To determine where Klar protein is expressed, monoclonal antibodies (Klar-N and Klar-M) were generated that recognize epitopes near the N terminus and in the center of the protein (exon 4 and exon 9, respectively). Both antibodies detected Klar by Western analysis. Only Klar-M gave a strong signal by immunostaining. Comparison with a klar mutant showed that this signal was Klar specific. Klar was expressed in cells where it is known to be important for transport: in early embryos during droplet motion and at high levels in eye imaginal discs posterior to the morphogenetic furrow, i.e., in the region of the disc where photoreceptor nuclei migrate apically. Unexpectedly, Klar also was present in many other cells in which no function for Klar had previously been reported, including in embryos of various stages, other larval imaginal discs, larval brains, and adult ovaries. This broad expression suggests that Klar has many as yet unknown roles during Drosophila development and may control a wide range of transport processes. Thus, insight into Klar function has the potential to shed light on the regulation of diverse developmental processes (Guo, 2005).
Based on the phenotype of klar mutants, it had been proposed that Klar is present on the cargoes it regulates, physically interacting with motors and coordinating their activity (Welte, 1998). Klar is indeed expressed at the correct time to directly regulate the motors that power motion of embryonic lipid droplets and of photoreceptor nuclei. Is it also present at the correct intracellular location to serve such a role? As Klar's function is best characterized for lipid-droplet transport, it was asked where Klar localizes relative to this cargo and whether this localization was functionally important (Guo, 2005).
In wild-type embryos, Klar-M revealed a large number of distinct dots. These Klar dots are evenly distributed in the periphery of phase I embryos and undergo basal accumulation during phase II. Because this Klar distribution mimics that of lipid droplets at these stages, Klar may be associated with lipid droplets (Guo, 2005).
Embryos for Klar and for lipid droplets were preserved by using a neutral lipid-specific dye. Fixation conditions that preserved neutral lipids were suboptimal for Klar preservation, but many cases were nevertheless found in which Klar dots were adjacent to lipid droplets, consistent with the notion that Klar was associated with the droplets (Guo, 2005).
Because these colocalization data were merely suggestive, two independent strategies were used to alter the intracellular location of lipid droplets. Klar distribution should change in the same manner if Klar is indeed physically attached to the droplets. In a genetic approach, embryos deleted for the halo gene were used. In phase II halo embryos, lipid droplets accumulate apically, below the nuclei, rather than basally; no other organelles are mislocalized. In such embryos, the distribution of Klar dots was indeed shifted apically. Because apical Klar accumulation in halo embryos is not as complete as that of lipid droplets, it is proposed that a fraction of Klar is associated with other cargoes whose distribution is undisturbed. A second approach takes advantage of the low buoyant density of lipid droplets. For many animal eggs, even mild centrifugation leads to stratification of the visible material, with lipid droplets on one side of the embryo, yolk granules on the other, and nuclei just below the droplet layer. It was verified by direct staining for lipid droplets and DNA that this was also true for early Drosophila embryos. By immunostaining, Klar was highly enriched in the droplet layer, just above the nuclei. In summary, it is concluded that a major pool of Klar in early embryos is present on lipid droplets, supporting the notion that Klar is a crucial component of the postulated motor coordination complex, directly regulating the activity of the attached motors (Guo, 2005).
Morphogenesis of a multicellular structure requires not only that cells are specified to express particular gene products, but also that cells move to adopt characteristic shapes and positions. Little is known about how these two aspects of morphogenesis are coordinated. The developing Drosophila compound eye is a monolayer, in which cells are suspended between apical and basal membranes and assemble sequentially into hundreds of unit eyes, or facets, guided by a series of cell interactions. Because cells are determined to join the facet, their nuclei and cell bodies rise apically and then settle into position in the cell group. The final nuclear positions determine the shape of the individual cells. A Drosophila gene has been identified called marbles which is required for the apical nuclear migrations that accompany cell determination during eye development. In marbles mutant eyes, the sequence of cell specification that leads to the formation of facets occurs almost normally despite the failure of nuclear migration in many cells. The marbles mutant phenotype reveals that during Drosophila eye development cell determination does not require nuclear migration (Fischer-Vize, 1994).
Formation of tubes of the correct size and shape is essential for viability of most organisms, yet little is understood of the mechanisms controlling tube morphology. A new allele of hairy has been identified in a mutagenesis screen. hairy mutations cause branching and bulging of the normally unbranched salivary tube, in part through prolonged expression of huckebein (hkb). Hkb controls polarized cell shape change and apical membrane growth during salivary cell invagination via two downstream target genes, crumbs (crb), a determinant of the apical membrane, and klarsicht (klar), which mediates microtubule-dependent organelle transport. In invaginating salivary cells, crb and klar mediate growth and delivery of apical membrane, respectively, thus regulating the size and shape of the salivary tube (Myat, 2002).
In Drosophila embryos, microtubules oriented along apical-basal directions support saltatory vesicle movement. Vesicle traffic includes lipid droplets whose distribution shifts twice during early embryogenesis. Using microscopy, optical tweezers, and a novel squashed-mount embryo preparation, single droplets were tracked and the forces these generated were measured. Droplet stalling forces change developmentally, in a roughly quantized fashion, consistent with variation in the number of active motors. A mutation, klarsicht, was characterized that affects droplet transport. Klar+ facilitates changes in force, possibly by coordinating the activity of multiple motors. Alterations in transport affected motion in both apical and basal directions, indicating tight coupling between motors of opposite polarity. Mutations in klar also affect nuclear migration during eye development, suggesting multiple roles for Klar-based transport (Welte, 1998).
One of the challenges of employing multiple motors is their coordination. If motors had a short duty cycle, producing force during only a tiny fraction of the time they are moving on MTs, it would be necessary to have some means of synchronizing their activity in order for their individual forces to summate. To avoid a tug-of-war situation, motors of opposite polarities may not remain continually bound and active on the surfaces of vesicles. It is hypothesized that the Klar protein solves these kinds of problems in the wild type by establishing a tightly coordinated complex of motor (Welte, 1998).
Several lines of evidence suggest that coordination breaks down in the absence of Klar. The difference in lateral displacement between motions in the apical and basal directions disappears in klar embryos. Next, the anticipated competition between motors of opposite polarities in klar embryos might be expected to affect both velocity and force deleteriously. Consistent with this, a reduction of velocity was observed for droplets in klar embryos to about half that of the wild type. This change is similar to the reduction in the velocity of gliding microtubules moving in vitro in a competition assay using both dynein and kinesin. Moreover, lipid droplets in klar embryos are readily stalled by forces of a magnitude close to the unitary force. This suggests that droplets are either (1) propelled by a single motorcomplex or (2) propelled by multiple motors of both polarities, but with a number excess of just one, on average, pulling in any given direction (Welte, 1998).
The model, then, is one where Klar+ enforces the coordination of same-direction motors and thus avoids a tug-of-war, by switching off the plus end-directed motors when minus end-directed motors are active, and vice versa. It is postulated that the Klar+ complex either alternates the presentation of plus end-directed or minus end-directed motors to the MT, or that it couples several bidirectional motors, such that they switch directions synchronously. Regulation of this complex alters the frequency of switching, affecting persistence times and thus determining the direction of net transport. At the same time, the complex also controls the number of actively engaged motors (Welte, 1998).
This model makes testable predictions: (1) the Klar protein should be found on lipid droplets and potentially in direct association with motors; (2) multiple motors should be found on droplets; (3) the motor-Klar complex should be subject to regulation that directly affects its physical properties (Welte, 1998).
As photoreceptor cells mature, their nuclei are transported over tens of microns, first in the basal and then in the apical direction, reminiscent of lipid droplet transport in phase II and III embryos, respectively. klar mutations disrupt both transport processes similarly, impairing the ability to switch from basal to apical transport. This parallel suggests the existence of a general system for bidirectional transport of organelles, including embryonic lipid droplets, photoreceptor nuclei, and possibly other cargoes. At least in the eye disc, this transport system appears to rely on dynein, because a dominant mutation in p150Glued (a subunit of dynactin, the dynein activating complex) causes basal mislocalization of photoreceptor nuclei that is strikingly similar to that of klar mutants (Welte, 1998).
How similar is the role of Klar in the transport of lipid droplets and nuclei? In fungi, two contrasting mechanisms have been proposed for nuclear migration. In one view, nuclei are uncoupled from any microtubule organizing center (MTOC) and are propelled along MTs by cytoplasmic dynein directly, just as any cargo. In the other, nuclei are fastened to the MT network through an MTOC, and dynein anchored in the cytoplasmic membrane reels in the nuclei by translocating the MTs, also promoting their depolymerization in the process. If the first mechanism operates in photoreceptor cells, nuclei could employ the same Klar-based multimotor complexes that are postulated for lipid droplets. In the second case, Klar may organize individual dyneins at the apical membrane into efficient multimotor machines, in whose absence too little force is generated to pull nuclei apically. Because these two mechanisms predict different locations of motors and MT ends relative to the nucleus, they may be distinguishable by determining the intracellular location of dynein and the polarity of MTs, for example, with kinesin fusion proteins (Welte, 1998).
In summary, the klar and wild-type phenotypes reveal a general, microtubule motor-based system, which regulates switching between different directions of transport. Using the tools introduced in this study to visualize and quantitate lipid droplet movement in embryo preparations, it should be possible to eventually dissect many of the physical properties of this general system. Of particular interest will be mechanisms of developmental regulation and the coordination of multiple motors, issues not readily addressed using simpler in vitro motility assays (Welte, 1998).
The temporally regulated, cell-type-specific transport of organelles has great biological significance, yet little is known about the regulation of organelle transport during development. The Drosophila gene klarsicht is required for temporally regulated lipid droplet transport in developing embryos and for the stereotypical nuclear migrations in differentiating cells of the developing eye. Klarsicht is thought to coordinate the function of several molecular motors bound to a single lipid droplet or to facilitate the attachment of dynein to the cargo, but it is not known whether Klarsicht affects motors directly or indirectly. The klarsicht gene has been cloned and shown to encode a unique large protein. Drosophila klarsicht null mutants are viable, with obvious defects only in adult eye morphology. Epitope-tagged Klarsicht expressed in the eye from a transgene is perinuclear. In flies carrying transgenes that express markers for microtubule plus and minus ends, microtubules in differentiating cells of the eye are oriented with their plus ends apical and their minus ends at the nucleus. Drosophila klarsicht null mutants are viable and fertile, demonstrating that klarsicht is essential only for specific motor protein functions. Perinuclear localization of Klarsicht protein indicates that Klarsicht has a direct mechanical role in nuclear migration. Taken together with the finding that the minus ends of the microtubules are associated with the photoreceptor nuclei, the observation that Klarsicht is largely perinuclear supports the idea that Klarsicht associates with dynein, consistent with a model in which Klarsicht assists dynein in 'reeling in' the nucleus (Mosley-Bishop, 1999).
Flies expressing the assembled cDNA klar1 in particular patterns (some transformant lines with two copies of glrs-klar1 (expressed in all cells posterior to the furrow) or one of the three UAS-klar1 lines driven by elav-Gal4, i.e. expressed in all R cells posterior to the furrow, or GMR-Gal4, i.e. expressed in all cells posterior to the furrow) show a rough external eye phenotype. In the retinas of these adult eyes, many facets appear to lack some photoreceptors). As elav-Gal4;UAS-klar1 expresses klar1 only in the R cells, the rough phenotype, at least in this case, is due to overexpression of klar1 in R cells rather than to expression outside R cells. The rough eye phenotypes of the transformants in which klar1 was overexpressed in R cells (glrs-klar1 and elav-Gal4; UAS-klar) were not due to the failure of the initial apical nuclear migration in developing photoreceptors or cone cells posterior to the furrow. Thus, overexpression of klar1 in photoreceptors results in a mutant phenotype that is unlike the klar loss-of-function phenotype and that may not have involved nuclear migration. It is proposed that the mutant phenotype is most likely due to the sequestration of proteins with which Klar normally interacts, thus preventing them from performing the functions that they have independent of Klar and nuclear migration. The glrs-klar1 and UAS-klar1 flies provide useful tools for performing genetic screens to identify genes encoding proteins that interact with Klar (Mosley-Bishop, 1999).
In order to understand the mechanism of Klar function, its subcellular localization in larval eye discs was investigated. Flies were transformed with a glrs-Myc6-klar1 construct, in which the glrs promoter drives the expression of klar1 with a Myc6 epitope tag located just downstream of the start codon. One copy of the transgene rescues the mutant phenotype of klarmCD4 homozygotes completely. Thus, the Myc6-Klar1 protein is functional and at least some of the protein must be localized similarly to endogenous Klar. The Myc6-Klar1 protein is detected perinuclearly (Mosley-Bishop, 1999).
Mechanisms for moving nuclei are thought be different from those that move other organelles; unlike other organelles, nuclei are usually associated with the microtubule-organizing center and thus are essentially stuck to the minus ends of microtubules. In yeast and filamentous fungi, genetic and biochemical experiments have shown that, like lipid droplet transport, nuclear transport requires dynein and microtubules. There are two simple models, generated from the results of experiments on nuclear migration in fungi, that could explain how photoreceptor nuclei might carry out their apical migration upon specification. In the 'reeling-in' model, dynein becomes attached to the apical surface of photoreceptor cells and moves along the microtubules toward their minus ends, located at the nuclei, thereby reeling in the nucleus to the apical surface of the cell. The alternative 'walking-up' model proposes that the nucleus becomes disassociated from the microtubule-organizing center and the microtubules are organized in developing photoreceptors such that their minus ends are apical. In this case, dynein, as it moves up toward the minus ends of microtubules, could transport the nucleus apically as it would any other organelle, like a lipid droplet (Mosley-Bishop, 1999).
Assuming that, as in lipid transport, Klar functions directly with dynein in nuclear transport, the fungal models for nuclear migration make distinct predictions about microtubule organization and Klar localization in the eye. The reeling-in hypothesis predicts that microtubules should be oriented with their plus ends apical and their minus ends attached to the nucleus. Also, at least some Klar should be localized apically in the cell, perhaps helping to tether dynein to the apical cell surface or to otherwise aid its function there. Conversely, the walking-up model predicts that microtubules would be oriented with their minus ends apical and that Klar should be associated with the nucleus, where it would play the same role proposed for its function in lipid droplet transport (Mosley-Bishop, 1999).
The results do not fall neatly into either set of predictions. Although it is found that the microtubules are oriented with their plus ends apical and their minus ends associated with the nucleus, as predicted by the reeling-in model, Klar mainly is associated with the nucleus, as predicted by the walking-up model. The results do suggest, however, that Klar associates with dynein and do not rule out the existence of a small amount of Klar protein (and dynein) tethered to the apical surface that could reel in the nucleus. Further experiments in which Myc6-Klar is expressed in the eye using promoters that are active at high levels in R cells before the apical migration of their nuclei may enable clear detection of any apical Klar protein. Similarly, the method for detection of Nod-ß-gal and kinesin-ß-gal proteins would not reveal small subpopulations of microtubules that run counter to the array. Further studies to determine the subcellular localization of other components of the nuclear migration pathway, including dynein and kinesin, as well as to identify other molecules that interact with Klar should help to distinguish among the possible models (Mosley-Bishop, 1999).
Klar has been shown by light microscopy to be associated with the nuclear membrane (Mosley-Bishop, 1999). The subcellular localization of Klar was investigated in greater detail and at higher resolution using confocal microscopy. To visualize Klar protein, an epitope-tagged form of Klar, 6Xmyc-Klar, was expressed in R-cells by using a UAS-6Xmyc-klar transgene and an elav-Gal4 driver (elav>6Xmyc-klar). The 6Xmyc-Klar protein has been shown to be functional (Mosley-Bishop, 1999) Otherwise wild-type eye discs expressing elav>6Xmyc-klar were labeled with anti-Myc and also with anti-Elav, which marks R-cell nuclei after they have risen apically. Klar is associated with the nuclear membrane, and in addition, dots of Klar are seen to extend from the nuclei toward the apical cell surface. The apical dots resemble the apical expression pattern of Futsch, also known as 22C10, a neural-specific microtubule-associated protein (Patterson, 2004).
There is a variety of experimental evidence that the KASH domains of Anc-1 and Syne-1 localizes those proteins to the nuclear envelope (Mislow, 2001; Mislow, 2002; Starr, 2002; Zhang, 2001; Zhen, 2002). Similarly, it was found that Klar is genuinely associated with the nuclear membrane, rather than appearing perinuclear only because it is associated with microtubules that extend around the nucleus. (1) Immunostaining with anti-Futsch reveals the microtubule cytoskeleton as it extends from the apical to basal cell surfaces, weaving around the nucleus. Although Futsch is bound to microtubules, it does not appear perinuclear as does Klar. (2) 6Xmyc-Klar colocalizes with the nuclear envelope protein lamin. (3) The two aspects of Klar localization are separable: when an isolated Klar KASH domain is expressed, only nuclear membrane localization, not apical microtubule localization, is observed. It is concluded that 6Xmyc-Klar localizes to the apical microtubules and to the nuclear envelope in R-cells (Patterson, 2004).
To probe the function of Klar in nuclear migration, it was asked whether the cytoskeleton is organized differently in klar mutants than in wild-type eye discs. The MTOC was marked by expressing a Nod-ßgal fusion protein, which accumulates at microtubule minus ends. The MTOC is the point in the cell from which the microtubules grow: the slow-growing minus ends gather at the MTOC and the rapidly growing plus ends emanate from it. Nod-ßgal was expressed using an elav-Gal4 driver and a UAS-nod-lacZ transgene. Otherwise wild-type and also klar mutant eye discs expressing elav>nod-lacZ were double-labeled with anti-Elav and anti-ßgal. In wild-type, Nod-ßgal is closely associated with the R-cell nuclei, and just apical to them (Patterson, 2004).
In klar mutants, most of the R-cell nuclei are basal, but all of the Nod-ßgal is apical. There are two possible interpretations of this result: (1) The klar mutant R-cells with basal nuclei (most of the cells) do not form an MTOC, or (2) the MTOC is separated from the abnormally basal nuclei of klar mutant R-cells. If most of the R-cells in klar mutant eye discs do not form an MTOC, then in klar discs fewer apical dots of Nod-ßgal would be expected in each ommatidial cluster than in wild-type. By contrast, it was observed that the Nod-ßgal dots in klar mutant discs appear wild-type in number and pattern. It is concluded that in klar mutant R-cells the MTOC forms normally, but usually separates from the nucleus (Patterson, 2004).
A transgene called glrs-klar overexpresses klar+ in the developing eye, resulting in defects in eye morphology (Mosley-Bishop, 1999). To identify additional genes that function in nuclear migration in the Drosophila eye, a mutagenesis screen was performed for dominant enhancers of the glrs-klar rough eye phenotype. Nine mutant alleles of a complementation group termed egk1 (enhancer of glrs-klar) were isolated. The nine egk1 alleles were divided into three groups based on the severity of their mutant phenotype: (1) four alleles are lethal as homozygotes or in trans to each other, (2) four alleles (Ari3, Ari7, K2, 83) are semiviable as homozygotes, and (3) one allele (A25) is homozygous viable. Initial observation of the egk1 mutants suggested that the egk1 gene has an essential role in eye development; adults homozygous for any of the semiviable or viable alleles have external eye defects. Meiotic mapping localized egk1 between the markers dp and b on chromosome 2, and subsequent physical mapping localized egk1 to polytene position 25E3-6, the region uncovered by the deletion chromosome Df(2L)cl-h4. In trans to Df(2L)cl-h4, the lethal egk1 alleles are lethal and the semiviable or viable egk1 alleles are semiviable. The weak egk1 alleles are shown to be loss-of-function mutants and they display nuclear migration defects. Because egk1 loss-of-function mutants have a similar mutant eye phenotype to klar mutants and egk1 interacts genetically with klar, it is concluded that the egk1 gene is likely to function in the klar pathway (Patterson, 2004).
Among the ~25 genes in 25E3-6, Lam Dm0 (Lam) was chosen as a candidate for egk1. Lam encodes type B nuclear lamin, an intermediate filament protein that is a major component of the inner nuclear envelope. To determine if egk1 is Lam, several of the egk1 alleles were tested for complementation with two previously identified homozygous lethal Lam mutants, Lam4643 and LamP. Neither Lam mutant complements any of the egk1 alleles tested. In addition, the DNA sequences were determined for the Lam genes in flies homozygous for each of the semiviable or viable egk1 alleles. In each case, a nonsense or frameshift mutation was found within the Lam coding region. An antibody to Lam (mAbADL84) recognizes no protein in immunostained eye discs carrying any one of the semiviable alleles in trans to Df(2L)cl-h4. This result is consistent with the DNA sequence analysis of the four semiviable Lam alleles, which predicts that severely truncated Lam proteins are the most likely gene products. Even if these truncated proteins are produced and stable, they need not contain the epitope recognized by mAbADL84. The weakest allele, A25, has a frameshift that results in the deletion of the C-terminal CaaX box, which localizes lamin to the inner nuclear membrane. Consistent with this observation, A25/Df(2L)cl-h4 eye discs immunostained with mAbADL84 reveal that Lam protein does not localize to the membrane, but instead is found throughout the nucleus. Finally, a P element containing Lam+ genomic DNA (Tw2-LamP) rescues the lethality and eye phenotypes of the egk1 alleles. It is concluded that egk1 is Lam (Patterson, 2004).
To determine if the eye morphology flaws in Lam mutants are due to nuclear migration defects, anti-Elav was used to label R-cell nuclei in Lam eye discs. All of the semiviable and viable Lam mutants were analyzed; LamA25, LamAri3, and LamAri7 homozygotes were assayed, and all five weak Lam alleles were analyzed in trans to Df(2L)cl-h4. With the exception of the weakest allele, LamA25, all homozygotes and hemizygotes showed similar phenotypes; as in klar mutants, R-cell nuclei are present throughout the apical/basal axis of the eye disc, and most of them are basal. In LamA25 homozygous discs the R-cell nuclear positions are indistinguishable from wild type. By contrast, LamA25/Df(2L)cl-h4 discs show nuclear migration defects, but they are less severe than those of the other alleles analyzed. The difference in severity of the nuclear migration defects in the different alleles is mirrored in their adult eye morphology. Adult retinas with R-cell nuclear migration defects, like those of klar mutants, typically have misshapen rhabdomeres (Fischer-Vize, 1999; Mosley-Bishop, 1999). Rhabdomeres are light-gathering organelles that project from each photoreceptor cell throughout the apical/basal plane of the eye disc. When R-cell nuclei fail to migrate apically, the cell shapes are aberrant, resulting in oddly shaped or missing rhabdomeres in tangential retinal sections. The retinas of LamA25 homozygotes are nearly wild type, LamA25 hemizygotes are defective, and the eyes of LamAri3 homozygotes or hemizygotes have more severe defects (Patterson, 2004)
Several results described above indicate that the five weak Lam mutant alleles are partial loss-of-function mutants, as opposed to gain-of-function mutants: 1) Both lethal and viable Lam alleles were isolated as enhancers of glrs-klar. This result indicates that all classes of Lam alleles have a similar (detrimental) effect on nuclear migration. 2) LamA25 homozygotes have a weaker phenotype than LamA25/Df(2L)cl-h4 hemizygotes. 3) All phenotypes of the weak and strong Lam mutants (lethality and eye phenotypes) are complemented completely by one copy of the transgene Tw2-LamP, which contains a Lam+ gene. Nevertheless, to be certain that the nuclear migration defects represent loss-of-function phenotypes, eye discs homozygous for Lam4346, a strong lethal, loss-of-function mutation caused by P element insertion, were observed. Lam4346 eye discs have nuclear migration defects similar to those of LamAri3 homozygotes or LamAri3/Df(2L)cl-h4. It is concluded that like klar+, the Lam+ gene is normally required for R-cell nuclear migration (Patterson, 2004).
The position of the MTOC was monitored in wild-type and Lam mutant eye discs with antibodies to gamma-tubulin, a constituent of a protein complex that binds the MTOC. In wild-type discs, dots of gamma-tubulin are observed just apical to the R-cell nuclei. Moreover, the gamma-tubulin dots are present only in differentiating cells, whose nuclei are normally apical. Undifferentiated cells that have not yet been recruited into ommatidial clusters surround the developing facets and their nuclei are basal. No gamma-tubulin is observed associated with the basal nuclei or at the apical surface of the undifferentiated cells; all of the gamma-tubulin dots are within the developing clusters. This result suggests that the cytoskeleton becomes organized and an MTOC forms in differentiating cells as they are recruited into the ommatidia (Patterson, 2004).
As in klar mutants, in Lam mutant discs the MTOCs of all of the R-cells are apical, even though most of the R-cell nuclei are basal. It is concluded that like Klar, lamin is required for nuclear migration and to link the MTOC to the nucleus (Patterson, 2004).
To determine if lamin and Klar function together, the localization of each protein was monitored in the mutant background of the other. In Lam mutant eye discs that express elav>6Xmyc-klar, Klar localization on microtubules apical to the nucleus appears normal. Perinuclear Klar, however, is largely absent in Lam mutants. In contrast, lamin localization appears normal in klar mutant eye discs. It is concluded that localization of Klar to the nuclear envelope requires nuclear lamin (Patterson, 2004).
It has been proposed that Klar facilitates nuclear migration in photoreceptors by linking the nucleus to the microtubule organizing center (MTOC). Genetic and immunohistochemical experiments have been performed that provide a critical test of this model. Mutants have been analyzed in the endogenous klar gene and also flies were examined that expressed deleted forms of Klar protein from transgenes. The KASH domain of Klar was found to be critical for perinuclear localization and for function. In addition, 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 (Fisher, 2004).
As a first step toward determining which portions of Klar protein are required for its function, the exon DNA sequences of six mutant klar alleles (Fisher-Vize, 1994) were determined. The results of genomic blotting experiments suggested that these alleles contain point mutations (Mosley-Bishop, 1999). All six alleles have either nonsense or frameshift mutations in coding exon. In addition, RNA blotting experiments were performed to detect klar transcripts in eye discs of third instar larvae homozygous for each mutant allele. Each mutant klar mRNA is present and at levels similar to those in wild type (Fisher, 2004).
Two alleles, klarmBX5 and klarmBX14, have frameshift mutations in the open reading frame after amino acids 79 and 915, respectively. The protein products of these alleles are difficult to predict. Although the simple expectation is that klarmBX5 and klarmBX14 would produce N-terminal Klar protein fragments, translation reinitiation could result in the production of C-terminal protein fragments (Fisher, 2004).
Four alleles (klarmCD4, klarmFQ19, klarmBX6, and klarmBX15) have premature stop codons late in the open reading frame. Since a Klar protein truncated somewhat more severely than KlarBX6 is stably produced by a transgene, these four alleles are likely to produce nearly full-length Klar proteins with C-terminal truncations. Notably, klarmCD4 and klarmBX15 should produce Klar protein lacking only the final 60 or 46 amino acids, respectively, which includes most of the KASH domain. These four mutant allele sequences suggest that the KASH domain is important for the function of Klar protein (Fisher, 2004).
To explore further the importance of the KASH domain, it was asked if klarmCD4, which has a stop codon just prior to the KASH domain, behaves as a strong loss-of-function allele genetically. Toward this end, the mutant eye phenotypes of klarmCD4 homozygotes and hemizygotes (klarmCD4/Df(3L)emcE12) were compared. The external eyes and R-cell morphology were observed in sectioned adult eyes and R-cell nuclear positions in developing larval eye discs. Wild-type external eyes appear smooth and crystalline. Homozygous klarmCD4 external eyes are subtly rougher than those of wild type. Sections through the wild-type retina reveal the R cells arranged in a trapezoid. The R cells are identified by their rhabdomeres, light-gathering organelles that project from each R cell into the center of the ommatidium. In klarmCD4 homozygotes, the R cells are present, largely in their normal positions, but the rhabdomeres are malformed. Since the rhabdomeres project out along the entire apical/basal axis of the R cells, the severe cell shape malformations resulting from the lack of an apical nucleus also result in rhabdomere malformations. The positions of the R-cell nuclei in discs were observed by labeling discs with antibodies to Elav, a neural nuclear protein. In wild-type eye discs, all of the R-cell nuclei are apical. By contrast, in klarmCD4 eye discs, most of the R-cell nuclei are basal and the remainder are randomly distributed throughout the apical/basal plane. The eye phenotypes of klarmCD4/Df(3L)emcE12 are qualitatively indistinguishable from those of klarmCD4 homozygotes. The mutant eye phenotypes of klarmBP/Df(3L)emcE12 were also examined. klarmBP is a translocation that breaks in the middle of the klar coding region and is thus very likely to be null (Fisher-Vize, 1994: Mosley-Bishop, 1999). The phenotypes of klarmBP/Df(3L)emcE12 and klarmCD4 are also indistinguishable. It is concluded that klarCD4 retains little or no function and thus that the KASH domain is critical to Klar activity (Fisher, 2004).
These results suggest that the KASH domain is essential for Klar function, perhaps because it localizes Klar to the nuclear envelope. However, the subcellular localization of the mutant Klar proteins cannot be determined. To circumvent this limitation and also to investigate the function of the region of Klar N-terminal to the KASH domain, transgenes were generated that express epitope-tagged partial Klar proteins. Previous results established expression vectors and an epitope tag useful for assays of Klar subcellular localization and for klar function. Localization of wild-type Klar was observed by generating transgenes that express a full-length 6xmyc-tagged Klar (6mKlarFL; Mosley-Bishop, 1999; Patterson, 2004). When 6mKlarFL is expressed in R cells using a neural-specific elav-Gal4 driver and a UAS-6mklarFL transgene, two distinct aspects of 6mKlarFL localization are observed; 6mKlarFL is detected at the nuclear membrane and also on microtubules apical to the nucleus (Patterson, 2004). When expressed from the GLRS vector, which is active in all cells posterior to the furrow in the eye disc, 6mKlarFL restores significant Klar function to klarCD4 homozygotes (Mosley-Bishop, 1999; Fisher, 2004).
To determine if the N- and C-terminal portions of Klar are differentially required for Klar localization and function, two klar gene constructs were generated that express different 6xmyc-tagged partial Klar proteins; 6mKlar3'DeltaS contains the 1774 N-terminal amino acids, and 6mKlar5'DeltaA contains the C-terminal 403 amino acids of Klar, which include the KASH domain. For assays of subcellular localization, transgenes were generated where expression of each construct is controlled by a UAS (UAS-6mklar3'DeltaS and UAS-6mklar5'DeltaA) and several transformant lines were generated with each. For assays of function, each construct was cloned into the GLRS vector (glrs-6mklar3'DeltaS and glrs-6mklar5'DeltaA) and several transformant lines were generated (Fisher, 2004).
Western blot experiments were performed to determine if 6mKlar protein is expressed stably by each transgene and to identify the transformant lines that express the highest levels of protein. 6mKlar proteins were detected in eye disc protein extracts from larvae with a single UAS transgene expressed by an eye-specific driver (GMR-Gal4) or from larvae with a single GLRS transgene. Anti-myc was used to detect 6mKlar and anti-tubulin was used as a control. One transformant line of each UAS construct and two lines of each GLRS construct with the highest expression levels were chosen for further analysis (Fisher, 2004).
To determine where the partial Klar proteins are located within R cells, eye discs with one copy of a UAS transgene (UAS-6mklarFL, UAS-6mklar3'DeltaS, or UAS-6mklar5'DeltaA) and one copy of an elav-Gal4 driver transgene were immunostained with anti-myc. The KlarFL protein localizes apically to the R-cell nuclei on microtubules and is also perinuclear (Patterson, 2004). 6mKlar5'DeltaA, the C-terminal Klar fragment, that contains the KASH domain, retains only one of the two aspects of 6mKlarFL localization; 6mKlar5'DeltaA localizes to the nuclear membrane, but not to the apical microtubules. Conversely, 6mKlar3'DeltaS, the N-terminal Klar fragment that lacks the KASH domain, localizes to the apical microtubules, but not to the nuclear membrane. Thus, distinct domains localize Klar to microtubules and the nuclear envelope (Fisher, 2004).
To determine if the 6mKlar3'DeltaS or 6mKlar5'DeltaA proteins retain significant levels of Klar function, glrs-6mklar3'DeltaS and glrs-6mklar5'DeltaA transgenes were tested, along with glrs-6mklarFL as a control, for complementation of the klarCD4 mutant eye phenotype. Complementation of the nuclear migration defects in eye discs and of the morphological defects in adult eyes were tested. A glrs-6mklarFL transgene rescues both defects significantly. By contrast, neither glrs-6mklar3'DeltaS nor glrs-6mklar5'DeltaA provides significant rescuing activity. This result is particularly striking given that the partial proteins, especially 6mKlar5'DeltaA, are produced at much higher levels than 6mKlarFL. Although the epitope tag does not affect the function of full-length Klar (Mosley-Bishop, 1999), it could affect the function 6mklar5'DeltaA. Aside from this caveat, these results indicate that neither the KASH domain nor the N terminus of Klar is sufficient for significant function. Rather, both the N-terminal and the C-terminal Klar fragments are necessary (Fisher, 2004).
Overexpression of KlarFL in the eye can result in morphological defects unrelated to nuclear migration (Mosley-Bishop, 1999; Patterson, 2004). Whether expression of the partial Klar proteins 6mKlar5'DeltaA or 6mKlar3'DeltaS also results in eye morphology defects was tested. None of the GLRS vector transgenes (3 lines of glrs-6mklarFL, 6 lines of glrs-6mklar5'DeltaA, or 11 lines of glrs-6mKlar3'DeltaS) cause mutant eye phenotypes when present in two copies. Similarly, none of the UAS transgenes, when expression is driven by elav-Gal4, results in a mutant eye phenotype. To boost the expression levels of the UAS transgenes, the GMR-Gal4 driver was used. Each of the 12 UAS-6mklarFL lines tested resulted in roughened external eyes when expressed using GMR-Gal4. By contrast, none of the 11 UAS-6mklar5'DeltaA lines produced a phenotype when expressed with GMR-Gal4, and only 1 of 5 UAS-6mklar3'DeltaS lines did. The failure of 6mKlar5'DeltaA overexpression to produce a mutant eye phenotype is particularly striking given that its expression level is considerably higher than that of KlarFL. It is concluded the overexpression phenotype caused by KlarFL is not due solely to the KASH domain or to the microtubule-localization domain, but requires an intact protein (Fisher, 2004).
Thus, when Klar is divided into N-terminal and C-terminal portions, the N terminus localizes to microtubules apical to the nucleus and the C terminus containing the KASH domain is perinuclear. Also, KASH domain is important for Klar function: (1) it was found that four independent klar mutant alleles have nonsense or frameshift mutations that are likely to result in deletion of the C-terminal KASH domain; (2) it was shown that one of these alleles retains little or no gene activity; (3) a transgene that expresses the N-terminal 1774 amino acids of Klar, which does not include the KASH domain, fails to retain significant klar gene function in vivo; (4) it was shown that the KASH domain alone is insufficient for Klar function. Even when overexpressed, a C-terminal 403-amino-acid Klar fragment that includes the KASH domain does not provide significant Klar function in vivo (Fisher, 2004).
Klar and Anc-1/Syne-1 (and Zyg-12) are unique in that they are held in the nuclear membrane through (probably indirect) interactions with nuclear lamin and they protrude into the cytoplasm. Starr (2002) reported that overexpression of the C. elegans Anc-1 KASH domain results in a dominant negative nuclear anchorage defect, presumably because the KASH domain competes with wild-type Anc-1 for a limited number of docking sites in the nuclear membrane. It was therefore surprising to find that overexpression of the Klar KASH domain does not result in a mutant phenotype. The difference between the current results and those of Starr (2002) could be due to technical differences in the two experimental systems. Alternatively, the different results could reflect a mechanistic difference in Anc-1 and Klar function (Fisher, 2004).
Among all of the genomes whose DNA sequences are known, Klar is unique to Drosophila and Anopheles. As nuclear migration and nuclear attachment to the MTOC are universal cellular phenomena, this result is surprising. Other metazoans appear to rely solely on alternative proteins for attaching the nucleus and MTOC and for nuclear migration. One of these proteins is C. elegans Zyg-12. In C. elegans embryos, Zyg-12 attaches nuclear membranes to centrosomes (the MTOC; Malone, 2003). Zyg-12 is present at the nuclear membrane and also at centrosomes and the mechanisms proposed for Klar and Zyg-12 function are similar. Yet, the two proteins have no obvious amino acid sequence similarity. Rather, Zyg-12 is a homolog of Drosophila Hook, which in Drosophila is proposed to link organelles other than the nucleus to the cytoskeleton. Hook is expressed in the eye, but a role for it in R-cell nuclear migration has not been reported. Perhaps the variety of mechanisms for connecting the nucleus to the MTOC reflects a requirement for regulation in diverse developmental contexts (Fisher, 2004 and references therein).
This article provides the first mechanistic insight into Klar, a multifunctional regulator of microtubule-based transport. In different cells, Klar localizes to distinct cargoes, due to alternatively expressed C-terminal-targeting modules. The newly identified LD domain is necessary and sufficient to target Klar to the surface of lipid droplets. The KASH domain is necessary and sufficient to target Klar to the nuclear envelope. Localization is crucial for Klar function and dictates which transport process Klar regulates in a given cell. Because Klar is widely expressed and likely localizes to additional cargoes, understanding Klar-based transport should shed light on a whole range of developmental processes (Guo, 2005).
The 60aa KASH domain of Klar is sufficient for perinuclear localization of a heterologous protein in cell culture and necessary for Klar's localization to the nuclear envelope in vivo. This function of the KASH domain is evolutionary conserved. For human Syne-1, the KASH domain directs nuclear envelope localization in COS-7 cells and C2C12 myoblasts (Zhang, 2001). For ANC-1, a 346aa region encompassing the KASH domain localizes to the nuclear envelope (Starr, 2002). And in Drosophila photoreceptor cells, an epitope-tagged Klar protein containing the KASH domain (corresponding to isoform alpha) localizes perinuclearly, in a lamin-dependent manner (Patterson, 2004). KASH domains are thought to bind via UNC-84/SUN to nuclear lamins (Starr, 2003; Guo, 2005).
The 112aa LD domain of Klar is sufficient to recruit a heterologous protein to the surface of lipid droplets in cultured cells, and Klar fragments without LD are diffusely cytoplasmic. In embryos, Klar proteins truncated N-terminal to LD fail to localize to droplets. Together, the LD domain of Klar seems to be both necessary and sufficient for droplet localization. Lipid droplets are specific organelles with their own distinct set of proteins, but how these proteins are targeted is not well understood. The small size of the Klar-LD domain makes it a good model to dissect which features of the protein mediate localization (Guo, 2005).
Previous studies of the klar mutant phenotype had identified Klar as a key regulator for bidirectional motion of lipid droplets (Welte, 1998). The current analysis shows that Klar is present at the correct time and the correct place to act as coordinator. Klar function depends on its localization: Klar proteins not localized to droplets fail to support normal droplet motion, and a Klar protein that lacks just the KASH domain is not sufficient for proper migration of photoreceptor nuclei (Guo, 2005).
In embryos, Klar is concentrated in a spot next to droplets, similar to the distribution of dynein, consistent with the model that Klar helps form a coordination complex that includes the plus- and minus-end motors (Welte, 1998). RFP-LD in S2 cells, in contrast, is present all over the droplet surface; there may be no motors bound to these droplets, or the tested Klar constructs may lack domains necessary to form complexes (Guo, 2005).
Isoforms alpha and ß are important for regulating the motors powering nuclear migration and droplet transport, respectively. Klar regions common to both isoforms likely mediate shared functions, such as motor coordination. To this core region, the cell can attach variable domains via alternative splicing. These variable components encompass not only the cargo-targeting domains but also additional regions that differ between alpha and ß. Variable regions might fine-tune Klar for particular tasks or might provide docking sites for specific transacting regulators, so that different cargoes could use the same motors and the same coordination machinery, yet still be regulated independently (Welte, 2004). This analysis has disentangled this elaborate organization of Klar and sets the stage for determining the detailed roles of these modules (Guo, 2005).
Dissecting Klar function will likely illuminate many transport processes throughout Drosophila development. Klar expression is detected in specific tissues throughout embryogenesis, in larval stages and in adults. Because many Klar dots have no close association with either lipid droplets or nuclei, there are likely undiscovered Klar cargoes. The developed antibodies should make it possible to identify these cargoes, by using colocalization or fractionation approaches (Guo, 2005).
The following framework is proposed for how cells are able to use a single regulator to control multiple transport processes. The various Klar isoforms are composed of distinct modules: (1) a shared core region important for motor coordination, possibly by physically interacting with the motors; (2) one of several cargo-targeting domains that recognize the identity of the cargo, and (3) variable accessory regions. A shared core makes conserved contacts with the motor complexes and allows cells to reuse the same coordination machinery in different transport processes. Cargo-targeting domains deliver Klar to the correct location. And variable regions fine-tune the biochemical properties of Klar to the particular transport process. In this model, Klar serves as a crucial bridge between the identity of the cargo and motor complexes. The molecular and genetic tools generated for Klar lay the groundwork for testing this model and for dissecting the mechanisms of Klar-based transport (Guo, 2005).
This regulatory strategy may be a general solution to the problem of how to employ a core regulator in a cargo-specific manner. For example, mammalian Syne-1 can localize to the Golgi, anchors nuclei to actin, and functions with the microtubule motor kinesin II in cytokinesis (Fan, 2004). Multiple transcripts, using three different transcription start sites, are predicted to encode at least four distinct protein isoforms (Gough, 2003) that contain either the KASH domain, Golgi binding sites, or both. For the C. elegans protein ANC-1, it is unknown whether different bands recognized by Western analysis represent functionally distinct isoforms (Starr, 2002). However, ANC-1-dependent anchoring of nuclei to actin requires the nuclear envelope protein UNC-84/SUN, whereas anchoring of mitochondria does not, consistent with the possibility that different ANC-1 isoforms interact with cargo-specific partners. The Drosophila dystrophin-related gene Msp300 produces at least two alternatively spliced transcripts, one that encodes the KASH domain and one that does not (Rosenberg-Hasson, 1996). Klar may therefore provide a general paradigm how a single core regulator can be deployed in many different processes yet be controlled independently (Guo, 2005).
Striated muscle fibers are characterized by their tightly organized cytoplasm. This study shows that the Drosophila melanogaster KASH proteins Klarsicht (Klar) and MSP-300 cooperate in promoting even myonuclear spacing by mediating a tight link between a newly discovered MSP-300 nuclear ring and a polarized network of astral microtubules (aMTs). In either klar or msp-300ΔKASH, or in klar and msp-300 double heterozygous mutants, the MSP-300 nuclear ring and the aMTs retracted from the nuclear envelope, abrogating this even nuclear spacing. Anchoring of the myonuclei to the core acto-myosin fibrillar compartment was mediated exclusively by MSP-300. This protein was also essential for promoting even distribution of the mitochondria and ER within the muscle fiber. Larval locomotion is impaired in both msp-300 and klar mutants, and the klar mutants were rescued by muscle-specific expression of Klar. Thus, these results describe a novel mechanism of nuclear spacing in striated muscles controlled by the cooperative activity of MSP-300, Klar, and astral MTs, and demonstrate its physiological significance (Elhanany-Tamir 2012).
Communication usually applies feedback loop-based filters and amplifiers to ensure undistorted delivery of messages. Such an amplifier acts during Drosophila melanogaster midoogenesis, when oskar messenger ribonucleic acid (mRNA) anchoring depends on its own locally translated protein product. This study found that the motor regulator Klar β mediates a gain-control process that prevents saturation-based distortions in this positive feedback loop. Like oskar mRNA, Klar β localizes to the posterior pole of oocytes in a kinesin-1-dependent manner. By live imaging and semiquantitative fluorescent in situ hybridization, it was shown that Klar β restrains oskar ribonucleoprotein motility and decreases the posterior-ward translocation of oskar mRNA, thereby adapting the rate of oskar delivery to the output of the anchoring machinery. This negative regulatory effect of Klar is particularly important for overriding temperature-induced changes in motility. It is concluded that by preventing defects in oskar anchoring, this mechanism contributes to the developmental robustness of a poikilothermic organism living in a variable temperature environment (Gaspar, 2014).
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 (Christophorou, 2015).
Rotations of nuclei have been described previously in somatic cells; their function remains however unclear. In germ cells, meiotic chromosome movements are thought to be required for homologue pairing, removing chromosome entanglements, promoting maturation of recombination intermediates, or for assessing chromosome homology before synapsis, in different model organisms. In Drosophila, a high temporal correlation was found between nuclear rotations and chromosome pairing occurring mainly in 8-cell cysts. This work uncovered a second interesting correlation between the speed of nuclear rotation and the degree of centromere pairing and clustering. Indeed, mutations in klaroid affected the least nuclear rotations and disrupted the least centromere associations and synapsis. Rotations were slowed down more significantly in klarsicht, sas-4 and asl mutant germ cells. Accordingly, strong defects were observed in the initial pairing of centromeres and in synaptonemal complex formation. Finally, nuclear rotations were completely abolished in the absence of Dynein or dynamic microtubules. In dynein mutant germ cells, an average of six centromeres were distinguished during pre-meiotic pairing, which is higher than any mutants tested previously, including null alleles of c(3)G. Similarly, five centromeres on average were coundted during clustering in region 2a, a mutant phenotype that is comparable to the strongest orientation disruptor (ord) or c(3)G mutations (lateral and central elements of synaptonemal complex respectively). Nuclear rotations thus play an important role in homologue chromosome pairing and synaptonemal complex formation (Christophorou, 2015).
It was found that microtubules could be nucleated from the fusome, the nuclear envelope and the centrosome in region 1 germ cells. On the basis of these observations and centrosome mutant analysis, it is speculated that the whip-like movements of microtubules could be the main forces creating cytoplasmic flows, as observed in many biological systems and demonstrated theoretically. In addition, microtubules nucleated by the centrosomes could also push on the nucleus and the cell membrane, which could bias nuclear movement towards one direction of rotation as proposed for the migration of this same oocyte. These two forces depend on microtubules and dynein, and would act redundantly for efficient and unidirectional nuclear rotations. However, even in the absence of dynamic microtubules, centromeres ended up paired, albeit much later in region 2b. Synapsis, on the other hand, was completely disrupted. It is thus believed that, as in yeast and worms, these movements are there to facilitate pairing, synapsis or recombination, but that at least chromosome pairing could occur slowly without motions by redundant mechanisms. In flies, Spag4 is a second SUN-domain protein, but it is only expressed in male testis and is thus not likely to play a role during oogenesis. There is also a second KASH-domain protein called MSP-300/Nesprin, which interacts with the actin cytoskeleton. In the absence of microtubules, nuclei were not ‘rolling’ anymore; however, they still showed some back and forth ‘rocking’ movements. It will be interesting to investigate whether MSP300/Nesprin and the actin cytoskeleton are involved in these rocking movements (Christophorou, 2015).
This study found that although mud mutant ovaries showed only mild defects in centromere dynamics, significant genetic interactions were uncovered with klaroid and klarsicht in this same process. Striking features of Mud in this study were its co-localization with centromeres in interphasic germline cysts and the formation of polycomplexes in mud mutant cysts. The formation of polycomplexes was associated with a lack of nuclear membrane and diffused DNA in the cytoplasm, suggesting that Mud is required to maintain nuclear envelope integrity. It is proposed that the disappearance of the NE in mud cysts is the primary defect leading first to the de-localization of DNA into the cytoplasm and then the formation of polycomplexes. Polycomplexes could thus be the result of self-assembly of synaptonemal complex components polymerizing in the absence of chromatin. Polycomplexes were also observed in klaroid and klarsicht mutants although at a lower penetrance than in mud mutants. Interestingly, large distortions of the NE were also observed in muscle cell nuclei mutant for unc-84, which encodes a C. elegans SUN protein. These deformations were particularly strong in these cells, because muscle cell nuclei are subjected to mechanical stress. It is likely that rolling nuclei of 8-cell cysts are also exposed to some mechanical forces. Klarsicht, Klaroid and Mud may all participate in maintaining the integrity of the nuclear envelope in these conditions. In their absence, the NE is weakened and cannot resist mechanical forces, which also leads to synaptonemal complex assembly defects. In the most extreme cases the NE completely disappears causing the formation of polycomplexes. Interestingly, Mud initially localizes at the NE of all germline cells in region 1, but then becomes localized only to the cells remaining in meiosis in region 2a, and finally only specifically at the NE of the oocyte. This may hint that the meiotic nucleus is subjected to specific mechanical forces during oogenesis (Christophorou, 2015).
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date revised: 26 December 2015
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