Halpin D et al. (2011), Mitotic spindle assembly around RCC1-coated bea...

XB-ART-44656

PLoS Biol 2011 Dec 01;912:e1001225. doi: 10.1371/journal.pbio.1001225.

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Mitotic spindle assembly around RCC1-coated beads in Xenopus egg extracts.

Halpin D , Kalab P , Wang J , Weis K , Heald R .

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During cell division the genetic material on chromosomes is distributed to daughter cells by a dynamic microtubule structure called the mitotic spindle. Here we establish a reconstitution system to assess the contribution of individual chromosome proteins to mitotic spindle formation around single 10 µm diameter porous glass beads in Xenopus egg extracts. We find that Regulator of Chromosome Condensation 1 (RCC1), the Guanine Nucleotide Exchange Factor (GEF) for the small GTPase Ran, can induce bipolar spindle formation. Remarkably, RCC1 beads oscillate within spindles from pole to pole, a behavior that could be converted to a more typical, stable association by the addition of a kinesin together with RCC1. These results identify two activities sufficient to mimic chromatin-mediated spindle assembly, and establish a foundation for future experiments to reconstitute spindle assembly entirely from purified components.

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Species referenced: Xenopus laevis
Genes referenced: mapre1 numa1 ran rcc1 tpx2

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	Figure 1. RCC1-coated beads induce spindle formation in Xenopus laevis egg extracts.(A) Schematic of a porous glass NeutrAvidin bead to which N-terminally biotinylated RCC1 is coupled, drawing not to scale. (B) Categories of microtubule structures formed around RCC1 beads. (C) Distribution of spindle structure categories formed around single RCC1 or chromatin beads. N = 3 extracts, 80–130 structures counted per extract. (D) Images comparing representative RCC1 and chromatin bead spindles. Weak signals in the DNA channel of RCC1 bead spindle and the Alexa Fluor 488-labeled Streptavidin (strept) channel of chromatin bead spindle are due to slight bead autofluorescence. (E) Quantification of differences between RCC1 and chromatin bead spindle lengths, overall spindle microtubule intensity, and pole/midzone ratio of spindle microtubule intensity. Intensity is in arbitrary units. 80–130 structures counted per extract. Microtubules are red, RCC1 beads are green, and DNA beads are blue. Scale bars, 10 µm.
	Figure 2. RCC1 beads fully activate the RanGTP spindle assembly pathway and share structural and dynamic features with chromatin bead spindles.(A) RanGTP induced cargo gradients visualized using the Rango FRET probe appear qualitatively similar around RCC1 and chromatin-coated beads and are enhanced by addition of exogenous Ran. Elevated FRET indicative of cargo release is red/yellow. (B) GFP-tagged importin β cargo TPX2 localizes similarly to RCC1 and chromatin bead spindles. Inhibition of cargo release by addition of importin β (71–876) similarly blocks microtubule assembly around both bead types. (C) RCC1 and chromatin bead spindles display similar sensitivity to disruption of pole formation upon addition of α-NuMA antibodies. Microtubules are red, beads are green or blue, and TPX2 is green. See Figure S2 for individual fluorescence image channels. Scale bars, 10 µm. Reactions in (B) and (C) were supplemented with 8–10 µM Ran.
	Figure 3. RCC1 beads oscillate pole-to-pole within the spindle and appear to be pushed by polymerizing microtubules.(A) Selected frames from Video S1 showing a stationary chromatin bead and an oscillating RCC1 bead. (B) Plots of bead position relative to one pole and spindle length over time. (C) Selected frames from Video S2 showing a forming monopolar RCC1 bead structure that pushes the bead, which appears to be trailed by polymerizing microtubules. Scale bars, 10 µm. Reactions were supplemented with 8–10 µM Ran.
	Figure 4. Kinesin or EB1 coupled together with RCC1 affect bead spindle dynamics and bipolarity.(A) Quantification of spindle structure categories when the conventional kinesin motor domain (kinesin-1, amino acids 1–360) is coupled to the bead together with RCC1, which predominantly yields a “push pole” phenotype (inset). N = 3 extracts, 80–130 structures counted per extract. Left panel shows frames from Video S3 and schematic showing how the push pole structure forms. (B) Plot of bead position and spindle length for RCC1 plus kinesin ATPase mutant R203K. See Video S4. (C) Comparison of spindle categories of RCC1 beads with or without kinesin-1 (R203K) after 1 h in egg extract shows that the kinesin ATPase mutant increases the percentage of bipolar and multipolar spindles. N = 3 extracts, 80–130 structures counted per extract. (D) Plot of bead position and spindle length for RCC1 plus EB1. See Video S5. (E) Comparison of spindle categories of RCC1 beads with or without EB1 after 1 h in egg extract shows that the EB1 increases the percentage of bipolar spindles. N = 3 extracts, 100–170 structures counted per extract. Scale bar, 10 µm. Reactions were supplemented with 8–10 µM Ran.

References [+] :

Akhmanova, Tracking the ends: a dynamic protein network controls the fate of microtubule tips. 2008, Pubmed