Sun, Yue et al. (2021) International Worm Meeting "A negative feedback mechanism regulates DLK-1 signaling in ciliated sensory neurons"

Sun, Yue, Jin, Yishi

[International Worm Meeting, 2021]

The conserved Dual-Leucine zipper Kinases (DLKs) are neuronal sensors to many forms of stress and traumatic injury. When activated under stress conditions, DLKs act as MAP3Ks upstream of JNK or p38 MAPK signaling to trigger neuroprotective responses. Constitutive activation of DLK, such as via overexpression, can lead to neurodegeneration in different disease models. Previous studies have revealed that under normal conditions, DLK protein activity is controlled by E3 ubiquitin ligase PHR(PAM/Highwire/RPM-1) mediated degradation and chaperones. However, it remains to be discovered whether there are other mechanisms regulating endogenous DLKs. To search for new regulators of C. elegans DLK-1, we performed a forward genetic screen using a GFP knock-in that tags endogenous dlk-1. We isolated a class of mutants showing ectopic accumulation of DLK-1 at dendritic endings of ciliated sensory neurons. We determined that multiple mutations cause loss-of-function in IntraFlagellar Transport (IFT) genes and impair cilia architecture and function in sensory neurons. The ectopic accumulation of DLK-1 at defective cilia activates signal transduction cascade, as we observed elevated expression of the transcription factor CEBP-1, a key downstream target of DLK-1 pathway, in many sensory neurons. However, the increased CEBP-1 expression shows selective dependency on dlk-1. To probe into the neuron-type specificity, we carried out quantitative analyses of CEBP-1 levels in several types of sensory neurons. We found that IFT defects in AWB and AWC cause increased CEBP-1 expression dependent on dlk-1, whereas similar IFT defects in ASI and ASH induce CEBP-1 elevation independent of dlk-1. Moreover, in AWC cebp-1 further represses expression of wild type DLK-1 but not a kinase-dead DLK-1, suggesting a negative feedback control of DLK-1 signaling via CEBP-1. Additionally, we found that in AWC neurons overexpression of DLK-1 causes dendrite overgrowth by activating CEBP-1. Together, we propose that in ciliated sensory neurons, DLK-1 signaling is regulated under a cell-specific feedback mechanism, which is coupled with IFT and may contribute to the maintenance of neuronal morphology.

Baudrimont A et al. (2010) PLoS Genet "Leptotene/zygotene chromosome movement via the SUN/KASH protein bridge in Caenorhabditis elegans."

Pasierbek P, Machacek T, Penkner A, Klein F, Fridkin A, Woglar A, Baudrimont A, Jantsch V, Gruenbaum Y, Gloggnitzer J, Wegrostek C

[PLoS Genet, 2010]

The Caenorhabditis elegans inner nuclear envelope protein matefin/SUN-1 plays a conserved, pivotal role in the process of genome haploidization. CHK-2-dependent phosphorylation of SUN-1 regulates homologous chromosome pairing and interhomolog recombination in Caenorhabditis elegans. Using time-lapse microscopy, we characterized the movement of matefin/SUN-1::GFP aggregates (the equivalent of chromosomal attachment plaques) and showed that the dynamics of matefin/SUN-1 aggregates remained unchanged throughout leptonene/zygotene, despite the progression of pairing. Movement of SUN-1 aggregates correlated with chromatin polarization. We also analyzed the requirements for the formation of movement-competent matefin/SUN-1 aggregates in the context of chromosome structure and found that chromosome axes were required to produce wild-type numbers of attachment plaques. Abrogation of synapsis led to a deceleration of SUN-1 aggregate movement. Analysis of matefin/SUN-1 in a double-strand break deficient mutant revealed that repair intermediates influenced matefin/SUN-1 aggregate dynamics. Investigation of movement in meiotic regulator mutants substantiated that proper orchestration of the meiotic program and effective repair of DNA double-strand breaks were necessary for the wild-type behavior of matefin/SUN-1 aggregates.

Minn I et al. (2009) Mol Biol Cell "SUN-1 and ZYG-12, mediators of centrosome-nucleus attachment, are a functional SUN/KASH pair in Caenorhabditis elegans."

Minn I, Hanna-Rose W, Rolls MM, Malone CJ

[Mol Biol Cell, 2009]

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.

Hao H et al. (2019) Nucleus "SUN/KASH interactions facilitate force transmission across the nuclear envelope."

Starr DA, Hao H

[Nucleus, 2019]

LINC complexes (Linker of Nucleoskeleton and Cytoskeleton), consisting of inner nuclear membrane SUN proteins and outer nuclear membrane KASH proteins, are essential for nuclear positioning, cell migration and chromosome dynamics. Crystal structures of the central SUN-KASH interaction domains revealed close interfaces predicted to mediate LINC complex formation and function. However, the in vivo significance of these conserved interactions were unclear. Cain et al (2018) used a combination of Caenorhabditis elegans genetics, imaging in cultured NIH 3T3 fibroblasts, and Molecular Dynamic simulations, to study SUN-KASH interactions in cells. The study found that a conserved aromatic residue at the -7 position of the C-terminal KASH domain and conserved cysteines in both the KASH and SUN domains play important roles in force transmission across the nuclear envelope. Other properties of LINC complexes may also play significant roles in transmitting mechanical forces generated by the cytoskeleton across the nuclear envelope. For example, the helicies preceding the SUN domain activate SUN proteins and may be important for activating KASH-SUN interactions or for association of SUN and/or KASH proteins with the membrane. The longer coiled-coils spanning the perinuclear space may also play a role in mediating force transmission. Finally, LINC complexes may be arranged in higher-order structures to facilitate high force-bearing processes.

Cain NE et al. (2014) J Cell Biol "The SUN protein UNC-84 is required only in force-bearing cells to maintain nuclear envelope architecture."

McDonald KL, Cain NE, Tapley EC, Starr DA, Cain BM

[J Cell Biol, 2014]

The nuclear envelope (NE) consists of two evenly spaced bilayers, the inner and outer nuclear membranes. The Sad1p and UNC-84 (SUN) proteins and Klarsicht, ANC-1, and Syne homology (KASH) proteins that interact to form LINC (linker of nucleoskeleton and cytoskeleton) complexes connecting the nucleoskeleton to the cytoskeleton have been implicated in maintaining NE spacing. Surprisingly, the NE morphology of most Caenorhabditis elegans nuclei was normal in the absence of functional SUN proteins. Distortions of the perinuclear space observed in unc-84 mutant muscle nuclei resembled those previously observed in HeLa cells, suggesting that SUN proteins are required to maintain NE architecture in cells under high mechanical strain. The UNC-84 protein with large deletions in its luminal domain was able to form functional NE bridges but had no observable effect on NE architecture. Therefore, SUN-KASH bridges are only required to maintain NE spacing in cells subjected to increased mechanical forces. Furthermore, SUN proteins do not dictate the width of the NE.

Il Minn et al. (2007) International Worm Meeting "Centrosome Attachment to the Nucleus Requires the SUN-1 Mediated Localization of ZYG-12 to the Outer Membrane of the Nuclear Envelope."

Il Minn, Christian J. Malone

[International Worm Meeting, 2007]

The localization of proteins specifically to the outer membrane of the nuclear envelope is thought to be required for attachment of the centrosome to the nucleus. The outer membrane of the nuclear envelope is contiguous with the ER and has been considered to be identical to the ER. Thus, the mechanism of specific outer nuclear membrane localization is unknown. We and others have recently identified a novel class of proteins with KASH domains that specifically localize to the outer membrane of the nuclear envelope via an interaction with SUN proteins. We have shown that two isoforms of the C. elegans protein ZYG-12, which contain KASH and transmembrane domains, depend on a SUN protein, SUN-1 (also called MTF-1), to localize to the nuclear envelope. ZYG-12 is required for attachment of the centrosome to the nucleus in C. elegans. To map the domains of ZYG-12 necessary for SUN-1 mediated localization, we expressed C-terminal fragments of ZYG-12, which contain KASH and transmembrane domains. These C-terminal fragments are sufficient for nuclear envelope localization in a SUN-1 dependent manner. We have investigated the membrane topology of both ZYG-12 and SUN-1. Both epitope accessibility and fluorescent protease protection assays using in vivo nuclei in combination with proteinase K protection assays using microsomes revealed that ZYG-12 and SUN-1 are type II membrane proteins of the outer and inner membrane of the nuclear envelope, respectively. In support of ZYG-12 being a type II membrane protein of the outer nuclear envelope, ZYG-12 physically interacts with cytoplasmic dynein via the N-terminus of ZYG-12 and is required for the localization of dynein to the nucleus. To investigate the molecular mechanism of SUN-1 dependent ZYG-12 localization, we asked if the proteins physically interact. Our data indicate that SUN-1 directly interacts with the KASH domain of ZYG-12. The interaction occurs between the KASH domain and the internal domain distinct from the SUN domain of SUN-1 as mapped by a membrane based two-hybrid assay and a pull-down assay. ZYG-12 at the outer nuclear membrane is necessary for centrosome attachment. Our results suggest that the inner nuclear membrane protein SUN-1 confines localization of ZYG-12 to the outer nuclear membrane via direct binding in the lumen of the nuclear envelope.

Penkner A et al. (2007) Dev Cell "The nuclear envelope protein Matefin/SUN-1 is required for homologous pairing in C. elegans meiosis."

Penkner A, Gruenbaum Y, Loidl J, Jantsch V, Tang L, Schweizer D, Novatchkova M, Fridkin A, Ladurner M

[Dev Cell, 2007]

We identify a highly specific mutation (jf18) in the Caenorhabditis elegans nuclear envelope protein matefin MTF-1/SUN-1 that provides direct evidence for active involvement of the nuclear envelope in homologous chromosome pairing in C. elegans meiosis. The reorganization of chromatin in early meiosis is disrupted in mtf-1/sun-1(jf18) gonads, concomitant with the absence of presynaptic homolog alignment. Synapsis is established precociously and nonhomologously. Wild-type leptotene/zygotene nuclei show patch-like aggregations of the ZYG-12 protein, which fail to develop in mtf-1/sun-1(jf18) mutants. These patches remarkably colocalize with a component of the cis-acting chromosomal pairing center (HIM-8) rather than the centrosome. Our data on this mtf-1/sun-1 allele challenge the previously postulated role of the centrosome/spindle organizing center in chromosome pairing, and clearly support a role for MTF-1/SUN-1 in meiotic chromosome reorganization and in homolog recognition, possibly by mediating local aggregation of the ZYG-12 protein in meiotic nuclei.

Penkner AM et al. (2009) Cell "Meiotic chromosome homology search involves modifications of the nuclear envelope protein Matefin/SUN-1."

Jantsch V, Ammerer G, Woglar A, Machacek T, Fridkin A, Pasierbek P, Gruenbaum Y, Penkner AM, Gloggnitzer J, Baudrimont A, Csaszar E

[Cell, 2009]

Genome haploidization during meiosis depends on recognition and association of parental homologous chromosomes. The C. elegans SUN/KASH domain proteins Matefin/SUN-1 and ZYG-12 have a conserved role in this process. They bridge the nuclear envelope, connecting the cytoplasm and the nucleoplasm to transmit forces that allow chromosome movement and homolog pairing and prevent nonhomologous synapsis. Here, we show that Matefin/SUN-1 forms rapidly moving aggregates at putative chromosomal attachment sites in the meiotic transition zone (TZ). We analyzed requirements for aggregate formation and identified multiple phosphotarget residues in the nucleoplasmic domain of Matefin/SUN-1. These CHK-2 dependent phosphorylations occur in leptotene/zygotene, diminish during pachytene and are involved in pairing. Mimicking phosphorylation causes an extended TZ and univalents at diakinesis. Our data suggest that the properties of the nuclear envelope are altered during the time window when homologs are sorted and Matefin/SUN-1 aggregates form, thereby controling the movement, homologous pairing and interhomolog recombination of chromosomes.

Matthew McGee et al. (2007) International Worm Meeting "KASH and SUN proteins bridge the nuclear envelope to transfer force from the cytoskeleton to the nucleoskeleton during nuclear positioning."

Matthew McGee, Daniel Starr

[International Worm Meeting, 2007]

Nuclear positioning, which includes both migration and anchorage of the nucleus, is an important process in C. elegans development. The outer nuclear membrane (ONM) KASH proteins UNC-83 and ANC-1 are required for nuclear migration and anchorage, respectively. The inner nuclear membrane (INM) SUN protein UNC-84 is required for both nuclear migration and anchorage. The UNC-83 and ANC-1 KASH domains are thought to interact with the UNC-84 SUN domain in the perinuclear space. This allows force to be transferred from the cytoskeleton to the nucleoskeleton during nuclear positioning. KASH protein localization is dependent on SUN proteins, but SUN protein localization is not dependent on KASH proteins. Using a membrane-bound yeast two-hybrid system (Igor Stagljar, Dualsystems Biotech), we have shown that the KASH domain directly interacts with two separable domains of UNC-84, the SUN and linker domains. This interaction is required for nuclear migration and anchorage. Using a molecular genetics approach, we found that the UNC-84 SUN domain is required for KASH protein localization and function in vivo. We also demonstrated that point mutations in the KASH domain disrupt the function but not the localization of UNC-83. This suggests a stronger interaction, presumably to allow a large transfer of force during nuclear positioning, is required to move or anchor the nucleus than is required to simply localize the KASH proteins to the ONM (McGee, et al. Mol Biol Cell. 2006:1790-801). This KASH and SUN interaction appears to be conserved across eukaryotes. To identify residues in the linker domain that are required for interaction with the UNC-83 KASH domain, we are performing a mutational analysis of the UNC-84 linker domain in the membrane-bound two-hybrid system. We are also using this two-hybrid system in a screen to test whether there are unidentified binding partners with the SUN domain of UNC-84. In order to test the conservation of function between human and C. elegans KASH and SUN domains, we are testing whether the human domains in the C. elegans KASH and SUN proteins localize and are functional. We have found that a human KASH domain in UNC-83 is able to partially rescue UNC-83 function in migrating hyp-7 nuclei and localize to the nuclear envelope. We have also found that this hyp-7 rescue is dependent on the UNC-84 SUN domain. This suggests that the human KASH domain is able to interact with the UNC-84 SUN domain in vivo. Further experiments are testing the functional rescue of human SUN domains in UNC-84.

McGee MD et al. (2009) J Cell Sci "KDP-1 is a nuclear envelope KASH protein required for cell-cycle progression."

Stagljar I, Starr DA, McGee MD

[J Cell Sci, 2009]

Klarsicht, ANC-1 and Syne homology (KASH) proteins localize to the outer nuclear membrane where they connect the nucleus to the cytoskeleton. KASH proteins interact with Sad1-UNC-84 (SUN) proteins to transfer forces across the nuclear envelope to position nuclei or move chromosomes. A new KASH protein, KDP-1, was identified in a membrane yeast two-hybrid screen of a Caenorhabditis elegans library using the SUN protein UNC-84 as bait. KDP-1 also interacted with SUN-1. KDP-1 was enriched at the nuclear envelope in a variety of tissues and required SUN-1 for nuclear envelope localization in the germline. Genetic analyses showed that kdp-1 was essential for embryonic viability, larval growth and germline development. kdp-1(RNAi) delayed the entry into mitosis in embryos, led to a small mitotic zone in the germline, and caused an endomitotic phenotype. Aspects of these phenotypes were similar to those seen in sun-1(RNAi), suggesting that KDP-1 functions with SUN-1 in the germline and early embryo. The data suggest that KDP-1 is a novel KASH protein that functions to ensure the timely progression of the cell cycle between the end of S phase and the entry into mitosis.