Figure 1. Selective Relative Sliding of MTs in Fission Yeast
Scale bars, 5 μm and 2 min.
(A) Whole-cell projected image (10 confocal planes spaced by 0.5 μm) of MT bundles with visible bright bundle midzones indicated by an 
.
(B) Top: single-plane confocal image of two MT bundles. Bottom: kymograph generated from the yellow-boxed area. The intensity variations along the MT bundle in the top image (yellow arrow) can be recognized in the first line of the kymograph. Growth and shortening of MTs gives rise to triangular patterns in the kymograph—two examples are colored red and green. See text for explanation of the labels. MT sliding (red arrows) occurred after the nucleation of new MTs (label 4). A schematic interpretation of the MTs in the bundle is depicted for selected time points (white bars) with MT plus ends indicated in green. MTs are nucleated antiparallel on top of existing MTs.
(C) Kymograph of a MT nucleated on a rare single MT. Green, red, and yellow dotted lines indicate plus and minus ends and speckles, respectively. After nucleation sliding occurred over 1.2 μm (red arrow). The cartoon shows that sliding effectively stopped before the new plus end passed the old minus end (yellow circle).
(D) Same as (C), but MT nucleation occurred close to an existing minus end. The encircled MT is described in the text; two similar events are observed at later time points (white arrowheads). The cartoon shows that MT sliding stopped when the new plus end passed the old minus end.
Here, we demonstrate that the MT bundling properties of ase1p and klp2p suffice to sort dispersedly nucleated MTs into bipolar MT arrays. We show that ase1p contributes to bipolarity by selectively crosslinking antiparallel MTs. Newly nucleated MTs are therefore oriented antiparallel to preexisting MTs and are transported toward the bundle midzone by klp2p-mediated sliding. Sliding and bundling forces are regulated such that only short, newly nucleated, MTs are transported. As part of this regulation, ase1p accumulated all along the length of bundled MTs, whereas klp2p gathered at MT plus ends in a length-independent manner. We argue that similar length-dependent and -independent forces more generally regulate MT overlap. Thus, our analysis identifies MT end binding and polarity specificity as key elements to the organization of MT arrays.
Results
Klp2p Only Pulls at MT Plus Ends
The movement and dynamics of individual MTs within bundles can be visualized using kymographs (Sagolla et al., 2003
). The kymographs shown in this article display the intensity of fluorescently tagged tubulin along straight or slightly bend MT bundles as a one-dimensional horizontal array of pixels with corresponding intensity variations. Repeating this procedure for each frame of a time-lapsed movie generates a two-dimensional image with time along the vertical dimension. Figure 1B shows a kymograph of a wild-type cell expressing small amounts of GFP-tubulin (atb2) in addition to endogenous tubulin expression. Typical MTs that grew from and subsequently shortened back to the cell center form triangles of increased intensity (Figure 1B, an example is colored red). Shortening continued until the MTs were completely disassembled. Appearing speckles (Figure 1B, yellow circle), points of increased and decreased intensity along MTs caused by the nonuniform incorporation of GFP-tubulin, form vertical lines in kymographs (Waterman-Storer et al., 1998) that often allowed for the identification of the three sides of the triangle: nondynamic minus end (Figure 1B, label 6), growing plus end (Figure 1B, label 1), and shortening plus end (
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