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. 2020 Mar 19;9:e53807. doi: 10.7554/eLife.53807

Figure 1. Microneedle manipulation can exert local forces with spatiotemporal control on the mammalian spindle.

See also Figure 1—figure supplement 1 and Figure 1—video 1. (A–B) Representative PtK2 cell (GFP-tubulin, yellow) and membrane label (CellMask Orange, magenta) (A) before (undeformed cell) and (B) during (deformed cell) microneedle (Alexa-647, blue) manipulation. x-y and y-z views displayed (left and right panels). y-z view taken along the white dashed line shown in the left panels. (C) Left: Overlay of the y-z view of the membrane labeled images before (undeformed, magenta) and during (deformed, green) microneedle manipulation, in order to compare membrane shape and cell height (white line) adjacent to the microneedle due to manipulation. Right: Cell height adjacent to the microneedle, measured using the membrane label, in its undeformed versus deformed state (n = 7 cells, Spearman R coefficient = 0.93, p=0.003, Pearson R coefficient = 0.94, p=0.002). Dashed line represents no change in cell height. Solid grey line is the linear regression fit to the data (r2 = 0.88). (D) Schematic showing a very local deformation of the cell by the microneedle during manipulation, based on (A–C). (E) Schematic of the microneedle (black circle) manipulation assay used throughout this study, pulling (arrow) on a spindle’s outer k-fiber for two different magnitudes and durations. (F) Microneedle displacement over time for two different manipulation datasets: 12 s (red, n = 7 cells) and 60 s (navy, n = 23 cells) pulls. Plot shows mean ± SEM. (G) Timelapse images of the representative response of a metaphase spindle in a PtK2 cell (GFP-tubulin, yellow), when its outer k-fiber is deformed by the microneedle (Alexa-647, blue, white circle) by 2.5 μm over 60 s. The spindle enters anaphase about 20 min after manipulation. Microneedle begins moving at 00:00 (first frame). Scale bar = 5 μm. Time in min:sec. (H) Overlay of the tubulin labeled images of the spindle (G) pre-manipulation (undeformed, magenta) and post-manipulation and microneedle removal (relaxed, cyan). The spindle’s structure is similar pre- and post-manipulation, after correcting for spindle movement.

Figure 1—source data 1. This spreadsheet contains the height of a PtK2 cell when it is undeformed versus deformed by the microneedle (Figure 1C), and the microneedle displacement over time for both 12 s and 60 s manipulations (Figure 1F).

Figure 1.

Figure 1—figure supplement 1. Propidium iodide remains outside cells during microneedle manipulation.

Figure 1—figure supplement 1.

(A) Representative images of two PtK2 cells (GFP-tubulin, yellow) with compromised membranes in which cell impermeable propidium iodide binds and labels DNA (magenta). Scale bar = 5 μm. (B) Timelapse images of a PtK2 spindle (GFP-tubulin, yellow) during a 60 s manipulation in which propidium iodide (magenta) in the media does not enter the cell, suggesting that the membrane is sealed and does not rupture due to the microneedle (Alexa-647, blue, white circle) during this process. Scale bar = 5 μm. Time in min:sec.
Figure 1—video 1. Microneedle manipulation of a mammalian mitotic spindle at metaphase showing spindle relaxation and anaphase entry post-manipulation.
Download video file (103.5KB, mp4)
Microneedle manipulation of a metaphase spindle in a PtK2 cell. The microneedle (Alexa-647, blue) pulls (time 00:00) on the spindle’s outer k-fiber (GFP-tubulin, yellow) over 60 s and deforms the spindle. Upon needle removal (time 00:51), the spindle typically returns to its original structure. About 20 min after manipulation, the spindle has progressed to anaphase (time 25:10), consistent with cell health post manipulation. Scale bar = 5 μm. Time in min:sec. Video was collected using a spinning disk confocal microscope, at a rate of 1 frame every 5 s before and during manipulation. Video has been set to play back at constant rate of 5 frames per second. Movie corresponds to still images from Figure 1G.