Drosophila klaroid encodes a SUN domain protein required for Klarsicht localization to the nuclear envelope and nuclear migration in the eye

KASH (Klarsicht/Anc-1/Syne homology) domain proteins are cytoskeleton-associated proteins localized uniquely to the outer nuclear membrane. Klarsicht is a KASH protein required for nuclear migration in differentiating cells of the Drosophila eye. The C-terminal KASH domain of Klarsicht resides in the perinuclear space, and the cytoplasmic moiety connects to the microtubule organizing center. In C. elegans and vertebrate cells, SUN (Sad1/UNC-84) domain proteins reside in the inner nuclear membrane and tether KASH proteins to the outer nuclear membrane. Is there a Drosophila SUN protein that performs a similar function, and if so, is it like Klarsicht, obviously essential for nuclear positioning only in the eye? Here, we identify Drosophila Klaroid, a SUN protein that tethers Klarsicht. klaroid loss-of-function mutants are indistinguishable phenotypically from klarsicht mutants. Remarkably, neither gene is essential for Drosophila viability or fertility, and even in klaroid klorsicht double mutants, the only obvious external morphological defect is rough eyes. In addition, we find that klaroid and klarsicht are required for nuclear migration in differentiating neurons and in non-neural cells. Finally, while perinuclear Klaroid is ubiquitous in the eye, Klarsicht expression is limited to differentiating cells and may be part of the trigger for apical nuclear migration.     

On the roles of the Drosophila KASH domain proteins Msp-300 and Klarsicht

KASH (Klarsicht, Anc-1, Syne-1 homology) domain-containing proteins anchor the nucleus to the actin cytoskeleton or to microtubules. KASH proteins thus play pivotal roles in a variety of developmental processes where nuclear positioning is critical. Two KASH proteins have been identified in Drosophila: Muscle-specific protein-300 (Msp-300) and Klarsicht (Klar). Msp-300 anchors nuclei to actin, and has been reported to be essential for positioning of nurse cell nuclei during oogenesis, and thus production of mature ooctyes. Klar is required for positioning of photoreceptor and cone cell nuclei in the developing eye, which is critical for proper eye morphology. Here, we asked whether KASH domain-containing forms of Msp-300 are required for nuclear positioning in the eye, and we found that they are not. Moreover, in the course of this work, we discovered that contrary to previous reports, KASH domain-containing forms of Msp-300 are not required for viability, nor for oogenesis. However, we did find that Msp-300 has a function in egg laying, normally redundant with a function of Klar.     

On the roles of the Drosophila KASH domain proteins Msp-300 and Klarsicht

KASH (Klarsicht, Anc-1, Syne-1 homology) domain-containing proteins anchor the nucleus to the actin cytoskeleton or to microtubules. KASH proteins thus play pivotal roles in a variety of developmental processes where nuclear positioning is critical. Two KASH proteins have been identified in Drosophila: Muscle-specific protein-300 (Msp-300) and Klarsicht (Klar). Msp-300 anchors nuclei to actin, and has been reported to be essential for positioning of nurse cell nuclei during oogenesis, and thus production of mature ooctyes. Klar is required for positioning of photoreceptor and cone cell nuclei in the developing eye, which is critical for proper eye morphology. Here, we asked whether KASH domain-containing forms of Msp-300 are required for nuclear positioning in the eye, and we found that they are not. Moreover, in the course of this work, we discovered that contrary to previous reports, KASH domain-containing forms of Msp-300 are not required for viability, nor for oogenesis. However, we did find that Msp-300 has a function in egg laying, normally redundant with a function of Klar.     

Organelle positioning in muscles requires cooperation between two KASH proteins and microtubules

Striated muscle fibers are characterized by their tightly organized cytoplasm. Here, we show 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-300(ΔKASH), 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, our 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.     

UNC-83 IS a KASH protein required for nuclear migration and is recruited to the outer nuclear membrane by a physical interaction with the SUN protein UNC-84

UNC-84 is required to localize UNC-83 to the nuclear envelope where it functions during nuclear migration. A KASH domain in UNC-83 was identified. KASH domains are conserved in the nuclear envelope proteins Syne/nesprins, Klarsicht, MSP-300, and ANC-1. Caenorhabditis elegans UNC-83 was shown to localize to the outer nuclear membrane and UNC-84 to the inner nuclear membrane in transfected mammalian cells, suggesting the KASH and SUN protein targeting mechanisms are conserved. Deletion of the KASH domain of UNC-83 blocked nuclear migration and localization to the C. elegans nuclear envelope. Some point mutations in the UNC-83 KASH domain disrupted nuclear migration, even if they localized normally. At least two separable portions of the C-terminal half of UNC-84 were found to interact with the UNC-83 KASH domain in a membrane-bound, split-ubiquitin yeast two-hybrid system. However, the SUN domain was essential for UNC-84 function and UNC-83 localization in vivo. These data support the model that KASH and SUN proteins bridge the nuclear envelope, connecting the nuclear lamina to cytoskeletal components. This mechanism seems conserved across eukaryotes and is the first proposed mechanism to target proteins specifically to the outer nuclear membrane.     

Structure of Sad1-UNC84 homology (SUN) domain defines features of molecular bridge in nuclear envelope

The SUN (Sad1-UNC-84 homology) domain is conserved in a number of nuclear envelope proteins involved in nuclear migration, meiotic telomere tethering, and antiviral responses. The LINC (linker of nucleoskeleton and cytoskeleton) complex, formed by the SUN and the nesprin proteins at the nuclear envelope, serves as a mechanical linkage across the nuclear envelope. Here we report the crystal structure of the SUN2 protein SUN domain, which reveals a homotrimer. The SUN domain is sufficient to mediate binding to the KASH (Klarsicht, ANC-1, and Syne homology) domain of nesprin 2, and the regions involved in the interaction have been identified. Binding of the SUN domain to the KASH domain is abolished by deletion of a region important for trimerization or by point mutations associated with nuclear migration failure. We propose a model of the LINC complex, where the SUN and the KASH domains form a higher ordered oligomeric network in the nuclear envelope. These findings provide the structural basis for understanding the function and the regulation of the LINC complex.     

KASH 'n Karry: the KASH domain family of cargo-specific cytoskeletal adaptor proteins

A diverse family of proteins has been discovered with a small C-terminal KASH domain in common. KASH domain proteins are localized uniquely to the outer nuclear envelope, enabling their cytoplasmic extensions to tether the nucleus to actin filaments or microtubules. KASH domains are targeted to the outer nuclear envelope by SUN domains of inner nuclear envelope proteins. Several KASH protein genes were discovered as mutant alleles in model organisms with defects in developmentally regulated nuclear positioning. Recently, KASH-less isoforms have been found that connect the cytoskeleton to organelles other than the nucleus. A widened view of these proteins is now emerging, where KASH proteins and their KASH-less counterparts are cargo-specific adaptors that not only link organelles to the cytoskeleton but also regulate developmentally specific organelle movements.     

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.     

A conserved KASH domain protein associates with telomeres, SUN1, and dynactin during mammalian meiosis

In yeasts and worms, KASH (Klarsicht/ANC-1/Syne/homology) domain and SUN (Sad-1/UNC-84) domain nuclear envelope (NE) proteins play a crucial role in meiotic chromosome movement and homologue pairing. However, although the vertebrate SUN domain protein SUN1 is involved in these processes, its partner has remained identified. Based on subcellular localization screening in mouse spermatocytes, we identified a novel germ cell-specific protein, KASH5, that localized exclusively at telomeres from the leptotene to diplotene stages in both spermatocytes and oocytes. KASH5 possesses hitherto unknown KASH-related sequences that directly interacted with SUN1 and mediated telomere localization. Thus, KASH5 is a mammalian meiosis-specific KASH domain protein. We show that meiotic chromosome movement depended on microtubules and that KASH5 interacted with the microtubule-associated dynein-dynactin complex. These results suggest that KASH5 connects the telomere-associated SUN1 protein to the cytoplasmic force-generating mechanism involved in meiotic chromosome movement. Our study strongly suggests that the meiotic homologue-pairing mechanism mediated by the SUN-KASH NE bridge is highly conserved among eukaryotes.     

The KASH domain protein MSP-300 plays an essential role in nuclear anchoring during Drosophila oogenesis

During late stages of Drosophila oogenesis, the cytoplasm of nurse cells in the egg chamber is rapidly transferred ("dumped") to oocytes, while the nurse cell nuclei are anchored by a mechanism that involves the actin cytoskeleton. The factors that mediate this interaction between nuclei and actin cytoskeleton are unknown. MSP-300 is the likely Drosophila ortholog of the mammalian Syne-1 and -2 and C. elegans ANC-1 proteins, contained both actin-binding and nuclear envelope localization domains. By using an antibody against C-terminus of MSP-300, we find that MSP-300 is distributed throughout the cytoplasm and accumulates at the nuclear envelope of nurse cells and the oocyte. A GFP fusion protein containing the C-terminal region of MSP-300 is also sufficient to localize protein on the nuclear envelope in oocytes. To eliminate the maternal gene activity during oogenesis, we generated homozygous germ-line clones of a loss-of-function mutation in msp-300 in otherwise heterozygous mothers. In the mutant egg chambers that develop from such clones, cytoplasmic dumping of nurse cells is severely disturbed. The nuclei of nurse cells and the oocyte are mislocalized and the usually well-organized actin structures are severely disrupted. These results indicate that maternal MSP-300 plays an important role in actin-dependent nuclear anchorage during cytoplasmic transport.     

Plant SUN domain proteins: Components of putative plant LINC complexes?

We have recently reported the identification and characterization of Sad1/UNC84 (SUN) domain proteins in various plant species. In animals and yeasts, SUN domain proteins are localized at the inner nuclear membrane and form a bridge across the nuclear envelope (NE) by interacting with outer nuclear membrane-localized Klarsicht/Anc-1/Syne-1 homology (KASH) domain proteins. This bridge physically connects cytoskeletal elements with chromatin and nucleoskeletal components. These multiprotein complexes are essential for various cellular and nuclear processes. The identification of SUN domain proteins provides the first evidence of putative NE bridging complexes in plants. Here we speculate on the composition and functions of these in regards to our current understanding of plant SUN domain proteins.Key words: SUN domain protein, LINC complex, plant nuclear envelope, cytoskeleton, KASH domain proteins, Arabidopsis     

Analysis of Meiosis in SUN1 Deficient Mice Reveals a Distinct Role of SUN2 in Mammalian Meiotic LINC Complex Formation and Function

LINC complexes are evolutionarily conserved nuclear envelope bridges, composed of SUN (Sad-1/UNC-84) and KASH (Klarsicht/ANC-1/Syne/homology) domain proteins. They are crucial for nuclear positioning and nuclear shape determination, and also mediate nuclear envelope (NE) attachment of meiotic telomeres, essential for driving homolog synapsis and recombination. In mice, SUN1 and SUN2 are the only SUN domain proteins expressed during meiosis, sharing their localization with meiosis-specific KASH5. Recent studies have shown that loss of SUN1 severely interferes with meiotic processes. Absence of SUN1 provokes defective telomere attachment and causes infertility. Here, we report that meiotic telomere attachment is not entirely lost in mice deficient for SUN1, but numerous telomeres are still attached to the NE through SUN2/KASH5-LINC complexes. In Sun1^−/− meiocytes attached telomeres retained the capacity to form bouquet-like clusters. Furthermore, we could detect significant numbers of late meiotic recombination events in Sun1^−/− mice. Together, this indicates that even in the absence of SUN1 telomere attachment and their movement within the nuclear envelope per se can be functional.     

Identification of unique SUN-interacting nuclear envelope proteins with diverse functions in plants

Although a plethora of nuclear envelope (NE) transmembrane proteins (NETs) have been identified in opisthokonts, plant NETs are largely unknown. The only known NET homologues in plants are Sad1/UNC-84 (SUN) proteins, which bind Klarsicht/ANC-1/Syne-1 homology (KASH) proteins. Therefore, de novo identification of plant NETs is necessary. Based on similarities between opisthokont KASH proteins and the only known plant KASH proteins, WPP domain–interacting proteins, we used a computational method to identify the KASH subset of plant NETs. Ten potential plant KASH protein families were identified, and five candidates from four of these families were verified for their NE localization, depending on SUN domain interaction. Of those, Arabidopsis thaliana SINE1 is involved in actin-dependent nuclear positioning in guard cells, whereas its paralogue SINE2 contributes to innate immunity against an oomycete pathogen. This study dramatically expands our knowledge of plant KASH proteins and suggests that plants and opisthokonts have recruited different KASH proteins to perform NE regulatory functions.     

Molecular analysis of the klarsicht gene and its role in nuclear migration within differentiating cells of the Drosophila eye.

BACKGROUND: 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. RESULTS: Here, we have cloned the klarsicht gene and shown that it encodes a unique large protein. Drosophila klarsicht null mutants were viable, with obvious defects only in adult eye morphology. Epitope-tagged Klarsicht expressed in the eye from a transgene was perinuclear. In flies carrying transgenes that express markers for microtubule plus and minus ends, microtubules in differentiating cells of the eye were oriented with their plus ends apical and their minus ends at the nucleus. CONCLUSIONS: Drosophila klarsicht null mutants were 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.     

Novel plant SUN-KASH bridges are involved in RanGAP anchoring and nuclear shape determination

Inner nuclear membrane Sad1/UNC-84 (SUN) proteins interact with outer nuclear membrane (ONM) Klarsicht/ANC-1/Syne homology (KASH) proteins, forming linkers of nucleoskeleton to cytoskeleton conserved from yeast to human and involved in positioning of nuclei and chromosomes. Defects in SUN-KASH bridges are linked to muscular dystrophy, progeria, and cancer. SUN proteins were recently identified in plants, but their ONM KASH partners are unknown. Arabidopsis WPP domain-interacting proteins (AtWIPs) are plant-specific ONM proteins that redundantly anchor Arabidopsis RanGTPase-activating protein 1 (AtRanGAP1) to the nuclear envelope (NE). In this paper, we report that AtWIPs are plant-specific KASH proteins interacting with Arabidopsis SUN proteins (AtSUNs). The interaction is required for both AtWIP1 and AtRanGAP1 NE localization. AtWIPs and AtSUNs are necessary for maintaining the elongated nuclear shape of Arabidopsis epidermal cells. Together, our data identify the first KASH members in the plant kingdom and provide a novel function of SUN-KASH complexes, suggesting that a functionally diverged SUN-KASH bridge is conserved beyond the opisthokonts.     

LINC complexes form by binding of three KASH peptides to domain interfaces of trimeric SUN proteins

Linker of nucleoskeleton and cytoskeleton (LINC) complexes span the nuclear envelope and are composed of KASH and SUN proteins residing in the outer and inner nuclear membrane, respectively. LINC formation relies on direct binding of KASH and SUN in the perinuclear space. Thereby, molecular tethers are formed that can transmit forces for chromosome movements, nuclear migration, and anchorage. We present crystal structures of the human SUN2-KASH1/2 complex, the core of the LINC complex. The SUN2 domain is rigidly attached to a trimeric coiled coil that prepositions it to bind three KASH peptides. The peptides bind in three deep and expansive grooves formed between adjacent SUN domains, effectively acting as molecular glue. In addition, a disulfide between conserved cysteines on SUN and KASH covalently links both proteins. The structure provides the basis of LINC complex formation and suggests a model for how LINC complexes might arrange into higher-order clusters to enhance force-coupling.     

Organelle-specific control of intracellular transport: distinctly targeted isoforms of the regulator Klar

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, we analyzed the Drosophila Klarsicht (Klar) protein that regulates distinct microtubule-based transport processes. We find that localization of Klar to its cargoes is crucial for Klar function. Using mutations, we identify functionally important regions of Klar that confer distinct cargo specificity. 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. Our analysis disentangles Klar's modular organization: we propose 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.     

The diverse functional LINCs of the nuclear envelope to the cytoskeleton and chromatin

The nuclear envelope (NE) is connected to the different types of cytoskeletal elements by linker of nucleoskeleton and cytoskeleton (LINC) complexes. LINC complexes exist from yeast to humans, and have preserved their general architecture throughout evolution. They are composed of SUN and KASH domain proteins of the inner and the outer nuclear membrane, respectively. These SUN–KASH bridges are used for the transmission of forces across the NE and support diverse biological processes. Here, we review the function of SUN and KASH domain proteins in various unicellular and multicellular species. Specifically, we discuss their influence on nuclear morphology and cytoskeletal organization. Further, emphasis is given on the role of LINC complexes in nuclear anchorage and migration as well as in genome organization.     

SUN1 interacts with nuclear lamin A and cytoplasmic nesprins to provide a physical connection between the nuclear lamina and the cytoskeleton

Nuclear migration and positioning within cells are critical for many developmental processes and are governed by the cytoskeletal network. Although mechanisms of nuclear-cytoskeletal attachment are unclear, growing evidence links a novel family of nuclear envelope (NE) proteins that share a conserved C-terminal SUN (Sad1/UNC-84 homology) domain. Analysis of Caenorhabditis elegans mutants has implicated UNC-84 in actin-mediated nuclear positioning by regulating NE anchoring of a giant actin-binding protein, ANC-1. Here, we report the identification of SUN1 as a lamin A-binding protein in a yeast two-hybrid screen. We demonstrate that SUN1 is an integral membrane protein located at the inner nuclear membrane. While the N-terminal domain of SUN1 is responsible for detergent-resistant association with the nuclear lamina and lamin A binding, lamin A/C expression is not required for SUN1 NE localization. Furthermore, SUN1 does not interact with type B lamins, suggesting that NE localization is ensured by binding to an additional nuclear component(s), most likely chromatin. Importantly, we find that the luminal C-terminal domain of SUN1 interacts with the mammalian ANC-1 homologs nesprins 1 and 2 via their conserved KASH domain. Our data provide evidence of a physical nuclear-cytoskeletal connection that is likely to be a key mechanism in nuclear-cytoplasmic communication and regulation of nuclear position.     

The Caenorhabditis elegans SUN protein UNC-84 interacts with lamin to transfer forces from the cytoplasm to the nucleoskeleton during nuclear migration

Nuclear migration is a critical component of many cellular and developmental processes. The nuclear envelope forms a barrier between the cytoplasm, where mechanical forces are generated, and the nucleoskeleton. The LINC complex consists of KASH proteins in the outer nuclear membrane and SUN proteins in the inner nuclear membrane that bridge the nuclear envelope. How forces are transferred from the LINC complex to the nucleoskeleton is poorly understood. The Caenorhabditis elegans lamin, LMN-1, is required for nuclear migration and interacts with the nucleoplasmic domain of the SUN protein UNC-84. This interaction is weakened by the unc-84(P91S) missense mutation. These mutant nuclei have an intermediate nuclear migration defect—live imaging of nuclei or LMN-1::GFP shows that many nuclei migrate normally, others initiate migration before subsequently failing, and others fail to begin migration. At least one other component of the nucleoskeleton, the NET5/Samp1/Ima1 homologue SAMP-1, plays a role in nuclear migration. We propose a nut-and-bolt model to explain how forces are dissipated across the nuclear envelope during nuclear migration. In this model, SUN/KASH bridges serve as bolts through the nuclear envelope, and nucleoskeleton components LMN-1 and SAMP-1 act as both nuts and washers on the inside of the nucleus.