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.     

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.     

SUN proteins facilitate the removal of membranes from chromatin during nuclear envelope breakdown

SUN proteins reside in the inner nuclear membrane and form complexes with KASH proteins of the outer nuclear membrane that connect the nuclear envelope (NE) to the cytoskeleton. These complexes have well-established functions in nuclear anchorage and migration in interphase, but little is known about their involvement in mitotic processes. Our analysis demonstrates that simultaneous depletion of human SUN1 and SUN2 delayed removal of membranes from chromatin during NE breakdown (NEBD) and impaired the formation of prophase NE invaginations (PNEIs), similar to microtubule depolymerization or down-regulation of the dynein cofactors NudE/EL. In addition, overexpression of dominant-negative SUN and KASH constructs reduced the occurrence of PNEI, indicating a requirement for functional SUN–KASH complexes in NE remodeling. Codepletion of SUN1/2 slowed cell proliferation and resulted in an accumulation of morphologically defective and disoriented mitotic spindles. Quantification of mitotic timing revealed a delay between NEBD and chromatin separation, indicating a role of SUN proteins in bipolar spindle assembly and mitotic progression.     

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 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.     

SUN-1 and ZYG-12, Mediators of Centrosome–Nucleus Attachment,Are a Functional SUN/KASH Pair in Caenorhabditis elegans

Klarsicht/ANC-1/Syne/homology (KASH)/Sad-1/UNC-84 (SUN) protein pairs can act as connectors between cytoplasmic organelles and the nucleoskeleton. Caenorhabditis elegans ZYG-12 and SUN-1 are essential for centrosome–nucleus attachment. Although SUN-1 has a canonical SUN domain, ZYG-12 has a divergent KASH domain. Here, we establish that the ZYG-12 mini KASH domain is functional and, in combination with a portion of coiled-coil domain, is sufficient for nuclear envelope localization. ZYG-12 and SUN-1 are hypothesized to be outer and inner nuclear membrane proteins, respectively, and to interact, but neither their topologies nor their physical interaction has been directly investigated. We show that ZYG-12 is a type II outer nuclear membrane (ONM) protein and that SUN-1 is a type II inner nuclear membrane protein. The proteins interact in the luminal space of the nuclear envelope via the ZYG-12 mini KASH domain and a region of SUN-1 that does not include the SUN domain. SUN-1 is hypothesized to restrict ZYG-12 to the ONM, preventing diffusion through the endoplasmic reticulum. We establish that ZYG-12 is indeed immobile at the ONM by using fluorescence recovery after photobleaching and show that SUN-1 is sufficient to localize ZYG-12 in cells. This work supports current models of KASH/SUN pairs and highlights the diversity in sequence elements defining KASH domains.     

The Drosophila KASH domain proteins Msp-300 and Klarsicht and the SUN domain protein Klaroid have no essential function during oogenesis

Proteins harboring a C-terminal KASH (Klarsicht/Anc-1/Syne Homology) domain, which attaches to the nucleus, have been identified in many different organisms. Two KASH proteins are known from Drosophila, Msp-300 and Klarsicht, the latter of which plays a role in nuclear migration during eye development. Here, we show that a complete deletion of Msp-300 leads to larval lethality. This lethality appears to be due to Msp-300 isoforms containing the N-terminal actin binding, but not the C-terminal KASH domain. Msp-300 and Klar are expressed during oogenesis and localize to the nuclear envelope of the germ line nuclei. However, neither Msp-300 single mutants nor Msp-300; klar double mutants cause defects in nuclear migration or anchoring during oogenesis. Germ line nuclear envelope localization of both KASH domain proteins depends on klaroid, the only Drosophila SUN domain homolog expressed in females. Like Msp-300 and klar, klaroid is also dispensable for normal ovarian development.     

Another way to move chromosomes

A typical way of moving chromosomes is exemplified by mitotic segregation, in which the centromere is directly captured by spindle microtubules. In this study, we highlight another way of moving chromosomes remotely from outside the nucleus, which involves SUN and KASH domain nuclear envelope proteins. SUN and KASH domain protein families are known to connect the nucleus to cytoskeletal networks and play a role in migration and positioning of the nucleus. Recent studies in the fission yeast Schizossacharomyces pombe demonstrated an additional role for the SUN–KASH protein complex in chromosome movements. During meiotic prophase, telomeres are moved to rearrange chromosomes within the nucleus. The SUN–KASH protein complex located in the nuclear envelope is involved in this process. Telomeres are connected to the SUN protein on the nucleoplasmic side, and the dynein motor complex binds to the KASH protein on the cytoplasmic side. Telomeres are then moved along the nuclear envelope using cytoplasmic microtubules. These findings illustrate a general mechanism for transmitting a cytoskeletal driving force to chromosomes across the nuclear envelope. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Note added in proof Recently, a related article on C. elegans SUN protein has been published: Penkner A, Tang L, Novatchkova M, Ladurner M, Fridkin A, Gruenbaum Y, Schweizer D, Loidl J, Jantsch V (2007) The nuclear envelope protein Matefin/Sun-1 is required for homologous pairing in C. elegans meiosis. Dev Cell 12:873–885     

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.     

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.     

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.     

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.     

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.     

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.     

Mammalian SUN Protein Interaction Networks at the Inner Nuclear Membrane and Their Role in Laminopathy Disease Processes

The nuclear envelope (NE) LINC complex, in mammals comprised of SUN domain and nesprin proteins, provides a direct connection between the nuclear lamina and the cytoskeleton, which contributes to nuclear positioning and cellular rigidity. SUN1 and SUN2 interact with lamin A, but lamin A is only required for NE localization of SUN2, and it remains unclear how SUN1 is anchored. Here, we identify emerin and short nesprin-2 isoforms as novel nucleoplasmic binding partners of SUN1/2. These have overlapping binding sites distinct from the lamin A binding site. However, we demonstrate that tight association of SUN1 with the nuclear lamina depends upon a short motif within residues 209–228, a region that does not interact significantly with known SUN1 binding partners. Moreover, SUN1 localizes correctly in cells lacking emerin. Importantly then, the major determinant of SUN1 NE localization has yet to be identified. We further find that a subset of lamin A mutations, associated with laminopathies Emery-Dreifuss muscular dystrophy (EDMD) and Hutchinson-Gilford progeria syndrome (HGPS), disrupt lamin A interaction with SUN1 and SUN2. Despite this, NE localization of SUN1 and SUN2 is not impaired in cell lines from either class of patients. Intriguingly, SUN1 expression at the NE is instead enhanced in a significant proportion of HGPS but not EDMD cells and strongly correlates with pre-lamin A accumulation due to preferential interaction of SUN1 with pre-lamin A. We propose that these different perturbations in lamin A-SUN protein interactions may underlie the opposing effects of EDMD and HGPS mutations on nuclear and cellular mechanics.     

Structural insights into SUN-KASH complexes across the nuclear envelope

Linker of the nucleoskeleton and the cytoskeleton (LINC) complexes are composed of SUN and KASH domain-containing proteins and bridge the inner and outer membranes of the nuclear envelope. LINC complexes play critical roles in nuclear positioning, cell polarization and cellular stiffness. Previously, we reported the homotrimeric structure of human SUN2. We have now determined the crystal structure of the human SUN2-KASH complex. In the complex structure, the SUN domain homotrimer binds to three independent “hook”-like KASH peptides. The overall conformation of the SUN domain in the complex closely resembles the SUN domain in its apo state. A major conformational change involves the AA''-loop of KASH-bound SUN domain, which rearranges to form a mini β-sheet that interacts with the KASH peptide. The PPPT motif of the KASH domain fits tightly into a hydrophobic pocket on the homotrimeric interface of the SUN domain, which we termed the BI-pocket. Moreover, two adjacent protomers of the SUN domain homotrimer sandwich the KASH domain by hydrophobic interaction and hydrogen bonding. Mutations of these binding sites disrupt or reduce the association between the SUN and KASH domains in vitro. In addition, transfection of wild-type, but not mutant, SUN2 promotes cell migration in Ovcar-3 cells. These results provide a structural model of the LINC complex, which is essential for additional study of the physical and functional coupling between the cytoplasm and the nucleoplasm.     

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.     

Dynamics of Arabidopsis SUN proteins during mitosis and their involvement in nuclear shaping

The nuclear envelope (NE) is a highly active structure with a specific set of nuclear envelope proteins acting in diverse cellular events. SUN proteins are conserved NE proteins among eukaryotes. Although they form nucleocytoplasmic linkage complexes in metazoan cells, their functions in the plant kingdom are unknown. To understand the function of plant SUN proteins, in this study we first investigated the dynamics of Arabidopsis SUN proteins during mitosis in Arabidopsis roots and cultured cells. For this purpose, we performed dual and triple visualization of these proteins, microtubules, chromosomes, and endoplasmic reticulum (ER) in cultured cells, and observed their dynamics during mitosis using a high-speed spinning disk confocal microscope. The localizations of SUN proteins changed dynamically during mitosis, tightly coupled with NE dynamics. Moreover, NE re-formation marked with SUN proteins is temporally and spatially coordinated with plant-specific microtubule structures such as phragmoplasts. Finally, the analysis with gene knockdowns of AtSUN1 and AtSUN2 indicated that they are necessary for the maintenance and/or formation of polarized nuclear shape in root hairs. These results suggest that Arabidopsis SUN proteins function in the maintenance or formation of nuclear shape as components of the nucleocytoskeletal complex.     

Polo kinases establish links between meiotic chromosomes and cytoskeletal forces essential for homolog pairing

During meiosis, chromosomes must find and align with their homologous partners. SUN and KASH-domain protein pairs play a conserved role by establishing transient linkages between chromosome ends and cytoskeletal forces across the intact nuclear envelope (NE). In C.?elegans, a pairing center (PC) on each chromosome mediates homolog pairing and linkage to the microtubule network. We report that the polo kinases PLK-1 and PLK-2 are targeted to the PC by ZIM/HIM-8-pairing proteins. Loss of plk-2 inhibits chromosome pairing and licenses synapsis between nonhomologous chromosomes, indicating that PLK-2 is required for PC-mediated interhomolog interactions. plk-2 is also required for meiosis-specific phosphorylation of SUN-1 and establishment of dynamic SUN/KASH (SUN-1/ZYG-12) modules that promote homolog pairing. Our results provide key insights into the regulation of homolog pairing and reveal that targeting of polo-like kinases to the NE by meiotic chromosomes establishes the conserved linkages to cytoskeletal forces needed for homology assessment.