Formation of nuclear splicing factor compartments is independent of lamins A/C

Abstract

Nuclear lamins are major architectural elements of the mammalian cell nucleus, and they have been implicated in the functional organization of the nuclear interior, possibly by providing structural support for nuclear compartments. Colocalization studies have suggested a structural role for lamins in the formation and maintenance of pre-mRNA splicing factor compartments. Here, we have directly tested this hypothesis by analysis of embryonic fibroblasts from knock-out mice lacking A- and C-type lamins. We show that the morphology and cellular properties of splicing factor compartments are independent of A- and C-type lamins. Genetic loss of lamins A/C has no effect on the cellular distribution of several pre-mRNA splicing factors and does not affect the compartment morphology as examined by light and electron microscopy. The association of splicing factors with the nuclear matrix fraction persists in the absence of lamins A/C. Live cell microscopy demonstrates that the intranuclear positional stability of splicing factor compartments is maintained and that the exchange dynamics of SF2/ASF between the compartments and the nucleoplasm is not affected by loss of lamin A/C. Our results demonstrate that formation and maintenance of intranuclear splicing factor compartments is independent of lamins A/C, and they argue against an essential structural role of lamins A/C in splicing factor compartment morphology.

Figure 1.

Absence of lamins A/C from LMNA -/- MEFs. Western blot (A) and (B and C) indirect immunofluorescence microscopy by using anti-lamin A/C polyclonal antibody. (B) In confocal sections, a bright rim staining at the nuclear periphery corresponding to the nuclear lamina can be recognized in LMNA +/+ cells. (C) The signal for lamin A/C is diffuse in LMNA -/- cells. The image in C is greatly overexposed. Bars, 10 μm.

Figure 2.

Distribution and matrix extraction properties of splicing factors are not affected by loss of lamins A/C. (A–F) Indirect immunofluorescence detection of several splicing factors. (G–L) Sequential extraction of SF2/ASF splicing factor. Bar, 10 μm.

Figure 3.

The dynamics of SF2/ASF in the presence/absence of lamins A/C. Time-lapse confocal microscopy of SF2/ASF-GFP in LMNA +/+ (A) and LMNA -/- (B) cells reveal no significant differences in the overall dynamics of SFCs. Time points are indicated in seconds. The overlay shows the same cell at the beginning and the end of the observation period (60 s). (C–H) FRAP analysis of GFP-SF2/ASF dynamics. Images were acquired before bleaching and immediately after and during recovery. Selected images are shown. (C and E) Bleaching of an SFC. (D and F) Bleaching of a nucleoplasmic area. The position of the bleaching spot is indicated by an arrowhead. (G and H) Quantitative analysis of recovery kinetics in a SFC (G) or a nucleoplasmic region (H) did not show any significant difference between LMNA +/+ cells (black circles) and LMNA-/- cells (white circles). Values represent averages ± S. D. from at least 12 cells from two or more experiments. Bars, 10 μm.

Figure 4.

Electron microscopy of cells lacking lamins A/C. (A and C) Control LMNA +/+ cells. (B and D) LMNA -/- cells. Samples were poststained using the EDTA-regressive method to visualize IGCs (dark granular areas). No differences in the morphology of stained IGCs in lamin A/C +/+ and lamin A/C -/- cells were observed. (A and B) Low magnification. (C and D) High magnification of the same cells. Nu, nucleolar area. Bars, 2 μm (A and B); 500 nm (C and D).

Figure 5.

Antibody LA-2H10 in LMNA -/- MEFs. (A) Indirect immunofluorescence microscopy by using LA-2H10 monoclonal antibody. Both in LMNA +/+ and -/- MEFs, the antibody recognizes splicing factor compartments and colocalizes with SF2/ASF. (B) Western blot analysis of LMNA +/+ and -/- MEFs with control anti-lamin antibody (sc-6215; left) and LA-2H10 (right). LA-2H10 recognizes multiple bands both in control LMNA +/+ and in -/- MEFs.