Coordinated Dynamics of RNA Splicing Speckles in the Nucleus - PubMed (original) (raw)

. 2016 Jun;231(6):1269-75.

doi: 10.1002/jcp.25224. Epub 2015 Nov 24.

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Coordinated Dynamics of RNA Splicing Speckles in the Nucleus

Qiao Zhang et al. J Cell Physiol. 2016 Jun.

Abstract

Despite being densely packed with chromatin, nuclear bodies and a nucleoskeletal network, the nucleus is a remarkably dynamic organelle. Chromatin loops form and relax, RNA transcripts and transcription factors move diffusively, and nuclear bodies move. We show here that RNA splicing speckled domains (splicing speckles) fluctuate in constrained nuclear volumes and remodel their shapes. Small speckles move in a directed way toward larger speckles with which they fuse. This directed movement is reduced upon decreasing cellular ATP levels or inhibiting RNA polymerase II activity. The random movement of speckles is reduced upon decreasing cellular ATP levels, moderately reduced after inhibition of SWI/SNF chromatin remodeling and modestly increased upon inhibiting RNA polymerase II activity. To define the paths through which speckles can translocate in the nucleus, we generated a pressure gradient to create flows in the nucleus. In response to the pressure gradient, speckles moved along curvilinear paths in the nucleus. Collectively, our results demonstrate a new type of ATP-dependent motion in the nucleus. We present a model where recycling splicing factors return as part of small sub-speckles from distal sites of RNA processing to larger splicing speckles by a directed ATP-driven mechanism through interchromatin spaces.

© 2015 Wiley Periodicals, Inc.

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Conflict of interest statement

Additional information

  1. ■
    None of the experiments involved live vertebrates.
  2. ■
    The authors declare no conflict of interest.

Figures

Figure 1

Figure 1

Directed motion of speckles in the nucleus. A) Time lapse fluorescence imaging of EGFP-SRm160 in an MCF-10A nucleus shows small speckles move toward and fuse with a larger speckle from five different directions (outlined by pseudo-color: red, high intensity; green, low intensity top). Bar, 2 μm. Colored arrowheads indicate individual, smaller speckles that merge with the large speckle. Plot shows the area of the large speckle as a function of time. The size of the large speckle does not change significantly despite the merging events. The directions of the merging speckles as they approach the large speckle are shown in the inset. B) Particle image velocimetry (PIV) was used to analyze movement patterns around a large speckle; five different nuclei are shown. The PIV results indicate that the small speckles move toward the large speckle from different directions and merge. Bar, 2 μm. C) Fluorescent images of EGFP-SRm160 labeled speckles (green) in an MCF-10A nucleus. Bar, 5 μm. The two speckles (indicated by arrows) in the white dashed rectangle move toward each other and merge into one speckle (enlarged view on the bottom, speckles indicated by arrowheads, Bar, 1 μm.) The speckles do not retain memory of their shapes after forming a new speckle, demonstrating the structural plasticity of speckles. D) Measurements of distance between merging speckles across several samples, N = 8 nuclei, and n = 18 pairs of speckles. The distance between the two merging speckles decreased slowly at first and then fused with an increased velocity as seen by the steeper slope of the curve just before the merging event.

Figure 2

Figure 2

Speckles move through conducting channels in the nucleus. A) Schematic shows the setup of the experiment in x-z view. A micropipette with tip diameter 0.5 μm was introduced into the nucleus and a known suction pressure was applied inside the micropipette. B) Fluorescent images of mRFP-SRm160 (splicing speckles) and GFP-histone H1.1 (histone) in an MCF-10A nucleus. Bar, 5 μm. Time lapse images show movements of the speckle (top panel), speckle outline (middle panel), and merged images (bottom). The speckle moves toward the suction point through a curved path (bottom panel, right image shows positions with time). Bar, 2 μm. The sketch at the bottom right summarizes all positions of the speckle during the process (yellow: start, red: end). C) Trajectories of speckles in three different nuclei under suction pressure applied at the point indicated with a black cross. Each color represents a different speckle. Some speckles moved in a random fashion, while multiple speckles moved toward the suction points through the same curved path forming speckle ‘trains’. D) Time lapse fluorescent images of mRFP-SRm160 and GFP-H1.1 shows clear elongation and deformation of speckles as they moved toward the suction point (indicated with white arrows). Bar, 5 μm. E), F), G) show enlarged view of speckle outlines in the blue, white and red insets in D, respectively. The outlines show deformations, translation and fusion of the speckles as they moved toward the suction point. Bar, 5 μm in E); bar, 2 μm in F) and G).

Figure 3

Figure 3

Speckles deform under applied pressure. A) Schematic shows the setup of the compression experiment in x-z view. A micropipette was gently pressed downward on the apex of the nucleus for short time periods of about 200 ms. B) Fluorescent images of EGFP-SRm160 (splicing speckles, arrowheads) in a representative MCF-10A show deformation and elongation through narrow channels upon pressure, and relaxation back to initial shape (enlarged view on the right, Bar, 1 μm). The white cross in the middle panel shows the point where compression applied. Bar, 2 μm.

Figure 4

Figure 4

Analysis of random motion of speckles. A) Trajectories of randomly moving speckles (corrected for nuclear drift) are shown for control cells, alpha-amanitin treated cells, Latrunculin A treated cells and ATP depleted cells (compare with control cells), shRNA control cells and BRG1 knockdown cells (compare with shRNA control cells), GFP control cells and GFP KASH4 cells (compare with GFP control cells). Each color represents the trajectory of an individual speckle. The trajectories were measured from time-lapse images acquired for 1 hr with 5 min time intervals. Bar, 1 μm. B) Plot of the average MSD of speckles in control cells as a function of time, and the corresponding regression fit line. The linear profile suggests that the speckle motion follows a simple random walk. Error bars represent the standard error of the mean, n ≥ 120 speckles.

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