|
FIGURE 1:. Pineapple assembly. (A) Typical field 1 h after addition of 5 μM Taxol and 5% DMSO to meiotic HSS containing tubulin (green) and NUMA (red) probes; 20à wide-field imaging. Note formation of similar assemblies across the whole field, comprising paired lines of microtubule bundles with NUMA aligned on the outside. (B) Round assemblies with open centers resembled a slice of canned pineapple (â¼2 h of assembly, 20à wide field). Tubulin (green), NuMA (red). (C) A 40à confocal image at 90 min showing localization of Aurora B (red) on the inside and NUMA (blue) on the outside. Note global alignment of both types of foci on curved planes, which appear as lines in optical sections. (D) A 100à confocal image taken at 90 min showing one side of an open assembly. NUMA (blue) was on the outside as usual. Both NUMA and Aurora B (red) accumulate at foci. Aurora B foci are smaller and more numerous. (E) Pineapple assembly in C-HSS, which is completely free of organelles; 120 min, 20à wide field. Tubulin (green), NUMA (red). (F) Pineapple assembly in solution. This image was taken by assembly in solution for 1 h, followed by 20à dilution into a Taxol-containing buffer without DMSO, squashing between a slide and coverslip, and imaging within â¼5 min (40à confocal). Other examples are shown in Figure 4. NUMA (blue), Aurora B (red), tubulin (green).
|
|
FIGURE 2:. Time-course of pineapple assembly. (A) Early stages of assembly, followed by 40Ã confocal imaging. NUMA (blue) started to concentrate on the outside around 10 min (see 3Ã field). Aurora B (red) started to concentrate on the inside around 20 min. Tubulin (green). (B) Later stages of assembly followed by 20Ã wide-field imaging. Once formed, pineapple width increased over tens of minutes and individual assemblies contracted lengthwise. Smaller assemblies tended to become round and then gradually disappear (blue arrows). (C) Assembly time course in the absence of glycogen. This reaction was run in parallel to the plus-glycogen reaction shown in B. Pineapple-like structures still formed, but assembly was slower, and final structures were less organized. NUMA (red), tubulin (green).
|
|
FIGURE 3:. Pineapple width depends on Taxol and DMSO concentrations. Concentrations as noted. Images taken from a set of reactions run in parallel and imaged at 90 min (40à confocal). Our standard condition was 5 μM Taxol, 5% DMSO (second and third panels). Tubulin (green), NUMA (blue), Aurora B (red).
|
|
FIGURE 4:. Microtubule length within pineapples. (A) Assembly reaction run in solution for 1 h, squashed, and imaged immediately for width measurement (40Ã confocal, tubulin probe only). (B) The same reaction as in A imaged 10 min after squashing, which allows time for assemblies to reorganize. (C) Single microtubules from the same reaction as in A and B after salt dissociation and fixation (60Ã wide-field imaging). For length comparison, AâC are presented at the same magnification. Note that the longest microtubule in C is similar in length to the widest width in B, but most microtubules in C are shorter. (D) Length histogram for dissociated microtubules from the reaction pictured in AâC. Mean pineapple width, estimated from images like those in A and B, is indicated by the blue arrow. (E) Typical pineapple image at 90 min with NUMA (blue) and Aurora B (red) probes selected for intensity profiling (40Ã confocal). Each color channel was linearly normalized between 0- and 256-Gy levels over the whole image. The white lines indicate a region of 100 pixels wide selected for an intensity profile parallel to the microtubule axis. (F) Fluorescence intensity profile from the line shown in E. Intensity was averaged over 100 pixels normal to the direction of the linescan to smooth out local variation. Note two NUMA peaks on the outside (blue) and an Aurora B peak on the inside (red). Average tubulin (green) intensity rose sharply just inside the two NUMA peaks and then decreased more gradually toward the center.
|
|
FIGURE 5:. Thin-section electron microscopy. Pineapples were assembled in perfusion chambers, fixed, stained with heavy metals, embedded, and thin sectioned. (AâD) Images from a control reaction. (EâI) Images from a reaction assembled in the presence of STLC to inhibit kinesin-5. (A) One side of a control pineapple with minus-end foci oriented to the left and plus end foci to the right. Arrows indicate regions where higher magnification views are shown in B and C. (B) Typical minus-end focus. Note the bundling of large numbers of approximately parallel microtubules. Minus-end foci lacked the electron-dense coating characteristic of plus-end foci, but we observed accumulation of small, dark puncta and filamentous material in some images. (C) Typical plus-end focus. Note fewer microtubules than in B and electron-dense coating near plus ends. Microtubules join the focus from a range of angles. (D) A line of plus-end foci from a different pineapple. Microtubules join each focus from a range of angles, mostly terminating there. A few appear to make side-on interactions with electron-dense material and bridge between foci. (E) Kinesin-5âinhibited pineapple. Note aster-like shape. From light-level images (Figure 6B) we know that plus-end foci are on the outside. (F) Inside of the pineapple shown in E. Note the dense microtubule bundles. Arrows indicate a bundle shown in higher magnification in G. (G) Bundle from the image in F rotated and enlarged. Note the periodic decoration of microtubules. (H, I) Examples of plus-end foci on the outside of kinesin-5 inhibited pineapples. Morphology and electron-dense coating near plus ends are similar to those for plus-end foci in control pineapples (C, D). One microtubule in I appears to make a side-on interaction.
|
|
FIGURE 6:. Role of spindle assembly motors. (A) Time-course of assembly when kinesin-5 (Eg5), dynein or both motors were inhibited in parallel reactions (20à wide field; tubulin probe only shown). Kinesin-5 was inhibited with 100 μM STLC. Dynein was inhibited with the CC1 fragment of dynactin (1 mg/ml). Note that STLC alone slightly accelerated initial aggregation, whereas CC1 slowed it considerably. (B) Final assemblies at 120 min from the reactions shown in A (40à confocal). In STLC alone, NUMA (blue) foci were on the inside and Aurora B (red) foci on the inside. In CC1 alone, minus ends and NUMA were less focused. In both inhibitors clusters of Aurora B foci were the dominant organizing feature. These combined with loose NUMA aggregates to organize microtubules into quasi-hexagonal arrays in some parts of the coverslip. The second panel in each pair is a 3à view of a region in the first panel. Each panel was normalized to 8 bits in each color channel to facilitate visualization of structure. This normalization overestimates local NUMA intensity in CC1 and CC1 + STLC, where it was much weaker than control and STLC alone. Aurora B foci were of similar intensity in all conditions.
|
|
FIGURE 7:. Cytokinesis midzone markers localize to pineapples. (A) Kif4 localization. A low, nonperturbing concentration of a functional Xenopus Kif4-GFP fusion (Bieling et al. 2010) was added to a pineapple assembly reaction. Shown is typical localization at 60 min (40Ã confocal). Kif4-GFP (red) was recruited to microtubules and accumulated at plus ends. (B) PRC1-like localization using a functional GFP fusion (see Materials and Methods). Shown is typical localization at 60 min (20Ã wide field). Note that PRC1L (red) localizes on the inside of the microtubule (green) structure. (C) Higher-magnification image of PRC1L localization. Shown is typical localization at 60 min (40Ã confocal). Note the microtubules (green) in the center of the pineapple staining for PRC1L (red). Inset, â¼2Ã of the colored image. (C) KIF23 (red) localization using labeled antibody raised to a C terminal peptide. Shown is typical localization at 60 min (40Ã confocal).
|
|
FIGURE 8:. Aurora B and Kif4 are required for plus-end alignment. (A) Aurora B and Kif4 depletion. HSS was depleted with anti-Aurora B, anti-Kif4, or control IgG and analyzed by immunoblotting. Depletion was >90% in both cases. In some experiments Kif4-GFP was added back to approximately endogenous levels. (B) Aurora B (red)âdepleted compared with control IgG-depleted pineapples at 60 min (40à confocal). Note that plus-end alignment is lost after Aurora B depletion, and the antibody probe to Aurora B (red) did not localize. NUMA (blue) localization was not affected by Aurora B depletion. (C) Aurora B inhibition. Aurora B activity was inhibited with 4 μM hesperadin in this experiment. At 20 min hesperadin-treated reactions were poorly organized compared with control. At 60 min minus ends always aligned and recruited NUMA. Plus ends aligned in some assemblies as shown, although others resembled Aurora B (red)âdepleted assemblies. Hesperadin inhibited, but did not completely block, recruitment of the Aurora B probe to microtubules (compare black and white panels in B and C, which were scaled the same). Plus-end accumulation of the probe was strongly inhibited. Similar results were obtained at 1.4 and 16 μM Hesperadin and with other small-molecule inhibitors (see text). (Câ²) Control reaction for C run in parallel with no drug. (D) Kif4 depletion. HSS was depleted with anti-Kif4 or control IgG, and pineapple assembly was run in parallel (20Ã, wide field, tubulin probe only). Depletion of Kif4 (bottom) had no effect on initial aggregation or minus-end alignment. Plus ends in the center appeared to grow longer than control and failed to align, resulting in assemblies that did not hollow out. Microtubules accumulated in whorls in the center in Kif4-depleted pineapples (35 min, 4à panel), suggesting disregulated plus-end growth. (E) Kif4-GFP addback (40à confocal, 100-min time point). Kif4 depletion (left) blocked plus-end focusing and alignment as in B. Aurora B was still recruited to microtubules but did not accumulate in obvious foci, probably because plus ends were distributed. Addback of Kif4-GFP (middle) rescued plus-end alignment and Aurora B accumulation and accumulated at plus ends as in A. Depletion with random IgG (right) had no effect.
|
|
FIGURE 9:. Model for pineapple assembly. Randomly oriented, Taxol-stabilized microtubules (left) recruit the spindle assembly factors dynein, NUMA, and kinesin-5. Dynein and NUMA cluster minus ends, whereas kinesin-5 slides antiparallel microtubules apart (middle, blue arrow). Together with parallel bundling factors, these activities cause the microtubules to sort into two parallel arrays with minus ends clustered on the outside (middle). Only the lower array is shown in the right for clarity. Plus ends recruit midzone assembly factors, including the CPC and Kif4. Plus ends tend to grow, but Kif4 restrains this growth. Lateral aggregation of midzone factors (purple zigzags) causes them to self-organize into a single plane (right). All these processes probably occur simultaneously and are only separated for clarity.
|
