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In 1995, Conly L. Rieder, Richard W. Cole, Alexey Khodjakov and Greenfield Sluder tested whether an unattached kinetochore supplies the inhibitory signal delaying anaphase. Living PtK1 biological cells on glass coverslips were mounted in Rose chambers at $35$–$37{}^{\circ }\mathrm{C}$. An inverted microscope with a $60\times$ objective recorded differential interference contrast microscopy video. A pulsed $532\mathrm{nm}$ laser focused through that objective destroyed selected chromosome regions. The readout was chromosome movement and anaphase onset before and after surgery.^[ref]

Destroying the last unattached kinetochore permitted anaphase without that chromosome aligning. Cutting chromosome arms served as a damage control: progression still awaited attachment of the last naturally one-pole-attached chromosome. Correlative electron microscopy checked the absence of the targeted kinetochore. Together these results located an inhibitory activity in or near the unattached kinetochore, rather than establishing a requirement for alignment itself.^[ref]

This intervention localized control; it did not identify every signaling molecule or guarantee faithful inheritance after damage. The cultured PtK1 preparation differs from the hypothetical $2n=4$ cartoon. Neither the cartoon nor this experiment establishes identical control in every species.

By Conly L. Rieder, Richard W. Cole, Alexey Khodjakov, Greenfield Sluder.

Online text: doi.org/10.1083/jcb.130.4.941

Experimental article published on 15 August 1995 in Journal of Cell Biology 130(4), pp. 941–948. The full author PDF was inspected at the separately linked copy.

Inspected PDF copy: pdfs.semanticscholar.org/d175/879a4000a67510e16d637c2cadea97250148.pdf

Cited passages: pp. 941–942, Abstract and Materials and Methods: Cell Culture; Laser Microsurgery and Video-enhanced Light Microscopy; pp. 943–947, arm-cutting control, Table I, Figures 1, 5 and 6; destruction of the last unattached kinetochore and correlative electron microscopy.