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. 2019 Jul 1;30(14):1705-1715.
doi: 10.1091/mbc.E19-01-0034. Epub 2019 May 8.

Force balances between interphase centrosomes as revealed by laser ablation

Jacob Odell  1 , Vitali Sikirzhytski  1 , Irina Tikhonenko  1 , Sonila Cobani  1 , Alexey Khodjakov  1 , Michael Koonce  1
Affiliations

Force balances between interphase centrosomes as revealed by laser ablation

Jacob Odell et al. Mol Biol Cell. .

Abstract

Numerous studies have highlighted the self-centering activities of individual microtubule (MT) arrays in animal cells, but relatively few works address the behavior of multiple arrays that coexist in a common cytoplasm. In multinucleated Dictyostelium discoideum cells, each centrosome organizes a radial MT network, and these networks remain separate from one another. This feature offers an opportunity to reveal the mechanism(s) responsible for the positioning of multiple centrosomes. Using a laser microbeam to eliminate one of the two centrosomes in binucleate cells, we show that the unaltered array is rapidly repositioned at the cell center. This result demonstrates that each MT array is constantly subject to centering forces and infers a mechanism to balance the positions of multiple arrays. Our results address the limited actions of three kinesins and a cross-linking MAP that are known to have effects in maintaining MT organization and suggest a simple means used to keep the arrays separated.

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Figures

FIGURE 1:
FIGURE 1:
Binucleate MT arrays manage discrete territories. (A) The distance is plotted between the two centrosomes in three representative wild-type cells as a function of time. Note the spacing between centrosomes varies, indicating that they are not rigidly connected. The left column in panel B shows individual frames of each cell, taken at or very near the onset of recording. The right column shows 2D maximum intensity projections of the 121 image stacks for each cell, showing the overall range of centrosome movements during the 10 min imaging period. Panel C presents a plot of the average distance between the centrosomes for eight WT cells. Error bars are SD. Panel D shows one example of MT tracking in a fixed binucleate cell using the FilamentTracer module in Imaris. MTs are color coded either magenta or green depending on their centrosomal origin. Additional examples can be viewed in Supplemental Figure S1. Scale bars in B and D = 5 μm.
FIGURE 2:
FIGURE 2:
Removal of one MT array results in centering of the undamaged array. (A) Individual frames of a binucleate cell pre and post laser ablation and at intervals through the 10-min imaging period. Numbers represent seconds post ablation. The two centrosomes in the targeted cell are marked with arrowheads in the prepanel. A second, mononucleated cell is also visible in this panel. (B) Maximum intensity projections of the first 100 s post ablation (top) and of the subsequent 500 s (bottom). In the top frame, the starting point of the centrosome is indicated with the arrowhead. Note the steady initial migration of the centrosome toward the cell center in the top frame, and then movements around the center in the lower frame (scale bar = 5 μm). (C) The centrosome track at 5-s intervals. The arrowhead marks the initial time point. Axes are in pixels. Scale bar = 2 um.
FIGURE 3:
FIGURE 3:
Centrosome centering in mutant backgrounds. Four representative examples are shown, one each of DdKif8, Kif9, Kif10, and Ase1A null cells. Pre and post columns represent single image frames before and after ablation; the adjacent two columns show maximum intensity projections of the first 100 s post ablation (left) and of the subsequent 500 s (right). The arrowheads mark the initial position of the centrosome, before its directed movement to the center. Scale bar = 5 μm. The rightmost column (Tracks) shows the frame-by-frame movement of the undamaged centrosome. Dots are separated by 5-s intervals. Scale bar = 2 μm.
FIGURE 4:
FIGURE 4:
Summary of centrosome movements. (A) Fitted plots of post ablation centrosome distance to cell center versus time for wild-type, DdKif8, Kif9, Kif10, and Ase1A null cells, and nonirradiated wild-type controls. Each curve represents the summation of 15 cells fitted to a Rodbard function using FIJI and normalized to a 1.0 starting point. (B) The raw averages, including error bars (SD) for each cell line.
FIGURE 5:
FIGURE 5:
Centrosome movement rates. Box plots for each cell type shown in A, the instantaneous rate of centrosome movement (frame by frame distance covered during the 100-s movement period toward the cell center), and in B, the average rate (straight line distance to cell center over same time frame) of centrosome centering. In A, the WT instantaneous rate of movement averages 4.6 μm/min ± 1.2 (SD) (n = 16); Kif8 = 4.2 ± 1.1 (n = 18); Kif9 = 3.7 ± 1.3 (n = 16); Kif10 = 4.2 ± 0.9 (n = 15); Ase1A = 4.5 ± 1.0 (n = 15). In B, the WT centering rate averages 1.8 μm/min ± 0.5 (SD); Kif8 = 1.1 ± 0.3; Kif9 = 1.9 ± 0.6; Kif10 = 1.6 ± 0.4; Ase1A = 1.6 ± 0.5 (n = 15 in all cases).
FIGURE 6:
FIGURE 6:
Nuclear engagements. Two sequences of binucleate cells, post laser ablation. The top row shows a wild-type cell where MTs from the surviving centrosome appear to engage the other nucleus for movement closer together. The bottom row is from a DdKif9 null cell where MT-nuclear engagements appear transient and not force productive. Time is in seconds; scale bar = 5 μm.
FIGURE 7:
FIGURE 7:
DdKif8 influences MT architecture, localizes to the cytoplasm and is a robust MT motor. (A) Two representative examples of interphase MT organization in mononucleate WT and in DdKif8 null cells (MTs in green, nuclei in blue). Panel B quantitates the average distance of the centrosome from the cell center in WT (1.4 μm ± 0.8 SD, n = 98) and Kif8 null (2.7 μm ± 1.2, n = 71). Panel C quantitates the number of MT segments encountered in similar line scans across cells. (WT = 18.0 ± 3.1 (SD), Kif8 null = 24.8 ± 4.9, n = 22 for both strains). (D) GFP-tagged DdKif8 distribution in the cytoplasm, forming punctate patterns during interphase and mitosis. No enrichment of the motor is seen at the MT tips, nor in the spindle midzone as has been reported for some isoforms of the Kinesin-4 family. Panels E and F illustrate DdKif8 MT binding and motility. (E) Three lanes of a Coomassie Blue stained protein gel, containing MW markers, the MT pellet, and supernatant after ATP extraction of MTs incubated with column fractions of DdKif8-GFP protein. The full-length 236 kDa fusion polypeptide is marked with an arrowhead. (F) Four sequential frames of MT gliding activity on a coverslip bound with the DdKif8 motor. Time in seconds. Scale bars in A, D, F = 5 μm.
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