Molecular genetic analysis of the nested Drosophila melanogaster lamin C gene

Abstract

Lamins are intermediate filaments that line the inner surface of the nuclear envelope, providing structural support and making contacts with chromatin. There are two types of lamins, A- and B-types, which differ in structure and expression. Drosophila possesses both lamin types, encoded by the LamC (A-type) and lamin Dm0 (B-type) genes. LamC is nested within an intron of the essential gene ttv. We demonstrate that null mutations in LamC are lethal, and expression of a wild-type LamC transgene rescues lethality of LamC but not ttv mutants. Mutations in the human A-type lamin gene lead to diseases called laminopathies. To determine if Drosophila might serve as a useful model to study lamin biology and disease mechanisms, we generated transgenic flies expressing mutant LamC proteins modeled after human disease-causing lamins. These transgenic animals display a nuclear lamin aggregation phenotype remarkably similar to that observed when human mutant A-type lamins are expressed in mammalian cells. LamC aggregates also cause disorganization of lamin Dm0, indicating interdependence of both lamin types for proper lamina assembly. Taken together, these data provide the first detailed genetic analysis of the LamC gene and support using Drosophila as a model to study the role of lamins in disease.

Figure 1.

(A) Diagram of the LamC and ttv genomic region. LamC (solid exons) is nested within the fifth intron of the essential gene tout velu (open exons). EP(2)2199, UM-8373, and G00158 are lethal P-element insertions in Lamin C. ttv⁰⁰⁶⁸¹ is a lethal insertion into the sixth intron of tout velu. (B) Diagram of LamC with the location of the P-element insert in stock G00158 indicated. G00158 is mutant for LamC and ttv. The diagram shows the position of the primers (arrows) used for PCR and sequencing (Table 3). (C) Southern analysis of LamC alleles. Bsu36I cleavage sites flank the LamC genomic region and generate a 7.2-kb fragment containing the wild-type LamC gene. The membrane was hybridized with full-length LamC cDNA.

Figure 2.

Western analysis of protein from LamC mutants used in rescue experiments. Western analysis of heterozygous adults carrying one mutant allele for LamC over the CyO balancer chromosome is shown. Protein extracts from a wild-type stock (y¹w^67c23) and heterozygous ttv⁰⁰⁶⁸¹ adults are also shown. LamC was detected with the antibody LC28.26 (Riemer et al. 1995). Quantitation and P-values (Student's t-test) for LamC levels from three independent samples per genotype are indicated on the histogram, and representative Western blots are depicted below. Values are normalized using the levels of α-tubulin and expressed as a ratio to the levels of LamC in the wild type, set at 1.0 (see materials and methods).

Figure 3.

Three LamC excision alleles appear to be protein nulls. Larval protein extracts were isolated from LamC excision alleles or the Df(2R)trix over the CyO-GFP chromosome. GFP⁻ samples represent LamC^EX/LamC^EX or LamC^EX/Df(2R)trix); GFP⁺ samples represent LamC^EX/CyO-GFP. Experimental triplicates are shown.

Figure 4.

Diagram of the mutant forms of LamC with principal domains labeled. NLS represents a putative nuclear localization signal. The top of the diagram shows the location of the P-element-encoded GFP insertion (present in stock G00158) and of the R401K amino acid substitution. The bottom of the diagram shows the extent of the N-terminal deletion; the first 48 amino acids are deleted, which removes the entire head domain and 8 amino acids from the first part of the rod domain.

Figure 5.

Nuclear defects associated with expression of mutant forms of LamC. (A) Tissues (salivary gland and epithelial) from second instar larvae stained with antibodies to LamC (red). Nuclei are indicated by DAPI staining (blue). (B) Salivary gland nuclei from third instar larvae stained with antibodies to LamC (red). (C) Salivary gland nuclei from third instar larvae stained with antibodies to lamin Dm₀ (red). Arrows indicate representative examples of nuclear defects (LamC O-ring aggregates and lam Dm₀ protrusions). Images in A were photographed using a 10× ocular and a 100× oil objective on a Leica DMLB microscope equipped with a Spot RT-Slider CCD camera (Diagnostic Instruments). B and C were photographed the same way, except using a 40× oil objective rather than the 100× objective.

Figure 6.

Western analysis of LamC levels in transgenic stocks used in rescue experiments and nuclear morphology assays. Protein extracts from a wild-type stock (y¹w^67c23) were used for normalization. Quantitation was carried out as described in Figure 3 and materials and methods.