Induction of asymmetrical cell division to analyze spindle-dependent organelle partitioning using correlative microscopy techniques.
This protocol describes an assay for the induction of asymmetrical cell division where the entire spindle is segregated into only one of the daughter cells. The procedure consists of four stages: (i) generation of asymmetrical monoasters by arresting cells in early mitosis with a kinesin Eg5 inhibitor; (ii) induction of cell division by microinjection of recombinant Mad1 protein or by the addition of a Cdk1 inhibitor; (iii) monitoring the division process by phase-contrast time-lapse microscopy; and (iv) processing for correlative immunofluorescence or correlative electron microscopy. This approach can be applied to determine the requirement for the mitotic spindle in organelle partitioning as well as to investigate the role of the monopolar spindle in cytokinesis. Moreover, the generated nucleus-lacking cytoplast provides an ideal environment to test the feasibility and activity of biological processes in the absence of genomic influence. The protocol takes 2-4 d to complete.
Wei
Jen-Hsuan
JH
Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
Seemann
Joachim
J
D033961:Adaptor Protein Complex 1 / Q000502:physiology / Q000648:ultrastructure; D000818:Animals; D018929:Cell Culture Techniques; D002455:Cell Division; D002460:Cell Line; D016547:Kinesins / Q000037:antagonists & inhibitors; D008858:Microscopy, Phase-Contrast; D048328:Potoroidae; D008941:Spindle Apparatus / Q000502:physiology / Q000648:ultrastructure*
D033961:Adaptor Protein Complex 1; D016547:Kinesins