The functions of Klarsicht and nuclear lamin in developmentally regulated nuclear migrations of photoreceptor cells in the Drosophila eye

Abstract

Photoreceptor nuclei in the Drosophila eye undergo developmentally regulated migrations. Nuclear migration is known to require the perinuclear protein Klarsicht, but the function of Klarsicht has been obscure. Here, we show that Klarsicht is required for connecting the microtubule organizing center (MTOC) to the nucleus. In addition, in a genetic screen for klarsicht-interacting genes, we identified Lam Dm(0), which encodes nuclear lamin. We find that, like Klarsicht, lamin is required for photoreceptor nuclear migration and for nuclear attachment to the MTOC. Moreover, perinuclear localization of Klarsicht requires lamin. We propose that nuclear migration requires linkage of the MTOC to the nucleus through an interaction between microtubules, Klarsicht, and lamin. The Klarsicht/lamin interaction provides a framework for understanding the mechanistic basis of human laminopathies.

Figure 1.

Nuclear migration in the Drosophila eye disc. A longitudinal section of a wild-type larval eye disc is diagrammed with anterior right (A) and posterior left (P). Most of the cell volume surrounds the nucleus. Nuclei of undifferentiated cells are randomly positioned in the monolayer anterior to the morphogenetic furrow (mf), which is moving anteriorly, in the direction of the arrow. In the furrow, the nuclei are basal, and posterior to the furrow, nuclei rise as cells are recruited into ommatidia. Ommatidial clusters are progressively more mature from anterior to posterior. R-cells are gray, and cone cells are white. (Adapted from Tomlinson and Ready, 1986.)

Figure 2.

Klar localization in eye discs. Shown are confocal images of eye discs double-labeled to reveal R-cell nuclei (anti-Elav; blue) and either 6Xmyc-Klar (anti-myc; green) or Futsch (mAb22C10; red), a microtubule-associated protein. (A, E, and I) Z-sections; (B-D, F-H, and J-L) progressively more basal XY-sections, whose positions correspond to the arrows in A, E, and I, respectively. (A-D) Wild-type eye discs expressing 6Xmyc-Klar in R-cells (elav>6Xmyc-klar). Klar is localized to microtubules apical to the nuclei (A-C) and is also perinuclear (D). (E-H) Lam⁸³/Df(3L)cl-h4 eye discs expressing 6Xmyc-Klar. Klar is localized to apical microtubules (E and F), but is not present in perinuclear rings in the apical R-cell nuclei (G) or the basal ones (H). (I-L) Wild-type eye discs revealing Futsch protein localization on microtubules is shown. Futsch is apical to the R-cell nuclei (I-K) and extends around the nuclei (I and L), to the basal surface of the disc (I). Size bar in L is ∼10 μm and applies to all panels.

Figure 3.

Klar colocalizes with nuclear lamin. Shown are confocal images of a single developing ommatidium from otherwise wild-type eye discs that express 6Xmyc-Klar in R-cells (elav >6Xmyc-klar). The eye discs were double-labeled with anti-Myc and anti-Lam. Size bar, ∼2 μm.

Figure 4.

Position of the MTOC and R-cell nuclei in eye discs. Confocal images of eye discs labeled to reveal R-cell nuclei (anti-Elav; blue or green) and the MTOC (anti-β-gal or anti-γ-tub; red) are shown. (A, C, E, and G) Z-sections; (B, D, F, and H) corresponding apical XY-sections, respectively. (A and B) Wild-type eye discs expressing Nod-β-gal in R-cells (elav>nod-lacZ). (C and D) klar^BX3/Df(3L)emc^E12 discs expressing Nod-β-gal in R-cells. (E and F) Wild-type eye discs. (G and H) Lam^Ari3/Df(3L)cl-h4 eye discs. The arrowheads in E and G indicate the MTOC. (Anti-γ-tub has some background membrane staining.) Size bar in H is ∼10 μm and applies to all panels.

Figure 5.

Identification and characterization of egk1 mutants. (A) Scanning electron micrographs of eyes of the genotypes indicated are shown. gk is glrs-klar. Y is the Y chromosome. The egk1 allele shown is Lam^Ari3. (B) The cross scheme used in the F1 mutagenesis screen for enhancers of the glrs-klar rough eye phenotype is shown. (C) The positions of the nonsense or frameshift mutations in each of the five homozygous viable Lam alleles isolated as egk1 mutations are shown. The allele names are at the top, and the number beneath each indicates the first amino acid affected. (The M residue of the start codon is 1.) NLS is the nuclear localization signal, and CaaX refers to the motif used to localize lamin to the cytoplasm at the inner nuclear membrane. (D) The precise nucleotide and predicted amino acid changes of the five mutant Lam alleles in C.

Figure 6.

Lamin localization in eye discs. Confocal images of eye discs double-labeled to reveal R-cell nuclei (anti-Elav; blue) and nuclear lamin (anti-Lam; red) are shown. (A and A′) Wild-type discs. (A) Lamin expression in apical nuclei; (A′) a merge of lamin and Elav. The apical nuclei in A′ that have lamin but no Elav are cone cell nuclei. The pink appearance of the R-cell nuclear lamin in A′ is due to colocalization of lamin and Elav within the nucleus. (B and B′) Lam^A25/Df(3L)cl-h4 discs. (B) Lamin expression; (B′) a merge of lamin and Elav. The pink is where lamin and Elav overlap. The purely red nuclei are of cone cells. The plane in B and B′ is more basal than in A and A′, in order to detect Lam mutant R-cell nuclei that are not as apical as in wild-type discs. Size bar in B′ is ∼10 μm.

Figure 7.

Eye phenotypes of Lam mutants. Light micrographs of apical tangential sections through adult compound eyes are shown in A, C, E, G, and I, and of sections through third instar larval eye discs immunostained with anti-Elav to label R-cell nuclei in B, D, F, H, J, and K. (A and B) Wild-type eyes and discs. The numbers in A refer to the seven R-cells in each ommatidium visible in apical sections. The R-cell nuclei are apical in B. (C and D) Lam^A25 homozygotes. In C, some ommatidia are defective. The disc in D is indistinguishable from wild-type. (E and F) Lam^A25/Df(2L)cl-h4 hemizygotes are shown. Severe eye morphology (E) defects and nuclear migration (F) defects are observed. (G and H) Lam^Ari3 homozygotes. The eye morphology (G) and nuclear migration (H) defects are more severe than in Lam^A25 hemizygotes. (I and J) Lam^Ari3/Df(2L)cl-h4 hemizygotes. The adult eye and disc defects are similar to those of Lam^Ari3 homozygotes. The red arrows in B, D, F, H, and J indicate the morphogenetic furrow. (K) A confocal image of a Lam⁴⁶⁴³ homozygous eye disc generated by mitotic recombination is shown, labeled with anti-Elav to mark R-cell nuclei, and phalloidin to mark apical and basal cell membranes. Size bar in J is ∼20 μm in all panels except B, D, and F, where it is ∼25 μm.

Figure 8.

Model for the roles of Klar and lamin in R-cell nuclear migration. A diagram showing how Klar links the nucleus to the MTOC is shown. INM and ONM are the inner and outer nuclear membranes, respectively. The two unfilled ovals indicate possible intermediate proteins that link the C-terminal KASH domain of Klar to lamin, and also link Klar to dynein. Dynein, in black, is walking in the direction of the arrow. (See text for details.)