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First published online 23 November 2004
doi: 10.1242/jcs.01568

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Multiple mechanisms regulate NuMA dynamics at spindle poles

Olga Kisurina-Evgenieva^1,*, Gary Mack^2,, Quansheng Du³, Ian Macara³, Alexey Khodjakov¹ and Duane A. Compton^2,

¹ Division of Molecular Medicine, Wadsworth Center, New York State Department of Health, Albany, NY 12201, USA
² Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA
³ Center for Cell Signaling, Box 800977, HSC, University of Virginia, Charlottesville, VA 22908, USA

View larger version (62K): [in a new window]   Fig. 1. Localization of endogenous NuMA in NRK 52E cells. (A) Early prophase. (B) Mid-prophase. (C) Late prophase. (D) Prometaphase. (E) Metaphase. (F) Anaphase. (G) Telophase. (H) Early G1. Immunostaining with anti-NuMA antibody (green), DNA is counterstained with Hoechst 33342. Maximal-intensity projections through the entire cell volume.

View larger version (60K): [in a new window]   Fig. 2. Behavior of GFP-NuMA in NRK52E cells. Selected frames from combinational 3D fluorescence (top half of each frame) / DIC (bottom half of each frame) time-lapse recording of an individual cell. (A) Early prophase. (B) Mid-prophase. (C) Prometaphase. (D) Anaphase onset. (E) Telophase. (F) Cytokinesis. Notice that the distribution of GFP-NuMA closely matches that of endogenous NuMA in the same cell type at all stages of mitosis (Fig. 1). Arrows in (F) indicate doubled NuMA-positive dots that remain visible in most cells during earlier G1. Time in minutes:seconds.

View larger version (60K): [in a new window]   Fig. 3. Intranuclear movements of GFP-NuMA during prophase in NRK 52E cells. (Same cell as in Fig. 2.) This sequence highlights NuMA movements as the cell progresses through late prophase. Selected frames from combinational 3D fluorescence (top half of each frame) / DIC (bottom half of each frame) time-lapse recording of an individual cell. (A-C) Early prophase, (D-E) late prophase. Notice that NuMA aggregates move inside of the nucleus, which still has intact nuclear envelope as revealed by DIC (bottom of each frame). Many NuMA aggregates are seen to move directly towards each other and then fuse (arrows in B,C and D,F). Time in minutes:seconds.

View larger version (171K): [in a new window]   Fig. 4. Polewards transport of GFP-NuMA. Similar to Fig. 3 except that this sequence focuses on NuMA movements after nuclear envelope breakdown (NEB). (A) At NEB, the separated centrosomes (arrows) do not contain appreciable amounts of NuMA. However, immediately after NEB, NuMA begins to `stream' towards the centrosomes (B,C, arrows). (D) Within 2-3 minutes of NEB, most NuMA becomes associated with the forming spindle poles. However, some NuMA aggregates can persist in the peripheral parts of the cell for much longer (A-E, arrowhead). These aggregates gradually decrease in size until they suddenly `stream' toward one of the spindle poles (arrowhead in F-H).

View larger version (102K): [in a new window]   Fig. 5. Dynamic exchange of pole-associated NuMA/GFP revealed by fluorescence recovery after photobleaching. (A-J) Selected frames from combinational time-lapse recording (similar to Figs 2, 3, 4). NuMA associated with one of the two spindle poles during late prometaphase was photobleached (A,B) by a 200 millisecond pulse of 488-nm laser light. The photobleached signal gradually recovered, whereas the intensity of the non-photobleached control pole decreased over time (C-H). Notice that the cytoplasmic double dots of NuMA that are seen in association with the control pole (I,J, arrow) did not appear in the photobleached pole. (K) Normalized intensity of NuMA-GFP fluorescence associated with the irradiated spindle pole over time.

View larger version (34K): [in a new window]   Fig. 6. Dynamic exchange of NuMA on microtubule asters. (A) Indirect immunofluorescence images of microtubule asters using a human NuMA-specific monoclonal antibody (Human NuMA) and a rabbit polyclonal antibody that recognizes both human and hamster NuMA (Total NuMA) immediately (0') or 40 minutes (40') after mixing with either buffer alone (KHMM) or hamster mitotic extract (BHK Extract). (B) Extracts were separated into 10,000 g soluble (S) or insoluble (P) fractions and immunoblotted with a human NuMA-specific monoclonal antibody and tubulin-specific antibody at various times (indicated in minutes) following dilution with either buffer alone (KHMM) or hamster mitotic extract (BHK Extract). (C) The proportion of human NuMA in the pellet fraction was determined using densitometry of immunoblots from three independent trials and is normalized to 100% using the 0-minute time point following dilution in KHMM buffer alone. Values represent the average and standard deviation. (D) Fluorescent images from the 40-minute time point were scanned with a horizontal line 1 pixel wide. Pixel intensities for both human NuMA (green) and total NuMA (red) are shown. Scale bar, 2 µm.

View larger version (35K): [in a new window]   Fig. 7. Staurosporine inhibits the dynamic exchange of NuMA on microtubule asters. (A) Indirect immunofluorescence images of microtubule asters using a human NuMA-specific monoclonal antibody (Human NuMA) and a rabbit polyclonal antibody that recognizes both human and hamster NuMA (Total NuMA) immediately (0') or 40 minutes (40') after mixing with either buffer alone (KHMM+STR) or hamster mitotic extract (BHK Extract+STR). (B) Extracts were separated into 10,000 g soluble (S) or insoluble (P) fractions and immunoblotted with a human NuMA-specific monoclonal antibody and a tubulin-specific antibody at various times (indicated in minutes) following dilution with either buffer alone (KHMM+STR) or hamster mitotic extract (BHK Extract+STR). (C) The proportion of human NuMA in the pellet fraction was determined using densitometry of immunoblots from three independent trials and is normalized to 100% using the 0-minute time point in following dilution in KHMM buffer alone. Values represent the average and standard deviation. (D) Fluorescent images from the 40-minute time point were scanned with a horizontal line 1 pixel wide. Pixel intensities for both human NuMA (green) and total NuMA (red) are shown. Scale bar, 2 µm.

View larger version (52K): [in a new window]   Fig. 8. LGN displaces NuMA from microtubule asters. (top) Indirect immunofluorescence images of microtubule arrangements in untreated extracts or extracts to which LGN has been added (+LGN). The LGN protein was added to samples before microtubule aster assembly (PRE), after microtubule aster assembly (POST) or after microtubule aster assembly along with staurosporine (POST+STR). (bottom) Extracts were separated into 10,000 g soluble (S) and insoluble (P) fractions, separated by size by SDS-PAGE and immunoblotted with antibodies specific to NuMA, Eg5, tubulin and LGN, as indicated. Scale bar, 5 µm.