The Role of Lamins in the Nucleoplasmic Reticulum, a Pleiomorphic Organelle That Enhances Nucleo-Cytoplasmic Interplay

Abstract

Invaginations of the nuclear membrane occur in different shapes, sizes, and compositions. Part of these pleiomorphic invaginations make up the nucleoplasmic reticulum (NR), while others are merely nuclear folds. We define the NR as tubular invaginations consisting of either both the inner and outer nuclear membrane, or only the inner nuclear membrane. Specifically, invaginations of both the inner and outer nuclear membrane are also called type II NR, while those of only the inner nuclear membrane are defined as type I NR. The formation and structure of the NR is determined by proteins associated to the nuclear membrane, which induce a high membrane curvature leading to tubular invaginations. Here we review and discuss the current knowledge of nuclear invaginations and the NR in particular. An increase in tubular invaginations of the nuclear envelope is associated with several pathologies, such as laminopathies, cancer, (reversible) heart failure, and Alzheimer's disease. Furthermore, viruses can induce both type I and II NR. In laminopathies, the amount of A-type lamins throughout the nucleus is generally decreased or the organization of lamins or lamin-associated proteins is disturbed. Also, lamin overexpression or modulation of lamin farnesylation status impacts NR formation, confirming the importance of lamin processing in NR formation. Virus infections reorganize the nuclear lamina via (de)phosphorylation of lamins, leading to an uneven thickness of the nuclear lamina and in turn lobulation of the nuclear membrane and the formation of invaginations of the inner nuclear membrane. Since most studies on the NR have been performed with cell cultures, we present additional proof for the existence of these structures in vivo, focusing on a variety of differentiated cardiovascular and hematopoietic cells. Furthermore, we substantiate the knowledge of the lamin composition of the NR by super-resolution images of the lamin A/C and B1 organization. Finally, we further highlight the essential role of lamins in NR formation by demonstrating that (over)expression of lamins can induce aberrant NR structures.

Keywords: STED microscopy; calcium regulation; electron microscopy; lamins; nuclear invaginations; nucleoplasmic reticulum.

FIGURE 1

Schematic overview of the different types of nuclear membrane invaginations. PNMI, Pleiomorphic nuclear membrane invaginations; NR, Nucleoplasmic reticulum; NF, Nuclear folds.

FIGURE 2

3D STED of normal human dermal fibroblast cells, stained for lamin A/C (red) and B1 (green). (A,E) 2D image of the middle of the nucleus. (B–D) Different 3D reconstruction views of the cell in (A) using ImageJ 3D viewer. (F–H) Different 3D reconstruction views of the cell in (E) using ImageJ 3D viewer. Scale bars indicate 5 μm. Also see Supplementary Figures S1,S2. Cell culture and immunostaining of NHDF was performed as described previously (Stiekema et al., 2021). STED images were taken with an abberior Instruments INFINITY LINE microscope equipped with an inverted IX83 microscope (Olympus), a 60× oil objective (UPlanXApo 60×/1.42 oil, Olympus), using pulsed excitation lasers at 561 nm (for secondary antibody Abberior STAR ORANGE) and 640 nm (for Abberior STAR RED) and a pulsed STED laser operating at 775 nm. All acquisition operations were controlled by the Lightbox Software. STED images were deconvoluted as described in Stiekema et al. (2021).

FIGURE 3

Effects of over-expression of different types of lamins on the nucleus of CHO-cells. (A) The over-expression of lamin A leads to nuclear membrane growth and the formation of a large number of tubular invaginations of the nuclear envelope. Scale bar indicates 10 μm. (B) The over-expression of lamin AΔ10 leads to nuclear membrane growth and the formation of a large number of tubular invaginations of the nuclear envelope. Scale bar indicates 10 μm. (C) The over-expression of lamin C leads to mainly aggregates in the nucleoplasm. Scale bar indicates 10 μm. (D) The over-expression of lamin B1 leads to nuclear membrane growth and the formation of a limited number of tubular invaginations of the nuclear envelope. Scale bar indicates 10 μm. (E) The over-expression of lamin B2 leads to enlarged nuclei with highly folded nuclear membranes and lobulations of the nuclear membrane. Scale bar indicates 10 μm. (F) EM-recording of tubular invaginations in lamin A over-expressing CHO cells. Scale bar indicates 5 μm. (G) Detailed recording of tubular invaginations in lamin A over-expressing CHO cells. Scale bar indicates 0.5 μm. CHO-cells were grown, transfected and imaged as described (Broers et al., 1999). Confocal fluorescence images (A‐E) show maximal Z-stack projections.

FIGURE 4

NR-like invaginations of the nuclear envelope in different cardiovascular cell types as seen at the electron microscopy level. (A) Cardiomyocyte in relaxation (goat in vivo atrial tissue), the nuclear membrane shows no invaginations or folds. (B) Cardiomyocyte in mild contraction (goat in vivo atrial tissue), the nuclear membrane shows some folds. (C) Cardiomyocyte in strong contraction (goat in vivo atrial tissue), the nuclear membrane is strongly folded. (D) Cardiomyocyte in relaxation (goat in vivo atrial tissue) demonstrating a large invagination of the nuclear envelope. (E) Mast cell in cardiac tissue (human ventricle), the nuclear membrane has large invaginations. (F) Capillary endothelial cell in cardiac tissue (human ventricle) with several invaginations of the nuclear envelope. (G) Fibroblast in cardiac tissue (human ventricle), the nuclear membrane is strongly folded. Scale bars indicate 1 μm. Goat atrial samples were fixed for 2 h in 3% glutaraldehyde buffered to pH 7.4 with 90 mM KH₂PO₄. Thereafter the samples were washed in the same buffer for 24 h and post-fixed for 1 h in 2% OsO₄ buffered to pH 7.4 with veronal acetate. Next, the samples were rapidly dehydrated through a graded series of ethanol and routinely embedded in Epon (Driesen et al., 2009). Ultra-thin sections were counterstained with uranyl acetate and lead citrate prior to examination in a Philips CM 100 electron microscope.

FIGURE 5

NR invaginations in human hematopoietic cells at the electron microscopy level. (A) Macrophage. (B) Mast cell. (C) T-lymphocyte. (D) B-lymphocyte. Scale bars indicate 1 μm. Human hematopoietic cells were cultured for at least 48 h in a culture dish after which the medium was discarded. Next, the cells were shortly rinsed with PBS and subsequently fixed at room temperature with 3% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) supplemented with 0.5 mM CaCl₂ for at least 24 h. The cells were then postfixed with 2% OsO₄ in cacodylate buffer (pH 7.4) containing 1.5% potassium ferricyanide at 48°C for 1 h. After a short rinse in cacodylate buffer, the cells were further dehydrated in graded ethanol series before embedding in epon. Ultra-thin sections were counterstained with uranyl acetate and lead citrate prior to examination in a Philips CM 100 electron microscope.

FIGURE 6

(A) Cardiomyocyte (human ventricular hibernating myocardium; reversible heart failure in vivo) demonstrating tubular invaginations of the nuclear envelope. Scale bar indicates 5 μm. (B) Cardiomyocyte nucleus (human ventricular hibernating myocardium; reversible heart failure in vivo) demonstrating tubular invaginations of the nuclear envelope. Scale bar indicates 2 μm. (C) Cardiomyocyte (human ventricular myocardium from dilated cardiomyopathy) demonstrating a large invagination of the nuclear envelope, engulfing contractile elements. Scale bar indicates 2 μm. Samples were prepared as described for Figure 4.

FIGURE 7

The effects of lovastatin treatment on the nucleus of CHO-cells at the electron microscopy (EM)-level. (A) EM recording of two types of tubular invaginations in CHO cells treated with lovastatin. Type I invaginations are marked by a single arrow, type II by a double arrow. Scale bar indicates 0.5 μm. (B) Detailed recording of type II NRin CHO cells treated with lovastatin. Scale bar indicates 0.1 μm. (C) Detailed recording of a type I NR in CHO cells treated with lovastatin. Scale bar indicates 0.1 μm. Ultrathin sections from fibroblasts of CHO cells were studied by electron microscopy (for sample preperations, see Figure 5) for nuclear membrane invaginations after 18 h of incubation with 40 μM of lovastatin (see also Verstraeten et al., 2006).