Sun, Jingru et al. (2021) International Worm Meeting "A novel in vitro Caenorhabiditis elegans transcription system"

Sun, Jingru, Wibisono, Phillip, Liu, Yiyong

[International Worm Meeting, 2021]

Caenorhabditis elegans is an excellent model organism for biological research, but its contributions to biochemical elucidation of eukaryotic transcription mechanisms have been limited. One of the biggest obstacles for C. elegans biochemical studies is the high difficulty of obtaining functionally active nuclear extract due to its thick surrounding cuticle. By employing Balch homogenization, we achieved effective disruption of larval and adult worms and obtained functionally active nuclear extract through subcellular fractionation. In vitro transcription reactions were successfully re-constituted using such nuclear extract. Furthermore, a PCR-based non-radioactive detection method was adapted into our system to either qualitatively or quantitatively detect transcription. Using this system to assess how pathogen infection affects C. elegans transcription revealed that Pseudomonas aeruginosa infection changes transcription activity in a promoter- or gene-specific manner. This in vitro system is useful for biochemically studying C. elegans transcription mechanisms and gene expression regulation. The effective preparation of functionally active nuclear extract in our system fills a technical gap in biochemical studies of C. elegans and will expand the usefulness of this model organism in addressing many biological questions beyond transcription.

Sun, Jingru et al. (2011) International Worm Meeting "G-protein coupled receptor OCTR-1 controls innate immunity by regulating non-canonical unfolded protein response genes."

Sun, Jingru, Aballay, Alejandro, Kajino-Sakamoto, Rie, Singh, Varsha

[International Worm Meeting, 2011]

The endoplasmic reticulum (ER) has developed specific signaling pathways called the unfolded protein response (UPR) to cope with ER stress and restore ER homeostasis. Recent studies indicate that the increased demand on protein folding in the ER, that may occur during bacterial infections, must be successfully alleviated by canonical and non-canonical UPR pathways for a complete immune response to be mounted. We found that the nervous system controls the activity of a non-canonical UPR pathway required for innate immunity in Caenorhabditis elegans. OCTR-1, a G-protein coupled receptor, functions in ASH and ASI neurons to actively suppress innate immune responses by down-regulating the expression of non-canonical UPR genes pqn/abu in non-neuronal tissues. Pseudomonas aeruginosa exposure up-regulates pqn/abu genes to levels comparable to those induced by treatment with the ER stressor tunicamycin. Our studies highlight pathways that may play important roles at dealing with pathogens directly and with the damages caused by the infection.

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.

Wibisono, Shawndra et al. (2021) International Worm Meeting "Neural G protein-coupled receptor OCTR-1 regulates temperature effects on lifespan in C. elegans"

Wibisono, Phillip, Wibisono, Shawndra, Sun, Jingru, Chen, Chia-Hui

[International Worm Meeting, 2021]

We have previously demonstrated that OCTR-1, an octopamine G protein-couple receptor, functions in the sensory neurons ASH to suppress the innate immune response in Caenorhabditis elegans by inhibiting the expression of immune genes (Sun et al. 2011 Science 332:729-732). Here we discover that OCTR-1 also regulates temperature effects on lifespan in C. elegans. At the normal growth temperature 20°C, octr-1(ok371) mutant animals have similar lifespan to wild-type animals. However, at higher temperature 25°C, octr-1(ok371) mutants live significant longer than wild-type animals. These results suggest that OCTR-1 may mediate temperature effects on lifespan. RNA-seq data analysis showed that 63 immune genes were significantly down-regulated in octr-1(ok371) mutants relative to wild-type animals at 25°C. We further demonstrated that inactivation of several most-downregulated genes by RNA interference in wild type animals significantly extended their lifespan, similar to the phenotype of octr-1(ok371) animals. These suggest a new molecular regulation mechanism that downregulation of immune genes extend the lifespan of C. elegans, which is opposite to the general belief that an increase in defense immunity extends lifespan. We are currently investigating the underlying molecular mechanism in detail and also examining if the thermosensory neurons AFD and the chemosensory neurons ASH are involved in this OCTR-1-mediated regulation on lifespan.

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.

Singh, Varsha et al. (2011) International Worm Meeting "Neuronal control of stress response and innate immunity in C. elegans."

Singh, Varsha, Aballay, Alejandro, Sun, Jingru

[International Worm Meeting, 2011]

Heat Shock Protein (HSP) and Unfolded Protein Response (UPR) genes are upregulated in response to various stresses including heat shock, oxidative stress and ER stress. In C. elegans, HSP and UPR genes are also required for an adequate immune response to pathogens. Recent studies have shown that thermosensory neurons can regulate HSP gene induction at the organismal level. Increasing evidence also indicates that the nervous system of C. elegans controls avoidance to certain pathogens and activation of innate immune pathways. To identify neurons capable of controlling the activation of HSP or UPR genes as well as innate immune responses to pathogens, we screened mutants, in genes involved in the function of sensory neurons, for expression of stress proteins in response to bacterial pathogens and their pathogen susceptibility. Mutation in thermosensory neurons affected not only the expression of stress proteins but also pathogen susceptibility. Additional sensory neurons suppressed innate immunity while others enhanced innate immunity.

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.