Microneedle manipulation of mammalian spindles enables sustained force application on k-fibers with spatial and temporal control. (A) Cartoon representation of microneedle (yellow) placement (3D and cross section) in a metaphase mammalian cell to exert sustained force on a k-fiber. (B) Plot of linear microneedle displacement over time during manipulation in metaphase PtK cell (mean ± SEM, n = 18 cells). This approach allows smooth, reproducible pulls on single mammalian k-fibers. (C) Representative z-stack reconstruction shows geometry of microneedle contact with the cell and metaphase spindle (tub; GFP-tubulin, magenta) as diagrammed in (A). The plasma membrane (PM; CellMask Orange dye, cyan) locally deforms around the microneedle (Alexa 647, yellow), but does not alter whole cell shape or puncture the cell. Scale bar, 4 µm. (D) Representative time-lapse images of microneedle (Alexa 555, yellow) manipulation to exert force on a k-fiber: it displaces the metaphase spindle (Cdc20-YFP, green; SiR-tubulin, magenta) and deforms the pulled k-fiber. Manipulated spindles can progress to anaphase (here at 10:04). Scale bar, 4 µm. See also Video 1.