Essential roles for Caenorhabditis elegans lamin gene in nuclear organization, cell cycle progression, and spatial organization of nuclear pore complexes

Abstract

Caenorhabditis elegans has a single lamin gene, designated lmn-1 (previously termed CeLam-1). Antibodies raised against the lmn-1 product (Ce-lamin) detected a 64-kDa nuclear envelope protein. Ce-lamin was detected in the nuclear periphery of all cells except sperm and was found in the nuclear interior in embryonic cells and in a fraction of adult cells. Reductions in the amount of Ce-lamin protein produce embryonic lethality. Although the majority of affected embryos survive to produce several hundred nuclei, defects can be detected as early as the first nuclear divisions. Abnormalities include rapid changes in nuclear morphology during interphase, loss of chromosomes, unequal separation of chromosomes into daughter nuclei, abnormal condensation of chromatin, an increase in DNA content, and abnormal distribution of nuclear pore complexes (NPCs). Under conditions of incomplete RNA interference, a fraction of embryos escaped embryonic arrest and continue to develop through larval life. These animals exhibit additional phenotypes including sterility and defective segregation of chromosomes in germ cells. Our observations show that lmn-1 is an essential gene in C. elegans, and that the nuclear lamins are involved in chromatin organization, cell cycle progression, chromosome segregation, and correct spacing of NPCs.

Figure 1

Ce-lamin encodes a 64-kDa protein. (A) Different lmn-1:gfp constructs used to generate Ce-lamin-GFP lines and clones used to produce dsRNA for the RNAi experiments. For lmn-1:gfp constructs, exons are presented as black boxes. The positions of promoter, GFP, and 3′ sequences are marked. The pDRCL2 construct lacks exons 4 to 6 of lmn-1. The pJKL380.4 construct was used to generate the integrated line PD4810. The pDRNL1 construct was used to generate the unintegrated line A12. The ends of the different constructs used for the RNAi experiments correspond to the positions in the lmn-1 insert in the pDRNL1 construct. (B) Western blot analysis of mixed stages: wild-type (WT), PD4810, and A12 animals probed with Ce-lamin antibodies. The position of Ce-lamin in wild-type nematodes is marked with an arrowhead. A band corresponding in size to the Ce-lamin-GFP fusion protein, which is present in the PD4810 line (marked with an arrow), is below the level of detection in the A12 line. The positions of the size markers in kilodaltons are shown on the right.

Figure 2

Ce-lamin protein distribution. Wild-type C. elegans (N2) were stained with affinity purified polyclonal antibodies to Ce-lamin (primary), and Cy3-conjugated antirabbit antibodies (secondary) (b,d,f,g). DNA was stained with DAPI (a,c,e). Embryo (a,b). Arrows in (a) are pointing toward nuclei in telophase where some Ce-lamin is dispersed in the cytoplasm (arrows in b). L1 larvae (c,d). The arrow in (e) points to a cluster of sperm cells in an adult hermaphrodite's gonad. Ce-lamin is detected only in the surrounding cells. Ce-lamin is expressed in all other cells in the gonad (g). Note the intense lamin staining in the distal tip cell (arrowhead in g). Expression of Ce-lamin-GFP can be detected in the nuclear envelope of all somatic cells in PD4810 embryo (h), larva (i), and adult (j). Arrow in (j) points toward germ cell nuclei in the gonad; Arrowhead points toward an oocyte nucleus. Bar in each panel represents 10 μm.

Figure 3

Ce-lamin is present in the nuclear interior. Top panel: embryos were double stained with monoclonal antibody MAb414 (red) and affinity-purified Ce-lamin antibodies (green), and viewed with a confocal microscope. Overlap between Ce-lamin and nucleoporins appears in yellow. Most embryonic nuclei contained internal Ce-lamin, while MAb414 staining was confined to the nuclear envelope and the cytoplasm. Examples of nuclei with internal Ce-lamin staining are marked with arrows. Middle panel: DNA staining, left image; lamin staining, all other images. Both lmn-1(RNAi) images show the same confocal image of Ce-lamin staining, of the corresponding DNA-stained embryos. Intensity levels in the right lmn-1(RNAi) image were elevated, in order to detect residual lamin staining (as compared with the normal intensity levels in the left lmn-1(RNAi) image). Ce-lamin staining was eliminated in lmn-1(RNAi) embryos from both the nuclear envelope and the nuclear interior. The right image shows lamin staining in the control wild-type embryos. Bottom panel: three consecutive confocal sections, (one micron apart, 0 to 2), of adult tissue in the A12 line, which expresses low levels of Ce-lamin-GFP (see Figure 1B). Examples of nuclei with internal Ce-lamin-GFP staining are marked with arrows. Bar in each panel represents 10 μm and applies to all images in that panel.

Figure 4

Arrested embryos injected with lmn-1 dsRNA. Ce-lamin indirect immunofluorescence staining of wild-type (a) and lmn-1 (RNAi) embryos (b). The lmn-1 (RNAi) embryos showed significantly reduced levels of Ce-lamin staining as compared with wild-type embryos. Fluorescence quantification of this difference revealed an effect > 60-fold (c). Nomarski image of a lmn-1 (RNAi) embryo (c). Note the uneven size of the nuclei and a cell with two nuclei (arrows in c). DAPI staining of lmn-1 (RNAi) embryos (d-i). At the time of arrest, abnormalities included nuclei with higher DNA content (black arrows in panel d and regions with fewer nuclei (our unpublished results). Abnormalities in nuclear morphology were already obvious in embryos undergoing the first nuclear divisions (e-i), although most embryos did develop further. These abnormalities included chromosome bridges between two nuclei (arrow in e), abnormal chromosome morphology (arrow in f), an unequal distribution of chromatin between daughter nuclei (arrows in g), and a chromatin or micronuclei left outside the daughter nuclei after nuclear division (arrows in i show the affected nuclei in h are magnified in j). The bar in panel (a) represents 10 μm and also applies to panels (b-h) and 2.5 μm in panel (i).

Figure 5

Nuclear morphology in lmn-1(RNAi) embryos. Five consecutive images of the AB cell nucleus in a two-cell embryo taken at 1-min intervals using 4-D time-lapse recording (Fire, 1994). The central focal plane for each nucleus is shown. The top panels are from a wild-type embryo, and the bottom two sets are from a lmn-1(RNAi) embryo, with the bottom set showing the outline of the nucleus in dotted line. Bar represents 5 μm.

Figure 6

Clustering of NPCs in lmn-1 (RNAi) embryos. Embryos were stained with monoclonal antibody MAb414 (a-c) and viewed with a confocal microscope. Wild-type embryo (a); lmn-1 (RNAi) embryos, (b-c). The single-head arrows in panel (b) point toward regions where NPCs are clustered on one side of the nucleus. The embryo in panel (c) has two nuclei, both of which have clustered NPCs, never seen in wild-type embryos. Most embryos also contain nuclei with normal spacing of NPCs (arrowhead). The lmn-1(RNAi) embryos were also stained with both lamin antibodies (d) and monoclonal antibody MAb414 (e). Bar in panel (a) represents 10 μm and applies to all panels.

Figure 7

Sterile and semisterile animals from the RNAi experiments. Nomarski images of progeny that escaped lmn-1(RNAi) lethality. These adults developed into either sterile (a) or semisterile animals (b,c). The adults had very few germ cells (arrow in b) and accumulated yolk. These embryos were laid in time windows 6 to 18 h, or > 36 h after injection, when RNAi effects are generally incomplete. These animals frequently produced abnormal oocytes with vacuoles (arrows in c). Occasionally, some of these animals gave rise to normal-looking embryos, which developed normally (double-headed arrow) (c). DAPI staining of wild-type (d), sterile (e) or semisterile animals (f-h). Some animals had few or no germ cells (e), whereas others had germ cells with both condensed nuclei and normal looking nuclei, starting from the most distal region of the germ line (arrows in f,g). The region shown in (h) is a higher magnification of the distal arm of the gonad shown in f. Arrow points to a condensed nucleus. Bar in panel c represents 20 μm for panels b-g, 40 μm for panel a, and 8 μm for panel h.