Groen AC et al. (2011), Microtubule assembly in meiotic extract require...

XB-ART-43527

Mol Biol Cell 2011 Sep 01;2217:3139-51. doi: 10.1091/mbc.E11-02-0158.

Show Gene links Show Anatomy links

Microtubule assembly in meiotic extract requires glycogen.

Groen AC , Coughlin M , Mitchison TJ .

???displayArticle.abstract???
The assembly of microtubules during mitosis requires many identified components, such as γ-tubulin ring complex (γ-TuRC), components of the Ran pathway (e.g., TPX2, HuRP, and Rae1), and XMAP215/chTOG. However, it is far from clear how these factors function together or whether more factors exist. In this study, we used biochemistry to attempt to identify active microtubule nucleation protein complexes from Xenopus meiotic egg extracts. Unexpectedly, we found both microtubule assembly and bipolar spindle assembly required glycogen, which acted both as a crowding agent and as metabolic source glucose. By also reconstituting microtubule assembly in clarified extracts, we showed microtubule assembly does not require ribosomes, mitochondria, or membranes. Our clarified extracts will provide a powerful tool for activity-based biochemical fractionations for microtubule assembly.

???displayArticle.pubmedLink??? 21737678
???displayArticle.pmcLink??? PMC3164461
???displayArticle.link??? Mol Biol Cell
???displayArticle.grants??? [+]

Species referenced: Xenopus laevis
Genes referenced: ckap5 dlgap5 kif2c numa1 rae1 ran tpx2

???attribute.lit??? ???displayArticles.show???

	FIGURE 1:. Microtubule assembly requires high-speed supernatant (HSS or cytosolic fraction) and particulate fractions. (A) The Ran-dependent microtubule-assembly assay used to quantify microtubule assembly. A 20× magnified field view of 16 asters (16 APF) after Ran(Q69L)GTP (1 mg/ml) addition to crude Xenopus extracts supplemented with X-rhodamine tubulin (50 μg/ml). Scale bar: 5 μm. (B) The four layers separated after high-speed centrifugation of 5 ml of crude Xenopus meiotic extract: (1) lipids, (2) soluble cytosol (or HSS), (3) membranes, and (4) yellow pellet. (C) Coomassie Blue–stained gel of fractions (1–4) presented in B. Input is crude Xenopus meiotic egg extracts (100 μg sample per lane). (D) Ran-dependent microtubule-assembly assay of each isolated fraction presented in B. The average number of microtubule asters per 20 fields after addition of 1 mg/ml of Ran(Q69L)GTP to crude extract (input) and each of the isolated fractions presented in B. None of the separated fractions contain microtubule-assembly (or aster) activity. Error bars = SD, n = 8. (E) Ran-dependent microtubule-assembly assay after mixing fraction presented in B. The average number of microtubule asters per 20 fields after addition of 1 mg/ml of Ran(Q69L)GTP to crude extract (input), soluble cytosol (HSS), and combinations of HSS plus lipids, HSS plus membranes, and HSS plus yellow pellet. Note the bulk of the activity returned in HSS plus yellow pellet combination. Error bars = SD, n = 7.
	FIGURE 2:. Glycogen is required for microtubule assembly. (A) Ran-dependent microtubule activity assays of yellow pellet after various treatments. Activity is amylase-sensitive. The average number of microtubule asters per 20× magnified field after addition of 1 mg/ml of Ran(Q69L)GTP to HSS plus yellow pellet (positive control), HSS (negative control), or HSS plus yellow pellet after treatment with: 10% Triton (Triton), phenol-chloroform extraction (phenol chlrfrm), RNase, or amylase. Error bars = SD, n = 4. (B) Ran-dependent microtubule assays with glycogen titration in HSS. Purified bovine glycogen restores Ran-dependent microtubule assembly to HSS and is titratable. The average number of microtubule asters per 20× magnified field after addition of 1 mg/ml of Ran(Q69L)GTP to crude extract (positive control), HSS (negative control) with no additional glycogen (0), and HSS plus purified bovine glycogen (5, 10, 20, 100, and 200 mg/ml). Higher concentrations of glycogen (20 and 100 mg/ml) increase bipolarity of structures (10 mg/ml = 0% bipolar structures, 20 mg/ml = 33% bipolar structures, and 100 mg/ml = 66% bipolar structures). Error bars = SD, n = 4. (C) Glycogen restores microtubule assembly to HSS. Microtubule pelleting assay: (1) Pellets of crude extract plus (R, Ran) and minus (C, control) Ran(Q69L)GTP, (2) HSS input (I), pellet of HSS plus Ran(Q69L)GTP (R), (3) pellet of HSS plus 20 mg/ml glycogen plus (R) and minus (C, control) Ran(Q69L)GTP, (4) pellet of HSS plus 200 mg/ml glycogen plus (R) and minus (C, control) Ran(Q69L)GTP. Note 200 mg/ml of glycogen induces ectopic microtubule assembly without Ran. (D) Representative images of microtubule structures assembled in Ran(Q69L)GTP-treated crude extracts (control) and Ran(Q69L)GTP-treated HSS plus 0, 5, 20, or 100 mg/ml glycogen. Note presence of more bipolar structures at 100 mg/ml of glycogen. Scale bar: 3 μm.
	FIGURE 3:. Membranes are not required for microtubule assembly. (A) FM4-64 staining of membrane structures present in C-HSS (left) and HSS (right). Scale bar: 1 μm. (B) Purification scheme of C-HSS. (C) C-HSS requires phosphate in dilution buffer for glycogen to restore microtubule assembly. Ran-dependent microtubule assembly in (1) HSS plus glycogen (30 mg/ml), (2) C-HSS diluted without a phosphate buffer (S-CSF-XB) plus glycogen (30 mg/ml), and (3) C-HSS diluted with a phosphate buffer (S-CSF-XB-phosphate) plus glycogen (30 mg/ml). Scale bar: 5 μm. (D) Electron micrograph of Ran-induced microtubules in crude extract (left) or C-HSS (right) supplemented with glycogen (30 mg/ml). Note the electron-dense mitochondria (red arrow) in crude extracts. Also, microtubules appear to bind more electron-dense material than asters assembled in C-HSS (right). Scale bar: 500 nm. (E) Ran-induced microtubules assemble in 0% (control), 0.1%, 0.5%, and 1% Triton-X–treated C-HSS supplemented with glycogen (30 mg/ml). Scale bar: 3 μm
	FIGURE 4:. Glycogen acts as a molecular crowding agent and metabolic source of glucose. (A) Ran-dependent microtubule-assembly assay showing glucose-6-phosphate (G-6-P) and dextran is required for activity in HSS. The average number of Ran-dependent microtubule asters per 20× magnified field after addition of 1 mg/ml of Ran(Q69L)GTP to crude extract (control), clarified HSS (C-HSS), C-HSS plus glycogen (20 mg/ml), C-HSS plus dextran (20 mg/ml), C-HSS plus dextran plus glucose-6-phosphate (5 mM). Note glucose-6-phosphate is required for microtubule assembly when nondegradable dextran is used in place of glycogen. Error bars = SD, n = 3. (B) Ran-dependent microtubule-assembly assay showing the activity of concentrated HSS. The average number of Ran-dependent microtubule asters per 20× magnified field after addition of 1 mg/ml of Ran(Q69L)GTP to crude extract (control), C-HSS (1X), and concentrated C-HSS (2X, 3X, or 4X). Average final protein concentrations (Bradford assay) of each extract are shown (in box below). Glucose-6-phosphate was added to dilution buffer in all C-HSS. Error bars = SD, n = 3. (C) Representative images of microtubule structures assembled in A. Scale bar: 3 μm. (D) Representative images of microtubule structures assembled in B. Scale bar: 3 μm. (E) Concentration of factors in C-HSS. (1) Coomassie Blue–stained PAGE of crude extract (0.5 μl), HSS (0.5 μl), and C-HSS (0.5 μl). (2) Western blot of PAGE with crude extract, HSS, C-HSS, and 4X-concentrated C-HSS (4X-C-HSS). Blots were probed with tubulin, TPX2, and XMAP215. Note the relative concentrations: crude extract is 2X as concentrated as C-HSS (1X), HSS is 3X as concentrated as C-HSS, and 4X-C-HSS is 4X as concentrated as C-HSS.
	FIGURE 5:. Glycogen is required for microtubule assembly. (A) Ran-dependent release of importin β cargoes does not depend on glycogen. Western blot of PAGE with C-HSS (input), Talon Dyna beads (beads) incubated in C-HSS and Talon Dyna beads bound to His-importin β incubated in C-HSS supplemented with either Ran(Q69L)GTP, Ran plus glycogen, or equal volume of buffer (CSF-XB for control). Blots were probed with antibodies for GST-RanGTP, TPX2, NuMA, importin α, and importin β. Importin β binds to TPX2, NuMA, and importin α; however, in the presence of RanGTP, importin β releases TPX2, NuMA, and importin α, and instead interacts with RanGTP. (B) MCAK antibody (300 μg/ml) induces microtubule assembly in 4X-concentrated C-HSS (4X-C-HSS; left), but not C-HSS (right). Scale bar: 3 μm. (C) The structures assembled around DNA in C-HSS plus glycogen (30 mg/ml and 100 mg/ml). Similar to Ran asters, structures appeared more bipolar at higher glycogen concentrations (100 mg/ml). No microtubule structures assemble around DNA in C-HSS without glycogen. Bipolar spindles assemble around DNA in crude extract; DNA is blue and microtubules are red. Scale bar: 10 μm. (D) Glycogen is required for microtubule assembly from Xenopus centrosomes in C-HSS. No microtubules assemble around centrosomes in C-HSS (left). Microtubules assemble in a radial array around centrosomes in C-HSS plus glycogen (30 mg/ml; right). Scale bar: 3 μm.
	FIGURE 6:. Glycogen is required for spindle and centrosome aster assembly in crude meiotic Xenopus extract. (A) Microtubule structures assembled around DNA (top) or centrosomes (bottom) in crude extracts treated with buffer (CSF-XB for control), amylase (1 mg/ml), or amylase (1 mg/ml) plus dextran (20 mg/ml). See (C) for quantification of spindle experiment. In control and amylase plus dextran condition, 100% of centrosomes nucleated microtubules, while 0% nucleated in amylase condition; DNA is blue and microtubules are red. Top, scale bar: 10 μm; bottom, scale bar: 3 μm. (B) Amylase treatment of interphase crude extract does not affect microtubule assembly around centrosomes. Microtubule structures assembled around centrosomes in crude extract with and without amylase (100% nucleated in both cases). Scale bar: 5 μm. (C) Spindle assembly in crude extract after amylase treatment. Average percentage of structures (bipolar spindles, half-spindles/monopoles, or no microtubules) assembled in cycled crude Xenopus meiotic extracts treated with buffer (CSF-XB for control), amylase (1 mg/ml), or amylase (1 mg/ml) plus dextran (20 mg/ml). Note that dextran can rescue spindle assembly defects of glycogen depletion by amylase. Error bars = SD, n = 4. (D) Images (5-min intervals) from real-time lapse imaging of cycled crude Xenopus meiotic extracts supplemented with X-rhodamine tubulin (50 μg/ml) and treated with buffer (CSF-XB for control) or amylase (1 mg/ml) after spindle assembly. Scale bar: 6 μm.

References [+] :

Askjaer, Ran GTPase cycle and importins alpha and beta are essential for spindle formation and nuclear envelope assembly in living Caenorhabditis elegans embryos. 2002, Pubmed