Chromosome- and spindle-pole-derived signals generate an intrinsic code for spindle position and orientation - PubMed (original) (raw)

Chromosome- and spindle-pole-derived signals generate an intrinsic code for spindle position and orientation

Tomomi Kiyomitsu et al. Nat Cell Biol. 2012.

Abstract

Mitotic spindle positioning by cortical pulling forces defines the cell division axis and location, which is critical for proper cell division and development. Although recent work has identified developmental and extrinsic cues that regulate spindle orientation, the contribution of intrinsic signals to spindle positioning and orientation remains unclear. Here, we demonstrate that cortical force generation in human cells is controlled by distinct spindle-pole- and chromosome-derived signals that regulate cytoplasmic dynein localization. First, dynein exhibits a dynamic asymmetric cortical localization that is negatively regulated by spindle-pole proximity, resulting in spindle oscillations to centre the spindle within the cell. We find that this signal comprises the spindle-pole-localized polo-like kinase (Plk1), which regulates dynein localization by controlling the interaction between dynein-dynactin and its upstream cortical targeting factors NuMA and LGN. Second, a chromosome-derived RanGTP gradient restricts the localization of NuMA-LGN to the lateral cell cortex to define and maintain the spindle orientation axis. RanGTP acts in part through the nuclear localization sequence of NuMA to locally alter the ability of NuMA-LGN to associate with the cell cortex in the vicinity of chromosomes. We propose that these chromosome- and spindle-pole-derived gradients generate an intrinsic code to control spindle position and orientation.

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Figures

Figure 1

Figure 1. Dynein and dynactin localize asymmetrically to the cell cortex during metaphase

(a) Left, time lapse images from a clonal HeLa cell line stably expressing GFP-LGN and mCherry-Arp1A. LGN localizes to the cell cortex prior to Arp1A, and displays symmetric localization. Arp1A displays asymmetric localization to the cell cortex when the spindle is mis-positioned (arrowhead). Right, graph of relative fluorescence intensity for a line scan of the indicated lines showing the spatial distribution of LGN and Arp1A. (b) Schematic showing the dependency relationships and symmetric-asymmetric behavior for cortical localization. (c) Kymographs showing DHC-GFP and chromosomes (Hoechst) generated from time lapse movies at 5 min intervals as indicated showing the oscillation of the spindle and its effect on cortical dynein localization in MG132 arrested control cells, LGN depleted cells, and cells treated with low dose nocodazole. Arrows indicate cortically localized DHC-GFP and arrowheads indicate spindle poles. (d) Graphs showing the relationship between spindle pole-cortex distance and dynein localization based on data from (c) for spindle poles moving towards the cell cortex. The numbers in parentheses indicate the average spindle pole-cortex distance when dynein localizes to the cortex (blue) or is delocalized (red). (e) Fluorescent images (left) and line scan (right) as in (a) showing GFP-LGN and mCherry-Arp1A localization in cells treated with the Eg5 inhibitor STLC to create monopolar spindles. Scale bars, 10 μm.

Figure 2

Figure 2. Plk1 negatively regulates the localization of cortical dynein

(a) Fluorescent images showing DHC-GFP and DNA (Hoechst) localization in cells treated with the Eg5 inhibitor STLC to create monopolar spindles with or without inhibition of Plk1 (BI2536). (b) Fluorescent images showing the localization of DHC-GFP or GFP-LGN (bottom), DNA (Hoechst, top), and the indicated membrane targeted mCherry fusions (top). Membrane targeted Plk1, but not Plk1 mutants, disrupt cortical dynein localization, but not LGN. (c) Graph showing quantification of the data in (a) for the frequency of cortical dynein localization +/− SD for the indicated conditions. Control; n=34, Wild type; n=50, Kinase dead; n=32, Polo-box mutant; n=19, Aurora-A; n=31, TPX2; n=11. ** indicates that the Plk1 targeted cells are statistically different from the other conditions with a 99.9% confidence interval based on a z test for a difference between proportions (d) Kymographs showing cortical dynein localization and spindle oscillations as in Fig. 1d for the indicated membrane targeted fusions. (e) Western blots showing the presence of selected proteins in the samples in Table1. (f) Model indicating the effect of Plk1 and spindle pole proximity on the localization of cortically localized dynein downstream of LGN. Scale bars, 10 μm.

Figure 3

Figure 3. The chromosome-derived Ran gradient negatively regulates LGN localization

(a) Fluorescent images of live cells showing the localization of GFP-LGN and DNA (Hoechst) for the indicated conditions. (b) Fluorescent image as in (a) showing the localization of GFP-LGN in cells treated with low dose nocodazole to generate unaligned chromosomes (arrow). (c) Graph showing the effect of chromosome position on cortical LGN localization. Chromosome-cortex distance was measured for cases where localization of LGN was (positive) or was not (negative) observed. (d) Fluorescent images of live cells showing the localization of GFP-LGN and DNA (Hoechst) in control cells, or cells expressing dominant negative mCherry-Ran T24N. (e) Enlargement of the indicated images in (d) showing the effect on LGN localization to the spindle midzone. (f) Quantification of the data from (d) showing the ratio of centrally localized and laterally localized LGN in the indicated conditions +/− SD (n=3). * indicates that the difference is statistically significant based on a student’s T test (p < 0.001) (g) Top, fluorescent images of tsBN2 (RCC1ts mutant) stably expressing GFP-LGN. Cells were arrested with nocodazole (n=30), and then either maintained at the permissive temperature (33°C; n=45) or shifted to the restrictive temperature (39.7°C; n=55). Bottom, graph showing the quantification of the localization data. (h) Fluorescent image showing the analysis of spindle orientation on L-patterned fibronectin coated coverslips. L-shaped fibronectin patterns cause cells to divide preferentially along the hypotenuse of the L. (i) Quantification of spindle orientation in control cells (Control RNAi; n=25, Mem-mCherry; n=48, untransfected control; n=60), or cells treated with the indicated conditions (LGN RNAi; n=21, p150 RNAi; n=11, Mem-mCherry Plk1; n=42, mCherry-RanT24N, n=31). Each treatment is statistically different from its paired control with either a 99% (*) or 99.9% (**) confidence interval based on a z test for a difference between proportions. (j) Images from time lapse movies of control cells (properly aligned), or cells expressing mCherry-Ran T24N (mis-aligned). Scale bars, 10 μm.

Figure 4

Figure 4. The Ran gradient regulates the association of the LGN-NuMA complex with membranes

(a) Diagram showing the interactions of different regions of LGN with NuMA and Gαi. (b) Fluorescent images showing the localization of full length GFP-LGN, or the LGN C-terminus. (c) Fluorescent images showing the localization of LGN-C and Gαi1 to the cell cortex in regions adjacent to individual aligned chromosomes in cells treated with low dose nocodazole. (d) Left, fluorescent images showing the localization of full length LGN (n=10), LGN-C (n=9), a fusion between the NuMA and LGN C-termini (n=6), and the NuMA-C-LGN-C fusion with a mutated NLS (n=14). In each case, endogenous LGN was depleted by RNAi. Right, graph showing the relative cortical enrichment of the fusion as in Fig. 3f. * indicates that the difference is statistically significant based on a student’s T test (p < 0.01) (e) Diagram showing a model for spindle pole and chromosome derived signals regulating cortical dynein localization. Spindle pole-localized Plk1 negatively regulates dynein localization downstream of LGN, and the chromosome-derived Ran-GTP gradient negatively regulates NuMA-LGN distribution. Together, these two intrinsic signals act to control spindle position and orientation in symmetrically dividing cells. Also see Supplemental Movie 1. Scale bars, 10 μm.

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