Dissecting mitosis by RNAi in Drosophila tissue culture cells

Abstract

Here we describe a detailed methodology to study the function of genes whose products function during mitosis by dsRNA-mediated interference (RNAi) in cultured cells of Drosophila melanogaster. This procedure is particularly useful for the analysis of genes for which genetic mutations are not available or for the dissection of complicated phenotypes derived from the analysis of such mutants. With the advent of whole genome sequencing it is expected that RNAi-based screenings will be one method of choice for the identification and study of novel genes involved in particular cellular processes. In this paper we focused particularly on the procedures for the proper phenotypic analysis of cells after RNAi-mediated depletion of proteins required for mitosis, the process by which the genetic information is segregated equally between daughter cells. We use RNAi of the microtubule-associated protein MAST/Orbit as an example for the usefulness of the technique.

Fig. 1. Preparation of dsRNA and specific protein depletion.

(A) PCR fragment of ~700 bp synthesized from MAST/Orbit cDNA. (B) Corresponding dsRNA obtained by in vitro transcription using the previous PCR fragment as template. The lower band corresponds to ssRNA that did not form duplexes. (C) Monitoring of protein levels upon addition of 30 µg of dsRNA to the cells by immunoblot using MAST polyclonal antibodies and anti-α-tubulin monoclonal antibodies as a loading control. (D) Determination of the amount of dsRNA necessary to deplete MAST from S2 and Dmel2 cells. Protein levels were monitored 120 h after addition of dsRNA. It is clear that for the case of MAST, protein depletion is significantly more effective in S2 cells. (E) Addition of dsRNA to S2 cells did not cause a significant effect upon cell viability throughout the experiment.

Fig. 2. Immunofluorescence analysis of mitotic S2 cells after RNAi.

Chromosomes were stained with DAPI (blue), microtubules were stained with an anti-α-tubulin antibody (green), centrosomes were stained with an anti-CP-190 antibody and the centromeres were stained with an anti-CID antibody (both white or red in the merged images). (A, A’ and E, E’) Control metaphase cells showing a well organized bipolar spindle with one centrosome at each pole and with the chromosomes correctly aligned at the metaphase plate. (B, B’ and F, F’) 72 h after RNAi, cells form monopolar spindles with the centrosomes clustered in the centre and the centromeres dispersed in the monoaster. (C, C’) Polyploid cell 96 h after RNAi showing multiple centrosomes. (G, G’) Cell showing abnormal chromosome congression 72 h after RNAi. (D, D’) Abnormal anaphase-like cells 120 h after RNAi showing a bipolar spindle where the centrosomes are clustered at only one of the poles. (E, E’) Another anaphase-like cell where non-disjoined chromosomes have attempted to segregate towards opposite poles. Scale bar is 5 µm.