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Dynamics of hnRNPs and omega speckles in normal and heat shocked live cell nuclei of Drosophila melanogaster

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Abstract

The nucleus limited long-noncoding hsrω-n transcripts, hnRNPs, and some other RNA processing proteins organize nucleoplasmic omega speckles in Drosophila. Unlike other nuclear speckles, omega speckles rapidly disappear following cell stress, while hnRNPs and other associated proteins move away from chromosome sites, nucleoplasm, and the disappearing speckles to get uniquely sequestered at hsrω locus. Omega speckles reappear and hnRNPs get redistributed to normal locations during recovery from stress. With a view to understand the dynamics of omega speckles and their associated proteins, we used live imaging of GFP tagged hnRNPs (Hrb87F, Hrb98DE, or Squid) in unstressed and stressed Drosophila cells. Omega speckles display size-dependent mobility in nucleoplasmic domains with significant colocalization with nuclear matrix Tpr/Megator and SAFB proteins, which also accumulate at hsrω gene site after stress. Instead of moving towards the nuclear periphery located hsrω locus following heat shock or colchicine treatment, omega speckles rapidly disappear within nucleoplasm while chromosomal and nucleoplasmic hnRNPs move, stochastically or, more likely, by nuclear matrix-mediated transport to hsrω locus in non-particulate form. Continuing transcription of hsrω during cell stress is essential for sequestering incoming hnRNPs at the site. While recovering from stress, the sequestered hnRNPs are released as omega speckles in ISWI-dependent manner. Photobleaching studies reveal hnRNPs to freely move between nucleoplasm, omega speckles, chromosome regions, and hsrω gene site although their residence periods at chromosomes and hsrω locus are longer. A model for regulation of exchange of hnRNPs between nuclear compartments by hsrω-n transcripts is presented.

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Acknowledgments

We thank Dr. H. Saumweber (Germany) for P11 and BX34 antibodies, Dr. Keith A. Maggert (USA) for UAS-SAFB-GFP stock, and Drs. Stephen W. Mckechnie (Australia) for hsrω 66, D. Corona (Italy) for ISWI 1 Bc/SM5 and ISWI 2; +/T(2;3)CyO, TM6B, Tb, Allan C. Spradling, (USA) for Hrb87F-GFP, and Alain Debec (France) for Squid-GFP and Hrb98DE-GFP stocks. This work was supported by the CEIB-II grant from Department of Biotechnology, Govt. of India and by the Board of Research in Nuclear Sciences (Department of Atomic Energy, Govt. of India) through Raja Ramanna Fellowship to SCL. We thank the Department of Science & Technology, Govt. of India (New Delhi) and the Banaras Hindu University for Confocal Microscopy facility in our laboratory. AKS has been supported as Senior Research Fellow by the Council of Scientific & Industrial Research (New Delhi) and as Research Associate by the Department of Biotechnology, Govt. of India.

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  1. Cytogenetics Laboratory, Department of Zoology, Banaras Hindu University, Varanasi, 221005, India

    Anand K. Singh & Subhash C. Lakhotia

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  1. Anand K. Singh
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Correspondence to Subhash C. Lakhotia.

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Fig. S1

Stable peripheral location of the hsrω gene site and reproducible accumulation and redistribution of hnRNPs during repeated HS and recovery periods. Time lapse live cell confocal images of Hrb87F-GFP expressing hsrω + SG nucleus at 5 min intervals (noted at upper left corner of each panel) during repeated HS and recovery periods. The on-stage incubation temperature for each row is indicated on left. (GIF 120 kb)

High resolution image (TIFF 1746 kb)

Fig. S2

FRAP of Hrb87F-GFP in nucleoplasm and at the 93D site in wild type cells. (A) FRAP in unstressed control peripodial cell at nucleoplasmic ROI (red circles). (B, C) FRAP after 30 min HS at nucleoplasmic ROI (B) or at 93D cluster ROI (C). (D) FRAP at ROI in the 74EF/75B developmental puffs in unstressed SG polytene nucleus. In all cases, confocal images of the same optical section are shown at prebleach stage, just after bleaching (t = 0.0 s) and at different time points (in s) thereafter (noted in top row of each column). Scale bar applies to all images. (GIF 221 kb)

High resolution image (TIFF 1138 kb)

Fig. S3

Hrb87F-GFP is exchanged between different nuclear compartments. (A) FLIP in unstressed control peripodial cell with ROI1 (red circle) and ROI2 (blue circle) being different sites in the nucleoplasm. (B) FLIP in 30 min heat shocked peripodial cell with ROI1 being nucleoplasmic (red circle), ROI2 being aggregate at 93D site (green circle) and ROI3 being a nucleoplasmic area (pink circle). (C) FLIP in 30 min heat shocked peripodial cell with the 93D cluster as ROI1 (red circle), ROI2 being the same region at 93D cluster (sky blue circle) and ROI3 being an area in nucleoplasm (purple circle). Each row shows confocal images of same optical section at different time points (in s) noted in top row of each column. The scale bar in 1st column of top row applies to all images. (GIF 171 kb)

High resolution image (TIFF 1641 kb)

Fig. S4

Association of hnRNPs with hsrω transcripts slows down their movement. FLIP in unstressed Hrb87F-GFP hsrω + (A) unstressed Hrb87F-GFP hsrω 66 peripodial cell (B) and heat shocked Hrb87F-GFP hsrω + peripodial cell (C). Green circles indicate photobleached nucleoplasmic ROI1 while red circles indicate nucleoplasmic ROI 2 region used to measure loss of fluorescence at different time points (in s) indicated in top row of each column. Genotype of cells is indicated on left of each row. The scale bar in 1st column of top row applies to all images. (GIF 203 kb)

High resolution image (TIFF 1957 kb)

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Singh, A.K., Lakhotia, S.C. Dynamics of hnRNPs and omega speckles in normal and heat shocked live cell nuclei of Drosophila melanogaster . Chromosoma 124, 367–383 (2015). https://doi.org/10.1007/s00412-015-0506-0

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