Shirasu-Hiza M , Coughlin P , Mitchison T .

GM39565 NIGMS NIH HHS , R01 GM039565 NIGMS NIH HHS , R37 GM039565 NIGMS NIH HHS

	Figure 1. There is a CPP MT–depolymerizing activity in Xenopus egg extract independent of XKCM1. (A) XKCM1 overlaps with the peak of depolymerizing activity on sucrose gradients. 50 μl of clarified CSF extract was sedimented over a 5–20% sucrose gradient. Western blot of fractions showed that XKCM1 is present in fractions 10–18. CPP MT–depolymerizing activity peaked in fractions 9–14 (see B). Arrows below the blot indicate sedimentation values for protein standards run on a parallel gradient. Active fractions are labeled with asterisks. (B) Inhibition of XKCM1 did not inhibit depolymerizing activity in sucrose gradient fractions. Fractions from the sucrose gradient shown in A were assayed for depolymerizing activity, using rhodamine-labeled CPP MTs as described in the Materials and methods. Each fraction was assayed in the absence of ATP and in the presence of random IgG or inhibitory amounts of α-XKCM1 antibody and fixed after 10 min. XKCM1 depolymerizing activity is ATP dependent. As shown, neither the absence of ATP nor the presence of αXKCM1 antibody blocked the depolymerizing activity of active fractions. Active fractions are labeled with asterisks. Bar, 10 μm.
	Figure 2. Proteins of 130 and 160 kD were enriched during the purification and consistently copeaked with activity. (A) Specific activity increased with each step of the purification, as did the prominence of p160 and p130 bands (arrows). Shown here is a silver-stained polyacrylamide gel containing fractions from the purification. Samples for the first seven lanes are listed as follows: mw, molecular weight markers; AS supe, 40% AS supernatant (5 μg protein; 4 U of specific activity); PS pool, phenyl sepharose pool of active fractions (4 μg; 10 U); hep pool, heparin pool (4.8 μg; 300 U); monoS 1, first monoS column (5 μg; 600 U); sup6, gel filtration/superose 6 (2.5 μg; 600 U). The remaining lanes (monoS2) represent fractions from the second monoS column; depolymerizing activity peaked in fractions 16, 17, and 18 (see B). Lanes loaded with these fractions each contain ∼1.3 μg of total protein and 1,260 U of specific activity. Arrows indicate p130 and p160 bands. Identity of the p130 bands (lower arrows) from these fractions was determined by mass spectrometry. (B) Activity profile for fractions for the monoS2 step. Relative activity was estimated for fractions 8–24 by serial titration in the depolymerization assay and is presented in this graph as arbitrary units per microgram protein. (C) p160 and p130 bands are NH2-terminal fragments of XMAP215. Western blots of high-speed supernatant (hss) and monoS2 fractions were probed for XMAP215 with NH2-terminal– or COOH-terminal–specific antibodies.
	Figure 3. XMAP215 contributes to CPP MT–depolymerizing activity in CSF extract. (A) Full-length XMAP215 copeaks with depolymerizing activity on a sucrose gradient. Sucrose gradient fractions were analyzed by Western blot with COOH-terminal–specific α-XMAP215 antibody. Active fractions are labeled with asterisks. (B) Crude extract was specifically depleted of full-length XMAP215, XKCM1, or both. Samples were depleted with random IgG, COOH-terminal α-XMAP215 antibody (ΔXMAP215), α-XKCM1 antibody (ΔXKCM1), or both α-XMAP215 and α-XKCM1 antibodies (Δboth). Western blots for the four major depolymerizers (XMAP215, XKCM1, katanin, and Op18) are shown for each condition. (C) Depletion of XMAP215 and XKCM1 from crude extract inhibited CPP MT depolymerization. Rhodamine-labeled CPP MTs were incubated in depleted extracts for 10 min. Representative fluorescence images of each sample are shown. Buffer used for negative control was CSF-XB (extract buffer used, see Materials and methods). Bar, 10 μm.
	Figure 4. Pure recombinant XMAP215 depolymerizes CPP MTs in vitro. (A) Full-length XMAP215 and an NH2-terminal fragment of XMAP215 both depolymerize CPP MTs, but a COOH-terminal fragment does not. Rhodamine-labeled CPP MTs were incubated for 15 min in buffer containing different concentrations of full-length XMAP215 (F), an NH2-terminal fragment (N), or a COOH-terminal fragment (C). Shown here are representative fluorescence images for four concentrations of each protein. Bar, 10 μm. (B) Full-length XMAP215 and the NH2-terminal fragment have depolymerizing activity between 6.25 and 200 nM. MT polymer was quantitated for each sample by calculating average fluorescent pixel area per field for each protein concentration of full-length (F), NH2-terminal (N), and COOH-terminal (C) XMAP215. MT polymer is expressed as percent median value of buffer control; error bars denote 10th and 90th percentile; the 75th, 50th, and 25th percentiles are represented by the top, middle, and bottom of each box.
	Figure 5. XMAP215 promotes CPP MT depolymerization at MT plus ends. (A) XMAP215 promotes end-dependent CPP MT depolymerization. Shown here are images from a time-lapse series of dim-bright CPP MTs (see Materials and methods) treated with buffer alone or buffer plus 19 nM XMAP215 (interval between still images is 40 s). In each sample, kinesin motility was used to determine MT polarity; translocation of the MT from left to right represents minus end leading and plus end lagging. In the XMAP215-treated sample, the plus end shortens while the minus end remains stable (see Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200211095/DC1). (B) Depolymerization by XMAP215 is specific to MT plus ends. Depolymerization rates were quantitated for each MT end from experiments as in A. Error bars denote 10th and 90th percentile; 75th, 50th, and 25th percentiles are represented by the top, middle, and bottom of each box.
	Figure 6. Mechanism of CPP MT depolymerization by XMAP215. (A) Nocodazole does not depolymerize CPP MTs in our assay. Dimer sequestration was tested by incubating rhodamine-labeled CPP MTs with buffer plus DMSO or 20 μM nocodazole. (B) XMAP215 does not accelerate hydrolysis of GMPCPP. TLC was used to detect the hydrolysis of γ-32P–labeled GMPCPP in CPP MTs treated with buffer alone, full-length XMAP215, NH2-terminal fragment of XMAP215, or Na-BRB80/60% glycerol (positive control). Time points were taken from each reaction at 0, 30, and 60 min. [γ-32P]GMPCPP (CPP), [γ-32P]ATP (ATP), and 32Pi (Pi) are loaded as markers. Release of Pi is seen by the appearance of a second spot in Na-BRB80/60% glycerol, but not in XMAP215 or NH2-terminal XMAP215 samples. Microscopy assays run in parallel demonstrated that treatment with XMAP215 and the NH2-terminal construct depolymerized CPP MTs by the final time point (not depicted). (C) XMAP215 causes protofilament curling on MT ends. CPP MTs were incubated in buffer alone or buffer plus 38.5 nM full-length XMAP215 for 2 min and imaged by negative stain EM. Bars: (left) 200 nm; (right) 50 nm.
	Figure 7. Model for the potential mechanism of XMAP215 as an antipause factor. Depicted here are the growing MT end (top) as a sheet-like structure of protofilament extensions and the shrinking MT end (bottom) with curled protofilaments (adapted from Miyamoto et al., 2002). The hypothetical paused MT end structure (middle) is positioned as an obligate intermediate between the two, drawn here with a blunt-ended, closed tube structure. We propose that XMAP215 destabilizes this pause state by weakening interprotofilament bonds and/or preventing tube closure, increasing transition to either the growing or shrinking state (arrows). Based on work by Cassimeris et al. (2001), we depict XMAP215 here as a long curved molecule that can bind protofilaments along their long axis.

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