Condensins exert force on chromatin-nuclear envelope tethers to mediate nucleoplasmic reticulum formation in Drosophila melanogaster

Abstract

Although the nuclear envelope is known primarily for its role as a boundary between the nucleus and cytoplasm in eukaryotes, it plays a vital and dynamic role in many cellular processes. Studies of nuclear structure have revealed tissue-specific changes in nuclear envelope architecture, suggesting that its three-dimensional structure contributes to its functionality. Despite the importance of the nuclear envelope, the factors that regulate and maintain nuclear envelope shape remain largely unexplored. The nuclear envelope makes extensive and dynamic interactions with the underlying chromatin. Given this inexorable link between chromatin and the nuclear envelope, it is possible that local and global chromatin organization reciprocally impact nuclear envelope form and function. In this study, we use Drosophila salivary glands to show that the three-dimensional structure of the nuclear envelope can be altered with condensin II-mediated chromatin condensation. Both naturally occurring and engineered chromatin-envelope interactions are sufficient to allow chromatin compaction forces to drive distortions of the nuclear envelope. Weakening of the nuclear lamina further enhanced envelope remodeling, suggesting that envelope structure is capable of counterbalancing chromatin compaction forces. Our experiments reveal that the nucleoplasmic reticulum is born of the nuclear envelope and remains dynamic in that they can be reabsorbed into the nuclear envelope. We propose a model where inner nuclear envelope-chromatin tethers allow interphase chromosome movements to change nuclear envelope morphology. Therefore, interphase chromatin compaction may be a normal mechanism that reorganizes nuclear architecture, while under pathological conditions, such as laminopathies, compaction forces may contribute to defects in nuclear morphology.

Keywords: chromatin compaction; chromatin force; nuclear architecture; nuclear envelope; nucleus.

Figure 1

Changes in nuclear architecture are induced by Cap-H2 overexpression. Individual salivary gland nuclei with different heat shock treatments are visualized with DAPI and anti-lamin marking the chromatin and nuclear envelope, respectively. Normal nuclear morphology can be seen in the heat shock inducible Cap-H2 overexpression line in the absence of heat shock treatment (A). Nuclear deformations can be observed in the heat shock−inducible Cap-H2 overexpression line with heath shock treatment (B), where the nuclear lamina has formed structures within the nuclear space. Normal morphology is observed in the HS > GAL4 control line without (C) and with (D) heat shock treatment. In the particular z-slice shown for the heat shock control (D), the nucleolus is especially prominent in the center of the nucleus but is not indicative of a global reorganization of the chromatin to the nuclear periphery. Scale bar is 10 microns for all panels.

Figure 2

Cap-H2 overexpression leads to remodeling of the nuclear membrane. The nuclear membrane of individual salivary gland nuclei is visualized with wheat germ agglutinin (WGA) and antibody to the nuclear pore complex (anti-Nup). Formation of nuclear membrane structures within the nucleus can be seen in Cap-H2 overexpression, induced by heat shock (A). Typical spherical nuclear membrane structure, with absence of intranuclear structures, can be seen in GAL4 control with heat shock treatment (B). Quantification of these structures shows an average number 1.88 per nucleus in Cap-H2 overexpressing cells and 0.04 per nucleus in cells not overexpressing Cap-H2 (C). This is a significant increase in frequency with Cap-H2 overexpression, P-value 7.1 e⁻⁷. Maximum diameter of these structures is not statistically different in Cap-H2 induction compared to those formed in the GAL4 control (D), P-value 0.59. Scale bar is 10 microns for all panels.

Figure 3

Cap-H2 stabilization leads to NR formation in cell culture. The nuclear membrane of Kc cell nuclei is visualized with wheat germ agglutinin (WGA) and antibody to the nuclear pore complex (anti-Nup). An increase in NR formation can be observed in cells with Cap-H2 stabilization, achieved through Slimb[RNAi] (A). Nuclei from control RNAi treatment maintain typical spherical configuration (B). Quantification of these structures shows significant increase in frequency with Cap-H2 stabilization (C), P-value 0.0025. Scale bars: 10 microns.

Figure 4

Transmission electron microscopy (TEM) imaging of nuclear envelope and nucleoplasmic reticulum (NR) in salivary gland nuclei. TEM of salivary glands reveal the ultrastructure of the nuclear envelope. Labeled in the panels are the following features: N (nucleus), C (cytoplasm), Ch (chromatin), NE (nuclear envelope), and NR (nucleoplasmic reticulum). The nuclear envelope can be seen lining the nuclear periphery of the GAL4 control (A). Cap-H2 overexpressing nuclei have additional structures within the nucleus, termed NR (B). Greater resolution images of the Cap-H2 overexpression nucleus, (C) and (D), reveal that the NR has an intact double membrane layer. Scale bars: (A) 2 microns, (B) 2 microns, (C) 500 nm, (D) 500 nm.

Figure 5

Chromatin associates with nucleoplasmic reticulum (NR) membrane. Transmission electron microscopy (TEM) of salivary glands reveal the ultrastructure of the nucleus in Cap-H2 overexpression tissues. Labeled in the panels are the following features: N (nucleus), C (cytoplasm), Ch (chromatin), NE (nuclear envelope), and NR (nucleoplasmic reticulum). Individual NR events are shown (A−C), with increased magnification on the right hand panels. In each instance, chromatin can be observed abutting the membrane of the NR. Scale bars are 500 nm in all panels.

Figure 6

Chromatin-envelope tethers associate with nucleoplasmic reticulum (NR). Salivary gland nuclei were examined for effects of ectopically expressed chromatin-envelope tether in Cap-H2 overexpression and control. The nuclear envelope is visualized with antibody to the nuclear pore complex (anti-Nup) and the chromatin-envelope tether tracked with green fluorescent protein (GFP)-LacI. In the heat shock control nucleus (A), the LacO array can be seen localizing to the nuclear periphery. The Cap-H2 overexpression nucleus has alternate localization, with the LacO array in the nuclear interior and associating with NR event (B). NR event can be observed associating with LacO array in greater detail in a greater magnification view (C). Total number of NR events was quantified for each cross (D). Both Cap-H2 overexpression crosses had increased NR events compared to the GAL4 control line, p-value <2.2e⁻¹⁶. Yet, there was no statistical difference between the Cap-H2 overexpression nuclei with and without chromatin-envelope tether, P-value 0.252. The frequency of association of the LacO array to NR was elevated in the Cap-H2 overexpression with chromatin-envelope tether (E), P-value 0.0031. Scale bars are 10 microns in all panels.

Figure 7

Time-lapse imaging of nucleoplasmic reticulum (NR) formation in Cap-H2 overexpressing salivary gland nucleus. Time lapse imaging of the nuclear envelope in Cap-H2 overexpressing nucleus used a fluorescent nuclear envelope, marked with a green fluorescent protein (GFP) tagged nuclear pore complex. Images are displayed in three-minute increments. The nucleus was imaged by capturing z-optical slices, with a step size of 2 microns, images shown are of a single z-slice. Structural changes of the nuclear envelope can be observed throughout the course of the experiment, including the budding of NR from the nuclear envelope at t = 45’. Arrowhead indicates the location of the NR budding event at initial and final time points. Scale bar is 10 microns. See File S1.

Figure 8

Weakening of the nuclear envelope enhances Cap-H2 induction of nucleoplasmic reticulum. Nuclear defects of individual salivary glands overexpressing the human progerin protein are visualized with DAPI and anti-Lamin. Overexpression of Cap-H2 yields an enhanced nucleoplasmic reticulum (NR) phenotype (A), compared to GAL4 control (B). Quantification of NR events in progerin background can increase the phenotype when paired with Cap-H2 overexpression (C). A significant increase is observed for overexpression of Cap-H2 and progerin compared to Cap-H2 overexpression alone, P-value 4.15e⁻⁷. Scale bars are 10 microns for all panels.

Figure 9

Model of chromatin compaction leading to nucleoplasmic reticulum (NR) formation. (A) Attachments between the chromatin (black and gray line) and nuclear envelope (red line) are established and maintained for normal cellular function. (B) As chromatin compaction shortens the chromatin, the nuclear envelope is pulled into the nuclear space through these attachments. (C) Formation of the NR occurs as the result of one or more chromatin-envelope attachments.