Live imaging screen reveals that TYRO3 and GAK ensure accurate spindle positioning in human cells

Live imaging screen for regulators of mitotic spindle positioning, screening a library of 1200 kinases, phosphatases and associated proteins (4 siRNAs per gene), in a 96 well format. Cells were incubated 48 hours in the presence of siRNA and than transferred to micropatterned 96 well plates containing L-shaped, fibronectin coated, micropatterns, helping to stereotype cell organelle positioning. On L-shapes, mitotic DNA (metaphase plates) are positioned in a 45° angle with respect to the arms of the Ls, thereby creating a ""reference"" phenotype. We screened for candidates whose depletion led to a significant offset from this ""reference"" or normal position during metaphase. We used HeLa cells carrying the mCherry::H2B transgene, visualizing DNA. The L-micropatterns were coated with fibronectin containing Alexa 555, thereby Ls were visible in the same fluorescent channel. After transfer of cells to micropatterned plates, cells attached within 6 hours to the micropattern. We imaged each plate for 24 hours (framerate 8min), imaging 2 visual fields per well. Each plate contained negative and positive control wells (8 scrambles siRNA wells, 8 LGN siRNA wells). Movies were analyzed using the ImageJ based algorithm TRACMIT.

Wolf B, Busso C, Gönczy P

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