Spindle assembly in the absence of a RanGTP gradient requires localized CPC activity - PubMed (original) (raw)

Spindle assembly in the absence of a RanGTP gradient requires localized CPC activity

Thomas J Maresca et al. Curr Biol. 2009.

Abstract

During animal cell division, a gradient of GTP-bound Ran is generated around mitotic chromatin. It is generally accepted that this RanGTP gradient is essential for organizing the spindle, because it locally activates critical spindle assembly factors. Here, we show in Xenopus laevis egg extract, where the gradient is best characterized, that spindles can assemble in the absence of a RanGTP gradient. Gradient-free spindle assembly occurred around sperm nuclei but not around chromatin-coated beads and required the chromosomal passenger complex (CPC). Artificial enrichment of CPC activity within hybrid bead arrays containing both immobilized chromatin and the CPC supported local microtubule assembly even in the absence of a RanGTP gradient. We conclude that RanGTP and the CPC constitute the two major molecular signals that spatially promote microtubule polymerization around chromatin. Furthermore, we hypothesize that the two signals mainly originate from discreet physical sites on the chromosomes to localize microtubule assembly around chromatin: a RanGTP signal from any chromatin and a CPC-dependent signal predominantly generated from centromeric chromatin.

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Figures

Figure 1

Bipolar spindles assemble around both DNA-coated beads and sperm nuclei although the kinetics of tubulin polymerization is faster around sperm nuclei. (A) Time-lapse fluorescence imaging of x-rhodamine tubulin during spindle assembly around DNA-coated beads (top row) and sperm nuclei with (bottom row) and without centrosomes (middle row). Note that the centrosome (arrow) becomes detached from the spindle structure during assembly. (B) Quantification of x-rhodamine tubulin fluorescence over time during bead (blue line) and sperm spindle (pink line) assembly. The bead spindle (from top row in 1A) reaches maximum fluorescence intensity ~30 minutes after microtubule polymerization begins. The sperm spindle (from middle row in 1A) achieves maximum fluorescence intensity within 10 minutes of the onset of microtubule polymerization. (C) Average tubulin fluorescence intensity of bead (N=8) and sperm (N=5) spindle assemblies over time. Samples were averaged after onset of tubulin polymerization because the lag phase for each spindle varied. Scale bars: 10 µm. Time in minutes:seconds.

Figure 2

Sperm nuclei, but not DNA-coated beads, support spindle assembly in the absence of a RanGTP gradient. (A) Representative micrographs of control, Ran T24N (T) and RanQ69L/RanT24N (Q/T) treated sperm and bead spindle assembly reactions. (B) Quantification of the structures assembled around sperm nuclei in control, Ran T24N and Q/T reactions. Control reactions had >90% bipolar spindles. Addition of T24N, which blocks RanGTP generation by RCC1 and therefore prevents SAF liberation from import receptors, severely compromised spindle assembly as only ~20% of structures were bipolar, while ~60% of nuclei had associated microtubule asters and ~20% had no associated microtubules. Nearly 80% of sperm nuclei formed bipolar spindles in the absence of a RanGTP gradient following Q/T treatment (N=3 independent experiments). Thus, global activation of SAFs in the absence of a chromatin localized RanGTP gradient still allows for localized microtubule polymerization and bipolar spindle assembly around sperm nuclei. (C) The YRC FRET probe exhibits reduced FRET efficiency (blue signal in the merged image) reflecting increased levels of GTP-bound Ran around chromatin in control reactions. Q/T treatment yields uniformly reduced FRET signal of the YRC probe revealing loss of a detectable RanGTP gradient even though spindles are assembled. (D) Addition of threefold molar excess of EB1 rescues sperm spindle assembly but not bead spindle assembly in the absence of a RanGTP gradient. (E) Quantification of the structures assembled around sperm nuclei in control, +T24N, +T24N+EB1 reactions. Control reactions had ~70% bipolar spindles while fewer than 10% of structures were bipolar following addition of T24N. The percentage of bipoles was elevated to 50% following addition of a threefold molar excess of EB1 to T24N-treated reactions. The images show tubulin in red and beads in blue. For each graph, 100 structures were counted for each condition from at least 3 independent experiments. Scale bars: 10 µm.

Figure 3

INCENP is required for RanGTP gradient-independent spindle assembly. (A) Representative micrographs of mock depleted, INCENP depleted and gradient-free (Q/T) INCENP-depleted sperm and bead spindle assembly reactions. The merged images show tubulin in red and DNA in blue. (B) Quantification of sperm nuclei structures in control, T and Q/T reactions in the presence and absence of INCENP (N=3 independent experiments). In mock-depleted extracts, ~90% of structures in control reactions are bipolar spindles, addition of T24N results in ~80% of structures having no associated microtubules and Q/T treatment yields mostly (~60%) bipolar spindles. INCENP depletion results in a range of microtubule structures around sperm nuclei including short bipolar spindles (~25%), asters (~40%) and naked DNA (~35%). 100% of sperm nuclei had no associated microtubules following the addition of T24N or Q/T to INCENP-depleted reactions. N=100 structures from at least 3 independent experiments for each condition. (C) INCENP levels are significantly reduced following immunodepletion with tubulin shown as a loading control. (D) Addition of an inhibitory MCAK antibody results in the formation of large microtubule arrays around sperm nuclei in both mock- and INCENP-depleted extracts. However, INCENP-depleted, RanGTP gradient-free (Q/T) extracts no longer assemble microtubule arrays in the vicinity of chromatin. Scale bars: 10 µm.

Figure 4

Artificial enrichment of INCENP confers RanGTP gradient-free spindle assembly activity to DNA-coated beads. (A) Representative image of a bipolar spindle assembled around a hybrid bead cluster consisting of DNA beads and α-INCENP coated beads. In the merged images tubulin is in red, DNA-coated beads are in blue and all beads (DNA- and α-INCENP coated beads) are in green. (B) Representative micrographs of RanGTP gradient-free structures assembled around sperm nuclei (bipolar spindle), IgG hybrids (no microtubules) and α-INCENP hybrids, which support the assembly of both microtubule arrays and bipolar spindles. (C) Quantification of the total number of microtubule arrays and bipolar spindles observed around hybrids in 2 µl samples of each condition (N= 2 independent experiments). ~4X as many microtubule arrays and >10X as many bipolar spindles were observed around α-INCENP hybrids relative to the control IgG hybrid clusters. (D) Comparable levels of the CPC components INCENP (upper panel) and Aurora B (middle and lower panels) are present on sperm and beads relative to the loading control the chromokinesin Xkid, which localizes throughout chromatin, as determined by western blot analysis. Both INCENP and Aurora B are highly enriched on the hybrid beads. Multiple higher molecular weight bands, one of which may be the IgG heavy chain, are evident in the shorter exposure of the Aurora B blot (lower panel) suggesting that Aurora B is hyper-phosphorylated on the anti-INCENP beads. Scale Bar: 10 µm.

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Spindle assembly in the absence of a RanGTP gradient requires localized CPC activity - PubMed (original) (raw)