histone H2Av) in either normoxic (+O2, top row) or anoxic (–O2, bottom row) conditions. Time in minutes starting in early prometaphase is indicated in the lower-left corners of the selected frames. Chromosome congression in anoxia is slow and incomplete at the time of anaphase onset. Subsequent chromosome segregation is also slow. Some of the frequent lagging centromeres are indicated by arrowheads. Bar, 10 µm. (C,D) Spindle checkpoint-competent embryos were incubated during the synchronous syncytial blastoderm cycles in either normoxic (+O2) or anoxic (–O2) buffer for 5 minutes before fixation and labeling with antibodies against -tubulin (Tub), Cenp-A (Cenp-A) and a DNA stain (DNA). The relative resistance of kinetochore fibers against anoxia-induced depolymerization is illustrated with representative metaphase figures (C). Bar, 5 µm. Moreover, inter-sister kinetochore distances were measured (n=125 sister kinetochore pairs from at least 18 different embryos) and the average (in µm ± s.d.) in prophase (pro) or metaphase (meta) is given in D. The average distance in normoxic (+O2) and anoxic (–O2) metaphase was found to be distinct (P<0.0001 in t-test; n=125). We did not detect significant variation in the average sister kinetochore separation with developmental stage (mitosis 11-13).

View larger version (61K): [in this window] [in a new window]   Fig. 3. Centrosome structure in anoxia. Syncytial blastoderm embryos were incubated (20 minutes) in either normoxic (+O2) or anoxic (–O2) buffer before fixation and immunolabeling. (A) Anti-Aurora A kinase (AurA), anti--tubulin (-Tub) and DNA (DNA) during metaphase. (B) Anti-Centrosomin (Cnn) and DNA (DNA) during metaphase and (C) during interphase. (D) GFP-D-TACC (TACC), anti--tubulin (-Tub) and DNA (DNA) during metaphase. Insets in the lower-left corners show a representative centrosome at higher magnification after labeling with anti-AurA (A) and anti-Cnn (B,C). Bars, 10 µm.

View larger version (90K): [in this window] [in a new window]   Fig. 4. Motor proteins in anoxia. Syncytial blastoderm embryos were incubated in either normoxic (+O2) or anoxic (–O2) buffer for 2 or 5 minutes before fixation and immunolabeling. (A) Anti-Kinesin 8/Klp67A (KLP67A), anti--tubulin (Tub) and DNA stain (DNA). Klp67A decreases rapidly in the central spindle region in response to anoxia. (B) Dynein light intermediate chain-GFP (DLIC) and DNA. Dynein accumulates rapidly at metaphase plates (arrowhead) in response to anoxia. Bars, 10 µm.

View larger version (78K): [in this window] [in a new window]   Fig. 5. Kinetochore proteins in anoxia. Syncytial blastoderm embryos were incubated in either normoxic (+O2) or anoxic (–O2) buffer for 20 minutes before fixation and immunolabeling. (A) Anti-Cenp-C (Cenp-C) and DNA stain (DNA). (B) EGFP-Nuf2 (Nuf2) and DNA stain (DNA) in gEGFP-Nuf2 embryos. In response to anoxia, Cenp-C and EGFP-Nuf2 accumulate on spindles predominantly in the centrosome-proximal region. Bar, 10 µm.

View larger version (100K): [in this window] [in a new window]   Fig. 6. Spindle checkpoint proteins in anoxia. (A) Selected frames after in vivo imaging of an embryo expressing EGFP-Mps1 (Mps1) and histone H2Av-mRFP (H2Av). Time in minutes is indicated in the frames showing the merged images in the bottom panel. Anoxia (–O2) was induced at t=1.75 minutes after the onset of chromosome condensation during entry into mitosis. The resulting EGFP-Mps1 accumulation within the spindle midzone region of metaphase figures (arrows) and in centrosome-associated filaments (arrowheads) is indicated. The embryo was reoxygenated (+O2) at t=9.75 minutes during the metaphase arrest, resulting in EGFP-Mps1 disappearance and completion of mitosis. Bar, 10 µm. (B) Frames showing EGFP-Mps1 at high magnification within the midzone region of a metaphase figure during the anoxia-induced mitotic arrest. Time in seconds is indicated within each frame. An EGFP-Mps1 particle moving along the spindle is indicated by arrowheads. (C) High-magnification views of single nuclei from syncytial gEGFP-Mps1 embryos during interphase. The embryos were fixed and labeled with anti--tubulin (-Tub) and DNA stain (DNA) after a 20-minute incubation in either normoxic (+O2) or anoxic (–O2) buffer. The weak centrosomal EGFP-Mps1 (Mps1) signals in normoxic embryos (arrowhead), as well as the strong signals in subcentrosomal dots in anoxic embryos (arrow), are indicated. Bar, 10 µm. (D) EGFP-Mps1 signals (Mps1) in cortical regions of preblastoderm embryos that were fixed after a 20-minute incubation in either normoxic (+O2) or anoxic (–O2) buffer. Anoxia induced filamentous or dot-like EGFP-Mps1 aggregates. Bar, 5 µm. (E) gEGFP-BubR1 embryos were fixed and labeled with anti--tubulin (-Tub) and DNA stain (DNA) after a 20-minute incubation in either normoxic (+O2) or anoxic (–O2) buffer. In response to anoxia, EGFP-BubR1 (BubR1) accumulates within the midzone region of metaphase figures and in peri-centrosomal filaments (arrowhead). Bar, 5 µm. (F) gEGFP-Mps1 embryos were fixed and labeled with anti-BubR1 (BubR1) and DNA stain (DNA) after a 20-minute incubation in anoxic (–O2) buffer. EGFP-Mps1 (Mps1) and anti-BubR1 signals largely overlap in the in peri-centrosomal filaments (arrowhead). (G,H,I) gEGFP-Bub3 (G), EGFP-Rod (H) or EGFP-Fzy (I) embryos were fixed after a 20-minute incubation in either normoxic (+O2) or anoxic (–O2) buffer and labeled with a DNA stain (G-I; DNA) and anti--tubulin (H; Tub). Bar, 5 µm.

View larger version (34K): [in this window] [in a new window]   Fig. 7. Mps1 modification in response to anoxia. gEGFP-Bub3 embryos were fixed and labeled with DNA stain (DNA) after a 20-minute incubation in either normoxic (+O2) or anoxic (–O2) buffer. Syncytial blastoderm embryos during interphase (i) or metaphase (m) were sorted using a microscope. Anoxic interphase embryos were further sorted into early interphase (ie) and late interphase (il) based on the appearance of chromatin that is condensed into a meshwork during early interphase and into distinct chromatids during late interphase. Sorted embryos were used for extract preparation and immunoblotting with anti-Mps1 (Mps1) and anti-GFP (Bub3).

View larger version (51K): [in this window] [in a new window]   Fig. 8. Spindle checkpoint activation by cytochrome oxidase inhibitor. gEGFP-Mps1 embryos were incubated in either absence (–CN) or presence (+CN) of cyanide for 5 (A) or 15 (B) minutes before fixation and labeling with anti--tubulin (Tub) and a DNA stain (DNA). Cyanide induces reorganization of EGFP-Mps1 (Mps1), microtubules and chromatin during metaphase (A) and interphase (B) that is indistinguishable from the effects of anoxia. Bar, 5 µm.