A comparative study of Drosophila and human A-type lamins

Abstract

Nuclear intermediate filament proteins, called lamins, form a meshwork that lines the inner surface of the nuclear envelope. Lamins contain three domains: an N-terminal head, a central rod and a C-terminal tail domain possessing an Ig-fold structural motif. Lamins are classified as either A- or B-type based on structure and expression pattern. The Drosophila genome possesses two genes encoding lamins, Lamin C and lamin Dm(0), which have been designated A- and B-type, respectively, based on their expression profile and structural features. In humans, mutations in the gene encoding A-type lamins are associated with a spectrum of predominantly tissue-specific diseases known as laminopathies. Linking the disease phenotypes to cellular functions of lamins has been a major challenge. Drosophila is being used as a model system to identify the roles of lamins in development. Towards this end, we performed a comparative study of Drosophila and human A-type lamins. Analysis of transgenic flies showed that human lamins localize predictably within the Drosophila nucleus. Consistent with this finding, yeast two-hybrid data demonstrated conservation of partner-protein interactions. Drosophila lacking A-type lamin show nuclear envelope defects similar to those observed with human laminopathies. Expression of mutant forms of the A-type Drosophila lamin modeled after human disease-causing amino acid substitutions revealed an essential role for the N-terminal head and the Ig-fold in larval muscle tissue. This tissue-restricted sensitivity suggests a conserved role for lamins in muscle biology. In conclusion, we show that (1) localization of A-type lamins and protein-partner interactions are conserved between Drosophila and humans, (2) loss of the Drosophila A-type lamin causes nuclear defects and (3) muscle tissue is sensitive to the expression of mutant forms of A-type lamin modeled after those causing disease in humans. These studies provide new insights on the role of lamins in nuclear biology and support Drosophila as a model for studies of human laminopathies involving muscle dysfunction.

Figure 1. Localization of human nuclear envelope proteins in Drosophila.

Polytene nuclei from salivary glands (top row z-series; middle row section) and diploid cells from imaginal discs (bottom row) were obtained from transgenic stocks and stained with antibodies specific for the human nuclear envelope proteins. All of the human proteins localized to the Drosophila nuclear envelope, with Lamin B2 showing aggregation.

Figure 2. Yeast two-hybrid analysis of nuclear protein interactions.

Interactions between human and Drosophila nuclear envelope proteins were tested using yeast two-hybrid analysis. Growth on selective media due to expression of the reporter gene indicates an interaction between the two proteins. + indicates growth; - indicates little to no growth.

Figure 3. Distribution of nuclear proteins in Lamin C nulls.

Polytene salivary glands and diploid imaginal disc tissue from wild type larvae and Lamin C nulls were stained with antibodies to nuclear envelope associated proteins, lamin Dm₀, Bocksbeutel and nuclear pores. Heterochromatin organization was determined by staining with antibodies against HP1. An enlarged version of the nuclear pore staining is shown to highlight the peripheral foci that appear in the Lamin C null, indicative of nuclear pore clustering.

Figure 4. Nuclear defects associated with loss of Lamin C.

EM images of nuclei from imaginal discs of second instar larvae. A typical image of a nucleus from a wild type stock (y,w^67c23) surrounded by intact nuclear envelope. Nuclei isolated the Lamin C null larvae show separation of the inner and outer membrane (small arrows) and large disruptions in the envelope (large arrows) that allow for chromatin leakage.

Figure 5. Nuclear and cytoplasmic defects in the larval body wall muscle of Lamin C nulls.

(A) Confocal images of dissected and fixed larval body wall muscle stained with antibodies to lamin Dm₀ (green), phalloidin (magenta) and DAPI (blue). (B) Confocal image of a muscle cell nuclei stained with antibodies to Lamin C (green) and phalloidin (magenta). Note the intense filamentous phalloidin staining within the nucleus. Other staining results from actin in the underlying contractile apparatus below the nucleus. (C) Single confocal slice of a muscle nuclei using 3D opacity to visualize lamin Dm₀ (green) and phalloidin (magenta). Lamin Dm₀ stains the nuclear periphery; phalloidin stains actin within contractile apparatus below the nucleus and filaments within the nucleus. (D) EM images of nuclei from wild type and Lamin C null larval body wall muscle. The boxed insert shows an enlarged cluster of circular particles within the nucleus of Lamin C nulls.

Figure 6. Diagram of mutant forms of Lamin C expressed in transgenic flies.

Top shows a diagram of the structure of human LMNA with disease-causing amino acid substitutions indicated. Bottom shows diagrams of the domain structure of Drosophila Lamin C and the mutant forms generated for analyses.

Figure 7. Levels of mutant forms of Lamin C expressed in larvae.

(A) Expression of mutant forms of Lamin C under control of three different Gal4-UAS drivers. The top shows a representative western of protein extract from third instar larvae containing the Mef2 or Act5 driver in combination with a transgene encoding NI95K, W520S or the N-terminal truncation (ΔN). Extract from the y,w67c23 host injection stock is used for comparison of endogenous Lamin C (end.). The bottom shows a graphical representation of quantitative analysis of westerns performed on three independently generated protein extracts for each genotype. The average value is plotted with error bars representing standard error of the mean. These results demonstrate that lethality caused by the N-terminal deletion and W557S is not a general result of over-expression. (B) Expression of W557S under control of five different drivers. The top representative western analysis of protein extracts from third instar larvae expressing a transgene encoding the W557S mutant form of Lamin C under the control of various Gal4 drivers. The bottom represents graphical representation of quantitative analysis of westerns performed on three independently generated protein extracts from larvae possessing the W557S expressing transgene and various drivers. The average value is plotted with standard error of the mean. These results indicate that the semi-lethal phenotype associated with W557S when expressed ubiquitiously or specifically in muscle does not correlate with levels of expression.

Figure 8. Nuclear localization of the mutant forms of Lamin C and lamin Dm₀.

Salivary gland nuclei from larvae expressing wild type or mutant forms of Lamin C under control of a hsp70 promoter were fixed and stained with antibodies to Lamin C (white), lamin Dm₀ (red) and TOPRO that detects DNA (blue).