Takagi J et al. (2013), Using micromanipulation to analyze control of v...

XB-ART-47536

Cell Rep 2013 Oct 17;51:44-50. doi: 10.1016/j.celrep.2013.09.021.

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Using micromanipulation to analyze control of vertebrate meiotic spindle size.

Takagi J , Itabashi T , Suzuki K , Kapoor TM , Shimamoto Y , Ishiwata S .

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The polymerization/depolymerization dynamics of microtubules (MTs) have been reported to contribute to control of the size and shape of spindles, but quantitative analysis of how the size and shape correlate with the amount and density of MTs in the spindle remains incomplete. Here, we measured these parameters using 3D microscopy of meiotic spindles that self-organized in Xenopus egg extracts and presented a simple equation describing the relationship among these parameters. To examine the validity of the equation, we cut the spindle into two fragments along the pole-to-pole axis by micromanipulation techniques that rapidly decrease the amount of MTs. The spheroidal shape spontaneously recovered within 5 min, but the size of each fragment remained small. The equation we obtained quantitatively describes how the spindle size correlates with the amount of MTs while maintaining the shape and the MT density.

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	Figure 1. Correlation between Spindle Size and Amount of MTs (A) 3D image of a metaphase spindle that self-organized in Xenopus egg extracts, labeled with fluorescent tubulin. 3D rendering was performed using a maximum-intensity projection technique. Scale bar, 10 Î¼m. (B) Relationship between spindle size (length [L], width [W], and volume [V]) and the amount of MTs (M) (n = 78 spindles). Black solid curves indicate the best fits (M = 2.0 Ã 10â3L4.0 [R2 = 0.53], M = 4.1 Ã 10â5W3.1 [R2 = 0.75], M = 0.61 Ã V [R2 = 0.93]). (C) Histograms on the left show the distribution of each parameter for an ensemble of 78 spindles that self-organized in six different Xenopus egg extracts. The thick purple bars on the left show the SD of each parameter for an ensemble of 78 spindles. The thin black bars on the left show the SD of each parameter for an ensemble of spindles in six different extracts. The histograms on the right show the distribution of each parameter within 30 min (n = 4 spindles, interval of the time lapse was 10 s [black and red] or 5 s [green and blue]). Colored bars on the right show the SD of the corresponding histograms. The values of the parameters are summarized in Table S1. See also Figure S1A and Table S2.
	Figure S2. Fragment without Chromosomes Disorganized within â¼5 Min, Related to Figure 3 (A) Schematic representation for cutting of the spindle. (B) Images of a spindle and its fragments using 2-D observation before and after cutting into 2 halves, labeled with fluorescent tubulin (red) and DNA (green). Numbers in images show the time (min) from the moment the spindle was cut. The scale bar represents 10 Î¼m.
	Figure 3. A Decrease in the Amount of MTs Decreases the Spindle Length (A) Fluorescence images of a spindle and its fragments before, during, and after it was cut into two halves, in which tubulins were labeled with a fluorescent dye. Needle positions are shown by arrowheads. Numbers on the top show the time (min:s) from the moment the spindle was cut. Images before and after cutting show the sum projection of the image stack. The scale bar represents 10 Î¼m. (B) Time courses of parameters before, during, and after the cutting. Blue open squares for M and V indicate the sum of the values of two fragments. (C) Relationship between spindle length (L) and the amount of MTs (M) before and after cutting (black solid squares [n = 10 spindles] and red open diamonds [n = 20 fragments], respectively; a black solid curve indicates the best fit). Gray solid squares show the relationship for the observed spindles as a control (n = 78 spindles; a gray solid curve indicates the best fit). (D) Relationship between spindle length (L) and MT density (D) before and after cutting (black solid squares [n = 10 spindles] and red open diamonds [n = 20 fragments], respectively). Gray solid squares show the relationship for the observed spindles as a control (n = 78 spindles). See also Figures S2 and S3AâS3C, and Movie S1.
	Figure 4. An Increase in the Amount of MTs Increases the Spindle Length (A) Fluorescence images obtained by 3D observation show how the two spindle fragments were fused. Needle positions are shown by arrowheads. Numbers on the top show the time (min:s) from the moment the fragments began to move. Images after contact represent the sum projection of the image stack. Scale bar: 10 Î¼m. (B) Time courses of parameters during fusion of two fragments. Blue dashed lines indicate the sum of values of two fragments at time 0. (C) Relationship between the spindle length (L) and the amount of MTs (M) before and after fusion (black solid squares [n = 18 fragments] and red open diamonds [n = 9 fused spindles], respectively; a black solid curve indicates the best fit). Gray solid squares show the relationship for the observed spindles as a control (n = 78 spindles, the gray solid curve indicates the best fit). (D) Relationship between spindle length (L) and MT density (D) before (black solid squares, n = 18 fragments) and after fusion (red open diamonds, n = 9 fused spindles). Gray solid squares show the relationship for the observed spindles as a control (n = 78 spindles). (E) Schematic representation showing the changes in the parameters in the cutting and fusion experiments. See also Figures S3DâS3F and Movie S2.

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Andersen, Spindle assembly and the art of regulating microtubule dynamics by MAPs and Stathmin/Op18. 2000, Pubmed