klarsicht: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References
Gene name - klarsicht

Synonyms -

Cytological map position - 61C1--3

Function - regulation of motor proteins

Keywords - nuclear migration, transport of lipid droplets, salivary gland, membrane biogenesis, dynein complex, eye

Symbol - klar

FlyBase ID: FBgn0001316

Genetic map position - 3-0.0

Classification - KASH (Klarsicht, Anc-1, Syne-1 homology) domain

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | Entrez gene | UniGene
BIOLOGICAL OVERVIEW

Recent literature
Myat, M.M., Rashmi, R.N., Manna, D., Xu, N., Patel, U., Galiano, M., Zielinski, K., Lam, A. and Welte, M.A. (2015). Drosophila KASH-domain protein Klarsicht regulates microtubule stability and integrin receptor localization during collective cell migration. Dev Biol [Epub ahead of print]. PubMed ID: 26247519
Summary:
During collective migration of the Drosophila embryonic salivary gland, cells rearrange to form a tube of a distinct shape and size. This study reports a novel role for the Drosophila Klarsicht-Anc-Syne Homology (KASH) domain protein Klarsicht (Klar) in the regulation of microtubule (MT) stability and integrin receptor localization during salivary gland migration. In wild-type salivary glands, MTs become progressively stabilized as gland migration progresses. In embryos specifically lacking the KASH domain containing isoforms of Klar, salivary gland cells fail to rearrange and migrate, and these defects are accompanied by decreased MT stability and altered integrin receptor localization. In muscles and photoreceptors, KASH isoforms of Klar work together with Klaroid (Koi), a SUN domain protein, to position nuclei; however, loss of Koi has no effect on salivary gland migration, suggesting that Klar controls gland migration through novel interactors. The disrupted cell rearrangement and integrin localization observed in klar mutants could be mimicked by overexpressing Spastin (Spas), a MT severing protein, in otherwise wild-type salivary glands. In turn, promoting MT stability by reducing spas gene dosage in klar mutant embryos rescues the integrin localization, cell rearrangement and gland migration defects. Klar genetically interacts with the Rho1 small GTPase in salivary gland migration and is required for the subcellular localization of Rho1. It was also shown that Klar binds tubulin directly in vitro. These results provide the first evidence that a KASH-domain protein regulates the MT cytoskeleton and integrin localization during collective cell migration.

Christophorou, N., Rubin, T., Bonnet, I., Piolot, T., Arnaud, M. and Huynh, J.R. (2015). Microtubule-driven nuclear rotations promote meiotic chromosome dynamics. Nat Cell Biol [Epub ahead of print]. PubMed ID: 26458247
Summary:
At the onset of meiosis, each chromosome needs to find its homologue and pair to ensure proper segregation. In Drosophila, pairing occurs during the mitotic cycles preceding meiosis. This study shows that germ cell nuclei undergo marked movements during this developmental window. It was demonstrated that microtubules and Dynein drive nuclear rotations and are required for centromere pairing and clustering. It was further found that Klaroid (SUN) and Klarsicht (KASH) co-localize with centromeres at the nuclear envelope and are required for proper chromosome motions and pairing. Mud (NuMA in vertebrates) was identified as co-localizing with centromeres, Klarsicht and Klaroid. Mud is also required to maintain the integrity of the nuclear envelope and for the correct assembly of the synaptonemal complex. These findings reveal a mechanism for chromosome pairing in Drosophila, and indicate that microtubules, centrosomes and associated proteins play a crucial role in the dynamic organization of chromosomes inside the nucleus.

The Klarsicht (Klar) protein is a crucial factor in the regulation of bidirectional transport of lipid droplets (Welte, 1998). Lipid droplets in early embryos move bidirectionally along microtubules, and the balance of plus- and minus-end travel distances changes twice over a 2-h period, resulting in switches in the direction of net transport. In the absence of Klar, travel distances, travel velocities, and stall forces are greatly reduced, for both plus- and minus-end travel. Without Klar the motors for plus- and minus-end motion are active indiscriminately, engaging in a tug-of-war (Welte, 1998). Thus, Klar seems to be central for understanding how the activity of opposite-polarity motors is coordinated during bidirectional transport. In this system, Klar controls the minus-end motor cytoplasmic dynein (Gross, 2000; Gross, 2002) and an as yet unknown plus-end motor (Guo, 2005 and references therein).

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 was 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; Guo, 2005 and references therein).

Klar is not only a central player in the mechanism of bidirectional transport of lipid droplets but also it controls motor activity for at least two other transport processes. In differentiating photoreceptors (R-cells) of wild-type animals, nuclei migrate first basally and then apically; in the absence of Klar, this switch in direction fails to occur, resulting in mispositioned nuclei (Fischer-Vize, 1994; Welte, 1998). In embryonic salivary glands, Klar controls minus-end-directed transport of secretory vesicles and thus modulates the growth of the apical membrane (Myat, 2002). Whether nuclei in photoreceptors and vesicles in salivary glands move bidirectionally like lipid droplets is not known, but both the minus-end motor dynein and the plus-end motor kinesin I are important for the correct positioning of photoreceptor nuclei. Whether Klar controls the motion of additional cargoes has not been determined (Guo, 2005 and references therein).

In its role in photoreceptor nuclear migration, Klarsicht is required for connecting the microtubule organizing center (MTOC) to the nucleus. In addition, in a genetic screen for klarsicht-interacting genes, Lam Dm0, which encodes nuclear lamin, was found. Like Klarsicht, lamin is required for photoreceptor nuclear migration and for nuclear attachment to the MTOC. Moreover, perinuclear localization of Klarsicht requires lamin. It is proposed that nuclear migration requires linkage of the MTOC to the nucleus through an interaction between microtubules, Klarsicht, and lamin (Patterson, 2004).

Nuclear migration in the developing eye is critical for shaping each individual cell and thus for normal morphology of the entire compound eye. The Drosophila compound eye develops within the larval eye imaginal disc, an epithelial monolayer. Within the eye disc, the morphogenetic furrow marks the initiation of eye assembly. Rows of identical facets, or ommatidia, assemble posterior to the furrow, starting with the eight photoreceptors (R-cells), followed by the lens-secreting cone cells, and finally the pigment cells. Anterior to the furrow, cells are undifferentiated and their nuclei are positioned randomly within the monolayer. The nuclei dive basally within the furrow and posterior to the furrow, migrate apically as they are recruited into ommatidia (Patterson, 2004 and references therein).

Two Drosophila genes, klarsicht (previously known as marbles) and Glued, are essential for the apical migration of nuclei in differentiating R-cells (Fischer-Vize, 1994; Fan, 1997). Glued encodes the large subunit of dynactin, a protein complex that regulates the minus-end-directed microtubule motor dynein. The requirement for dynactin suggests that R-cell nuclear migration is a dynein- and microtubule-dependent process. Consistent with this idea, two other Drosophila genes, Bicaudal-D and Lis1, both of which may regulate dynein, are implicated in R-cell nuclear migration, although their mutant phenotypes are weak compared with klarsicht and Glued. Lis-1, a WD40 repeat protein, is the homolog of the human disease gene Lissencephaly-1. Lissencephaly, or smooth brain, is a disorder resulting from defects in neuronal migrations essential for normal human brain development. Neuronal migration requires nuclear migration, and the involvement of Lis-1 in Drosophila R-cell nuclear migration suggests that the two processes may be in part analogous. It is now clear that a connection between the MTOC and the nucleus is necessary for nuclear migration and that this connection is mediated by Klar and nuclear lamin. In addition to suggesting a specific role for Klar in nuclear migration, the results propose a general mechanistic explanation for the cytoplasmic effects of nuclear lamin, including human laminopathies (Patterson, 2004).

To understand the role of Klar in R-cell nuclear migration, Klar subcellular localization and the position of the MTOC was investigated in klar mutant eye discs. In addition, genetics was used to identify nuclear lamin, which functions in the same pathway with Klar. Klar was found to be perinuclear and associated with microtubules apical to the nucleus. In addition, in klar and Lam mutant discs, MTOCs form normally in R-cells, but are often not associated with the nucleus as they are in wild-type eyes. Finally, Lam+ was found to be required for Klar localization to the nuclear membrane. These observations, taken together with previous results, suggest a model for the function of Klar in nuclear migration where Klar, held in the nuclear envelope by nuclear lamin, links the nucleus to the MTOC (Patterson, 2004).

The interaction between Klar and lamin may be indirect, but it is likely to be specific, rather than a generalized failure of nuclear envelope assembly in Lam mutants. Although most R-cell nuclei fail to migrate apically even in weak, viable Lam mutants, >90% of nuclear envelopes are intact even in stronger, lethal Lam mutants (Patterson, 2004 and references therein).

It is proposed that one or more proteins may form a bridge between the KASH domain of Klar, present in the outer nuclear membrane, and nuclear lamin, in the inner nuclear envelope. The observation that in addition to its perinuclear localization, Klar is cytoplasmic (on apical microtubules) supports the idea that Klar is in the outer, as opposed to the inner, nuclear membrane. Similarly, C. elegans Anc-1 is present in the cytoplasm as well as the nuclear membrane, and a model has been proposed where the Anc-1 KASH domain is held in the outer nuclear membrane by an inner nuclear membrane protein, Unc-84 (Malone, 1999; Starr, 2002). Although nuclear lamin has not been shown directly to be required for Anc-1 nuclear membrane localization, nuclear envelope localization of Unc-84 requires lamin (Lee, 2002). For Syne-1, the vertebrate homolog of Anc-1, experiments where the detergent digitonin was used to allow antibody access to the outer but not the inner nuclear membrane provide direct evidence that the KASH domain is in the outer nuclear membrane (Zhen, 2002). There is, however, some conflicting data (Zhang, 2001; Mislow, 2001; Mislow, 2002; Patterson, 2004 and references therein).

It is speculated that the N-terminal portion of Klar is linked to microtubules by dynein. At present, it is not possible to test for colocalization of Klar and dynein because there are no available reagents that allow detection of dynein or dynactin in the eye disc. Nevertheless, there is much evidence to support an essential role for dynein in R-cell nuclear migration and Klar function. Dynactin, a regulator of dynein, is essential for R-cell nuclear migration in the eye; mutants in the p150 dynactin subunit (Glued) have a phenotype similar to that of klar mutants in the eye disc. In addition, dynein linkage could explain why Klar is localized to microtubules only apical to the nucleus; Klar that escapes the hold of the nuclear envelope, still attached to dynein, could walk along microtubules to the MTOC. Finally, Klar has been implicated as a regulator of dynein in Drosophila embryos (Welte, 1998). In addition to its role in R-cell nuclear migration, Klar is required for developmentally regulated migration of lipid storage vesicles during embryogenesis. Lipid droplets at the center of the cellular blastoderm embryo normally migrate cortically during gastrulation. In embryos from klar mutant mothers, the lipid droplets fail to migrate. A variety of data support a model where dynein transports the lipid droplets along microtubules, whose minus ends are at the cell periphery. The results of biophysical experiments has led to a model where Klar may attach the appropriate types of motor to lipid droplets, control the number of actively engaged motors on a droplet, or coordinate the activities of kinesins and dyneins bound simultaneously to the same droplet (Jackle, 1998; Welte, 1998; Gross, 2000). Notably, dynein is required for nuclear attachment to centrosomes (the MTOCs) during mitosis in the Drosophila embryo. Klar, however, is not essential for this process (Fischer-Vize, 1994; Patterson, 2004 and references therein).

The observation that the MTOC is normally apical to the R-cell nuclei, at the leading edge of nuclear movement, suggests that a force pulls on the MTOC from above. It is speculated that the mechanism for this force could be analogous to the means by which the nucleus of budding yeast are pulled into the bud neck before cell division. One pathway for migration of the nucleus into the bud neck involves dynein, anchored at the cell cortex to which the nucleus is moving. Cortically tethered dynein 'reels in' the nucleus by walking along microtubules whose plus ends are at the cortex, toward the MTOC, which is anchored to the nucleus. In support of this idea, microtubule plus-ends are present apically in R-cells (Mosley-Bishop, 1999), and dynactin is essential for R-cell nuclear migration (Patterson, 2004 and references therein).

Whether a force emanating from the apical membrane pulling on the MTOC would drive nuclear migration or serve as an anchor after the nucleus has migrated depends on where the MTOC initially forms. The gamma-tubulin antibody detects MTOCs only apically in differentiating cells. Transiently basal MTOCs associated with nuclei that are about to rise could have escaped detection. However, if the MTOC does form apically, then the force that drives nuclear migration would come from below the nucleus, that is, dynein, linked to the nuclear membrane by Klar and lamin, walking on microtubules up toward the MTOC (Patterson, 2004).

The model proposed whereby Klar forms a bridge between nuclear lamin in the inner nuclear membrane and cytoplasmic microtubules provides a general framework for explaining how nuclear lamin affects cytoplasmic events. Drosophila Lam mutations result in D/V polarity defects in eggs, and tracheal branching defects in embryos. Moreover, a variety of human diseases are the result of mutations in the LMNA gene, which encodes lamin A. The Drosophila Lam Dm0 gene encodes type B lamin, whereas the Drosophila LamC gene encodes lamin C, which is most similar to human lamin A. The A/C- and B-type lamins are similar proteins, with some different structural features, and some expression pattern differences. LMNA-associated human diseases affect the heart, skeletal muscles, and the nervous system (Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, cardiomyopathy, and Charcot-Marie-Tooth disorder), and metabolism (Dunnigan-type lipodystrophy). The two main hypotheses as to how nuclear lamin defects can result in these disease phenotypes are that the mutations either result in nuclear envelope fragility or result in changes in gene expression. An alternative hypothesis is that the inner nuclear envelope interacts with the cytoplasm through proteins like Klar or Anc-1/Syne-1, which connect the inner nuclear envelope to the microtubule, or actin cytoskeletons, respectively (Patterson, 2004 and references therein).


GENE STRUCTURE

The single klar gene gives rise to at least three messages and three Klar protein isoforms. A proximal promoter before exon 0 and a distal promoter before exon G are ~80 kb apart. The originally published klar cDNA (exon 0-18) encodes isoform alpha that can partially rescue the klar nuclear migration defect in photoreceptors (Mosley-Bishop, 1999). The message for the droplet-specific isoform beta likely starts at the proximal promoter and ends with exon 15X that encodes the LD domain. Isoform gamma prominent in ovaries is encoded by a message transcribed from the distal promoter. No gross defects have been detected in ovaries when this isoform is disrupted by class II alleles. Other isoforms may exist. A comprehensive description of all Klar isoforms and of their expression pattern will be important for understanding the full role Klar plays in transport regulation (Guo, 2005).

cDNA clone length - 8312 bp

Bases in 5' UTR - 662

Exons - 18

Bases in 3' UTR - 861

PROTEIN STRUCTURE

Amino Acids - 2262

Structural Domains

klarsicht encodes a large protein, unique except for its small N-terminal KASH (Klarsicht, Anc-1, Syne-1 homology) domain, which localizes proteins to the nuclear membrane (Mosley-Bishop, 1999; Apel, 2000; Zhang, 2001; Starr, 2002; Zhen, 2002). The KASH-domain-containing protein Anc-1 and its vertebrate homolog, Syne-1 (also known as Myne-1, Nesprin, and NUANCE) are dystrophin-related proteins that anchor the nucleus to the actin cytoskeleton (Apel, 2000; Mislow, 2001: Mislow, 2002; Zhang, 2001; Starr, 2002; Zhen, 2002; Starr, 2003). In addition to its role in nuclear migration in the eye, klar is required for the developmentally regulated migrations of lipid droplets during embryogenesis. In this role, it has been proposed that Klar regulates dynein and also the plus-end-directed motor kinesin (Patterson, 2004 and references therein).

Comparing the klar genomic region and the reported klar cDNA (Mosley-Bishop, 1999) predicts 19 exons (exon 0 to exon 18). The only obviously conserved region in the predicted Klar protein (~250 kDa) is the C-terminal 60-amino acid Klarsicht, ANC-1, Syne-1 homology (KASH) domain (Starr, 2002). KASH domains are present in many proteins, including the actin-binding proteins ANC-1 and Syne-1, and the dystrophin-related Msp300. ANC-1 and Syne-1 are perinuclear and tether nuclei to the actin cytoskeleton (Apel, 2000; Starr, 2002). For Syne-1, the KASH domain is both necessary and sufficient to target the protein to the nuclear envelope (Zhang, 2001). A Myc-tagged Klar protein expressed ectopically in photoreceptors is also perinuclear (Mosley-Bishop, 1999), but the localization of endogenous Klar is unknown, in photoreceptors or any other tissue (Guo, 2005).


klarsicht: Regulation | Developmental Biology | Effects of Mutation | References

date revised: 20 June 2005

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