Surprisingly, perinuclear Klar-C signal is detected in ovaries of flies homozygous for the promoter deletion allele klarYG3. This observation suggests that cells might generate Klar variants not only by alternative splicing but also by using different promoters. To map the approximate position of the promoter responsible for Klar expression in ovaries, Klar-C signal was examined for all klar alleles. Because all class I alleles displayed perinuclear signal, this promoter must be located 3' to exon 14 (the location of the chromosomal break in klarmBX3). Certain cDNA clones from BDGP (e.g., GH05536 and GM02433) start with a small, alternative exon (G) located within intron 15. Promoter prediction programs indicate a high probability for a promoter 130 bases 5' to G, and the open reading frame in G is highly conserved in other Drosophila species. It is suggested that a klar message starting with exon G gives rise to the perinuclear Klar isoform in ovaries (Guo, 2005).
Western analysis with Klar-C detected a protein of apparent 75 kDa in wild-type and class I ovaries, consistent with a Klar protein of 62 kDa if cDNA GH05536 is translated. In alleles klarmBX12 and klarmBX13, no corresponding band was detectable, and klarmCD4 ovaries had a band of slightly lower molecular weight, consistent with the lack of the KASH domain predicted from DNA sequencing (Guo, 2005).
Microtubule-based transport in cells is powered by a small set of distinct motors, yet timing and destination of transport can be controlled in a cargo-specific manner. The mechanistic basis for this specificity is not understood. To address this question, the Drosophila Klarsicht protein that regulates distinct microtubule-based transport processes was analyzed. Localization of Klar to its cargoes is crucial for Klar function. Using mutations, functionally important regions of Klar that confer distinct cargo specificity were identied. In ovaries, Klar is present on the nuclear envelope, a localization that requires the C-terminal KASH domain. In early embryos, Klar is attached to lipid droplets, a localization mediated by a novel C-terminal domain encoded by an alternatively spliced exon. In cultured cells, these two domains are sufficient for targeting to the correct intracellular location. This analysis disentangles Klar's modular organization: it is proposed that a core region integral to motor regulation is attached to variable domains so that the cell can target regulators with overlapping, yet distinct functions to specific cargoes. Such isoform variation may be a general strategy for adapting a common regulatory mechanism to specifically control motion and positioning of multiple organelles (Guo, 2005).
Lack of Klar also disrupts the developmental regulation of droplet transport. In the wild type, these organelles are initially distributed throughout the periphery of the embryo (syncytial blastoderm, phase I), constantly moving back and forth along microtubules. At the beginning of cellularization (phase II), plus-end travel distances are up-regulated, causing net inward motion; the droplets accumulate basally, near microtubule plus-ends. One hour later (gastrulation, phase III), plus-end travel lengths decrease and droplets redistribute outward, apically. In embryos from klar mutant females (referred to as "klar embryos"), this switch from net inward to net outward motion in phase III fails to occur because the balance of plus- and minus-end motion does not change correctly. Based on these phenotypic analyses, it has been proposed that Klar may form a complex between the plus- and minus-end motors, controlling the response to transacting signals and coordinating motor activity (Welte, 1998). Therefore it was asked whether localization of Klar to lipid droplets is important for its function. To address this question, the effects were compared of 13 mutant klar alleles that had been isolated in several independent genetic screens. It was determined whether the mutant Klar proteins could support normal droplet motion and whether they were able to localize correctly (Guo, 2005).
Bulk movement of droplets can be assessed by changes in embryo transparency. In the wild type, net inward transport in phase II gives rise to a clear periphery, and net outward movement in phase III turns the periphery opaque. For all klar alleles tested, droplets accumulated basally in phase II. For 10 of the alleles, the mutant embryos had a transparent periphery in phase III because lipid droplets remained basally, around the yolk sac. These alleles with aberrant droplet transport will be referred to as class I. For the remaining three alleles (class II), lipid droplets spread apically in phase III, resulting in an opaque periphery just as in the wild type. Thus, class II alleles support normal net droplet transport, whereas class I alleles do not (Guo, 2005).
Immunolocalization revealed that all 10 class I alleles had aberrant Klar staining: Klar dots were either evenly distributed in the cytoplasm (klarmBX3 and klar1 or not detectable (klarmFN1, klarD, and klarB). For the class II alleles (klarmBX12), Klar dots accumulated basally as in the wild type. These results suggest that Klar localizes to lipid droplets in class II, but not in class I alleles, and that if Klar fails to localize to lipid droplets, it cannot carry out its regulatory function (Guo, 2005).
To confirm this conclusion, it was determined how centrifugation affected Klar distribution. For the class II allele klarmBX12, Klar was highly enriched in the droplet layer, just as for the wild type. The Klar dots present in class I alleles klarmBX3 and klar1 were not recruited to the droplet layer, but they were instead broadly distributed throughout other regions of the embryo. Lack of signal in klarB embryos demonstrated the specificity of Klar staining. Thus, Klar association with lipid droplets is disrupted in class I alleles and maintained in class II alleles (Guo, 2005).
Because Klar proteins encoded by class I alleles fail to localize correctly, the molecular lesions in these alleles should give insight into the mechanism that controls Klar localization. For those alleles in which no distinct Klar dots were detectable, the proteins might simply not be expressed. Alternatively, the mutant proteins might be present, but diffusely throughout the cytoplasm, not concentrated in distinct locations. To distinguish between these possibilities, Klar expression was monitored in early embryos by Western analysis. In the wild-type and for class II alleles, Klar-M detected several high-molecular-weight bands, with a major band above 250 kDa. In class I mutants, this Klar band was absent (klarmBP, klarmBX18, klarB, and klarYG3), shifted to lower molecular weight (klar1, klarD, and klarmFN1), or grossly increased in intensity (klarmBX3). Several of the alleles (klarD and klarmFN1) in which immunostaining did not reveal localization still expressed protein. These findings were corroborated by Western analysis with Klar-N. This antibody detected the same major form of Klar above 250 kDa, and the apparent size of this major Klar band changed similarly in various mutants. The fact that both antibodies recognized multiple Klar-specific bands (since they are all absent in several of the mutant klar alleles) might indicate that embryos express several distinct forms of Klar (Guo, 2005).
Guided by the apparent size of proteins on Westerns and by previous rough mapping of the chromosomal breakpoints in some alleles (Mosley-Bishop, 1999), candidate sections of the klar genomic region for the mutant alleles were sequenced to identify the underlying molecular lesions. Allele klarYG3 had a deletion of the putative promoter region. In all other cases, lesions were identified (nonsense mutations or chromosomal breaks) predicted to result in C-terminally truncated Klar proteins (Guo, 2005).
Class II alleles have lesions in exon 18; they are wild-type in Klar distribution and net droplet transport. Class I alleles have more N-terminal lesions, before and within exon 14, and encode truncated proteins that neither localize correctly nor support normal transport. It is concluded that a C-terminal region of Klar (between the beginning of exon 14 and the middle of exon 18) is necessary to localize Klar to lipid droplets (Guo, 2005).
Why do lesions in exon 18 not disrupt droplet transport? One possibility is that droplets are associated with a Klar isoform that lacks exon 18. Two klar cDNAs (LD08331 and LD24441) characterized by the Berkeley Drosophila genome project (BDGP) suggest that such an isoform exists. These cDNAs have an alternative to exon 15 at their 3' ends: this exon "15X" is identical to exon 15 in its 5' sequences but contains 640 additional bases from the start of intron 15. This putative extension is referred to as 15ext (exon 15X = exon 15 + 15ext). Translation of 15X would result in an alternative C terminus of Klar, because 15ext contains an in-frame stop codon. Because both cDNAs end 17 bases downstream of a potential PolyA signal (AATAAA), they may represent the genuine 3' end of certain klar messages (Guo, 2005). Klar messages that contain exon 15X are indeed expressed. In situ hybridization with a 15ext-specific probe detected messages in wild-type embryos, but not in embryos of two klar alleles for which chromosomal breaks should prevent transcription of 15X. The sequence of the predicted translation product of 15ext is significantly conserved between fly species representing an evolutionary divergence of 250 Mya. A Klar isoform encoded by messages ending with exon 15X would not be disrupted by class II alleles and thus may be the form of Klar that regulates lipid-droplet transport (Guo, 2005).
Expression of Klar is likely even more elaborate. Both alternative splicing of other exons and use of distinct promoters contribute to the generation of multiple Klar isoforms. Whether it might be possible to recreate Klar's localization to lipid droplets was analyzed in a simpler experimental system (Guo, 2005).
It was found that Drosophila S2 cells often contain numerous conspicuous particles that specifically stained with the neutral lipid-specific dye BODIPY493/503 and therefore represent lipid droplets. S2 cell lipid droplets look perfectly round and vary greatly in size, even within a single cell, unlike in early embryos where droplets are consistently ~0.5 µm in diameter (Guo, 2005).
To test for Klar localization, a set of constructs was generated in which Klar fragments were fused in frame to red fluorescent protein (RFP). These constructs were expresssed in S2 cells and their intracellular distribution was monitored by fluorescence microscopy (Guo, 2005).
RFP by itself was present diffusely throughout the cytoplasm. In contrast, a fusion construct containing the Klar region from exon 13 to the end of 15ext localized specifically to the periphery of lipid droplets, as shown by double labeling with BODIY493/503. All RFP-fusion constructs that contained 15ext localized to lipid droplets, whereas two constructs without 15ext were present throughout the cytoplasm. In particular, the Klar fragment encoded by 15ext was sufficient to target to lipid droplets. The cell culture and embryo data combined suggest that it is this region of Klar (the lipid droplet or LD domain) that is responsible for associating Klar with lipid droplets (Guo, 2005).
If targeting via the LD domain is so exquisitely specific, how does Klar associate with other cargoes? Next to droplet transport, Klar's best-characterized function is its role in the migration of nuclei in developing photoreceptors. To identify the regions of Klar necessary for this process, it was examined how this migration was affected by the collection of klar alleles. In wild-type eye imaginal discs, photoreceptor nuclei were uniformly positioned in clusters near the apical surface, whereas in discs of klar mutant animals nuclei were missing from this focal plane and were displaced basally (Fischer-Vize, 1994; Welte, 1998). Nuclei were mispositioned for all 13 alleles tested, from both class I and class II. Thus, the functions of Klar in droplet transport and nuclear migration are overlapping, but not identical (Guo, 2005).
Nuclear migration requires in particular exon 18 sequences that are not important for droplet transport. The three class II alleles truncate the open reading frame in exon 18 before or within the KASH domain. Because KASH domains of other proteins are implicated in targeting to the nuclear envelope (Zhang, 2001), it was hypothesized that this domain targets Klar to nuclei (Guo, 2005).
By Klar-M immunostaining, Klar localization relative to nuclei was difficult to analyze because many cells had abundant Klar dots both throughout the cytoplasm and near nuclei, possibly because several different forms of Klar are present, associated with different cargoes. To specifically reveal Klar forms with exon 18 sequences, antibody Klar-C was generated, that recognizes an epitope just N-terminal to the KASH domain (Guo, 2005).
By immunostaining, Klar-C revealed striking association of Klar with nuclei in several tissues, including the larval and adult gut, larval salivary glands, and adult ovaries. To examine this perinuclear Klar localization in detail, focus was placed on ovaries because of the large size of the polytene nurse cell nuclei. No Klar-C signal was detected in early embryos, supporting the conclusion that Klar isoforms at this developmental stage lack exon 18 sequences (Guo, 2005).
In wild-type ovaries, Klar-C revealed strong perinuclear signal in nurse cells, oocytes, and follicle cells. Klar was present in or on the nuclear envelope because Klar signal colocalizes with the nuclear envelope marker wheat germ agglutinin. Klar was distributed unevenly over the nuclear envelope, enriched in multiple distinct foci (Guo, 2005).
Klar-C signal is specific because no staining was detected in two class II alleles (klarmBX12 and klarmBX13). For the class II allele klarmCD4, Klar protein was mislocalized throughout the nurse-cell and oocyte cytoplasm. This result strongly suggests that the KASH domain is necessary for localization to the nuclear envelope, since this allele has a premature stop codon just before the KASH domain. When an RFP-KASH fusion was expressed in S2 cells, the protein was enriched perinuclearly, demonstrating that this domain also is sufficient for targeting to the nuclear envelope. It is concluded that Klar association with nuclei is mediated by the KASH domain (Guo, 2005).
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