Comparison of Nuclear Matrix and Mitotic Chromosome Scaffold Proteins in Drosophila S2 Cells—Transmission of Hallmarks of Nuclear Organization Through Mitosis

Rahul Sureka,Rashi Wadhwa,Suman S Thakur,Rashmi U Pathak,Rakesh K Mishra

doi:10.1074/mcp.ra118.000591

Abstract

Chromatin condenses several folds to form mitotic chromosomes during cell division and decondenses post-mitotically to reoccupy their nuclear territory and regain their specific transcriptional profile in a precisely lineage specific manner. This necessitates that the features of nuclear architecture and DNA topology persist through mitosis. We compared the proteome of nuclease and high salt resistant fraction of interphase nucleus known as nuclear matrix (NuMat) and an equivalent biochemical fraction in the mitotic chromosome known as mitotic chromosome scaffold (MiCS). Our study elucidates that as much as 67% of the NuMat proteins are retained in the MiCS indicating that the features of nuclear architecture in interphase nucleus are retained on the mitotic chromosomes. Proteins of the NuMat/MiCS have large dynamic range of MS signal and were detected in sub-femtomolar amounts. Chromatin/RNA binding proteins with hydrolase and helicase activity are highly enriched in NuMat as well as MiCS. Although several transcription factors involved in functioning of interphase nucleus are present exclusively in NuMat, protein components responsible for assembly of membrane-less nuclear bodies are uniquely retained in MiCS. Our study clearly indicates that the features of nuclear architecture, in the structural context of NuMat, are retained in MiCS and possibly play an important role in maintenance of cell lineage specific transcriptional status during cell division and thereby, serve as components of cellular memory.

Highlights

Genetic information that is encoded in a linear DNA sequence, needs to be organized in three-dimensional space of the nucleus in the form of chromatin for appropriate expression of genes [1,2,3]
The representative silver stained SDS-PAGE pictures showed that nuclear matrix (NuMat)/mitotic chromosome scaffold (MiCS) were enriched in high molecular weight proteins (Fig. 2, Lanes 2 and 6)
After in vivo NuMat/MiCS preparation, when bulk of chromatin and soluble proteins had been removed, the antibody continues to stain the NuMat proteins that undergo a striking reorganization into mitotic chromosomes and spindle like structure, which colocalizes with Lamin Dm0, during mitosis (Fig. 6A, NuMat, MiCS panels)

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—S2 cells were cultured in Schneider’s Media (GIBCO, Gaithersburg, MD) with 10% heat inactivated FBS at 25 °C. The solution was mixed by pipetting and incubated at room temperature for 5 mins. 4 ␮l of effectene reagent was added to the tube, mixed by pipetting, and incubated at room temperature for 10 mins. 100 ␮g/ml hygromycin was added to each well and the plates were further incubated in 25 °C incubator for 48 h. Following this the media was changed every 48 h with fresh media containing 100 ␮g/ml hygromycin. The expression of proteins was induced by adding 500 ␮M CuSO4 in the media and incubating the cells in 25 °C incubator for 24 h. Arrested S2 cells were pelleted, washed and resuspended in RSB (10 mM Tris pH 7.4, 10 mM NaCl, 5 mM Mgcl).

The abbreviations used are

RESULTS

DISCUSSION