Drosophila klarsicht has distinct subcellular localization domains for nuclear envelope and microtubule localization in the eye

Abstract

The Drosophila klarsicht (klar) gene is required for developmentally regulated migrations of photoreceptor cell nuclei in the eye. klar encodes a large ( approximately 250 kD) protein with only one recognizable amino acid sequence motif, a KASH (Klar, Anc-1, Syne-1 homology) domain, at its C terminus. It has been proposed that Klar facilitates nuclear migration by linking the nucleus to the microtubule organizing center (MTOC). Here we perform genetic and immunohistochemical experiments that provide a critical test of this model. We analyze mutants in the endogenous klar gene and also flies that express deleted forms of Klar protein from transgenes. We find that the KASH domain of Klar is critical for perinuclear localization and for function. In addition, we find that the N-terminal portion of Klar is also important for function and contains a domain that localizes the protein to microtubules apical to the nucleus. These results provide strong support for a model in which Klar links the nucleus to the MTOC.

Figure 1.—

KASH domain sequence comparisons. The KASH domain amino acid sequences of Klarsicht in fruit flies (Drosophila melanogaster) and mosquito (Anopheles gambiae) are aligned with the KASH domains of Msp-300, Anc-1, and Syne-1, which are homologs in fruit flies and mosquitoes, nematodes (C. elegans), and mice (Mus musculus). The shaded areas indicate transmembrane domains, and the boxed amino acids are identical in all KASH domains. The KASH domains constitute the final amino acids of their respective proteins. Accession numbers for the sequences are as follows: Dm Klarsicht, AAD43129 (Mosley-Bishop et al. 1999); Ag Klarsicht, XP_310059.1 (Anopheles Genome Sequence Consortium, 2003); Dm Msp-300, NP_723065 (Adams et al. 2000); Ag Msp-300, XP_310133 (Anopheles Genome Sequence Consortium, 2003); Ce Anc-1, NP_491353 (Starr and Han 2002); and Mm Syne-1, AAG24393 (Apel et al. 2000).

Figure 2.—

Diagram of the model for Klar function in R-cell nuclear migration. The large hatched bar is Klarsicht, the black rods are lamin, and the dark gray oval is the MTOC. The light gray oval and circle are hypothetical proteins that link Klarsicht with lamin and dynein. The black oval with the legs is dynein and the arrow indicates the direction of its movement, toward the MTOC. Plus and minus indicate plus and minus ends of microtubules.

Figure 3.—

(A) klar mutant allele sequences. The positions of the putative truncations in Klar protein resulting from the DNA lesions in six different mutant klar alleles are shown. Full-length Klar protein is 2262 amino acids (Mosley-Bishop et al. 1999). The solid box at the C terminus is the KASH domain. The numbers adjacent to the DNA sequences affected by the mutations indicate the nucleotide number of the cDNA, where number 1 is the A of the ATG start codon. (B) RNA blots of klar transcripts. Shown is a blot of eye disc total nucleic acid from wild-type (wt) larvae and from larvae homozygous for each of the klar mutant alleles indicated. klar mRNA and 18S rRNA were detected sequentially. The klar band corresponds to at least two mRNAs of similar size (∼8.0–8.5 kb; Mosley-Bishop et al. 1999).

Figure 4.—

klar^CD4 has little or no klar⁺ function. External adult eyes (A, D, G, J), light microscope images of adult apical retinal sections (B, E, H, K), and confocal microscope images of larval eye discs (C, F, I, L) are shown. The genotypes indicated in C, F, I, and L apply to the entire horizontal row. The black numbers in B indicate the seven R cells (R1–7) visible in apical sections of the retina. Eye color differences in A, D, G, J are due to eye color mutations in the backgrounds, not to the klar alleles. The larval eye discs are labeled with anti-Elav to highlight R-cell nuclei (blue) and phalloidin to highlight cell membranes (red). Five eyes of each genotype were observed and representative data are shown.

Figure 5.—

Proteins expressed by klar transgenes. (A) The structures of the complete and partial Klar proteins expressed by transgenes are shown. The hatched bars are 6xmyc epitope tags, and the dark gray bars are KASH domains. The numbers indicate the first and final amino acid of Klar present in each construct. (B) Western blots of larval eye disc extracts showing 6mKlar proteins expressed by three different UAS transgenes (left) and three different GLRS transgenes (right). Two lines of each of the GLRS transgenes are shown and the line numbers are indicated above each lane. Blots were probed with anti-Myc and anti-β-tubulin. 6mKlarFL is predicted to be ∼258 kD, 6mKlar3′ΔS ∼204 kD, and 6mKlar5′ΔA ∼53kD. 6mKlar5′ΔA appears larger than its predicted size. Perhaps the KASH-domain-containing Klar fragment is modified in vivo.

Figure 6.—

Localization of 6mKlar proteins in wild-type eye discs. (A) A diagram of a Z section through a developing eye disc is shown. The morphogenetic furrow (mf) is moving in the direction of the arrow. A, anterior; P, posterior. Photoreceptor cells are shaded gray; their nuclei and most of the cell cytoplasm migrate apically as the cells differentiate. The unshaded apical cell bodies are cone cells. Purple arrows at the left mark the XY planes of the confocal images indicated (after Tomlinson and Ready 1986). (B–J) Confocal images of eye discs that express the 6mKlar transgene indicated in R cells are shown, labeled with anti-Elav to highlight R-cell nuclei (purple) and anti-myc to reveal 6mKlar proteins. B, E, and H are Z sections, and C, D, F, G, I, J are XY sections. The 6mKlar protein apical to the nuclei is thought to be associated with microtubules because it closely resembles the localization pattern of the microtubule-associated protein Futsch (Patterson et al. 2004).

Figure 7.—

Complementation of klar^CD4 by glrs-6mklar transgenes. External adult eyes (A, D, and G), light micrographs of adult retinal sections (B, E, H), and confocal images of larval eye discs (C, F, and I) are shown. Eye discs in C, F, I are labeled with anti-Elav to highlight R cells (blue) and phalloidin to mark cell membranes (red). (A, B, C) Two copies of a glrs-6mklarFL transgene in a klar^mCD4 background. (D, E, F) Two copies of a glrs-6mklar5′ΔA transgene in a klar^mCD4 background. (G, H, I) Two copies of a glrs-6mklar3′ΔS transgene in a klar^mCD4 background. Five eyes of each genotype were observed and representative data are shown.