Sun, Lin et al. (2015) International Worm Meeting "Activity dependent modulation of neuronal regeneration."

Shay, James, Sun, Lin, Chung, Sam, Gabel, Christopher

[International Worm Meeting, 2015]

Neuronal activity plays a critical role during development modulating neurite outgrowth and the formation of synaptic connections. Following traumatic cellular damage, neuronal activity can also have a profound effect on the ability of a neuron to regenerate. Employing laser surgery, fluorescent calcium imaging and optogenetic techniques in C. elegans, we can optically damage individual nerve processes, measure and manipulate their activity levels and quantify the regenerative response. We find highly varied results dependent on neuron type, damage location and pattern of excitation. Periodic excitation of laser damaged ALM and PLM neurons with Channelrhodospin-2 results in an increase in regeneration that is dependent on the frequency of stimulation. In addition this enhance regeneration is link to cellular calcium signaling as it is block by disruption of either unc-68/RyR (encoding an ER calcium release channel) or L-type voltage gated calcium channels (VGCC). In contrast we find that innate sensory signaling of the ASJ amphid neuron restricts a DLK-1 independent regenerative pathway. Disruption of this sensory activity through laser severing of the sensory dendrite or genetic disruption of egl-19/VGCC results in robust neurite sprouting following axotomy. Interestingly, the mechanisms modulating this outgrowth appear similar to that of activity dependent ectopic outgrowth observed in the same neurons during development. Taken as a whole our work is elucidating how activity mediated mechanisms controlling regeneration are highly dependent on temporal characteristics and cellular context. .

Sun, Lin et al. (2011) International Worm Meeting "Characterization of intracellular calcium signaling within damage C. elegans neurons ."

Gao, Fay, Roodhouse, Kevin, Sun, Lin, Chung, Samuel, Gabel, Christopher V.

[International Worm Meeting, 2011]

Traumatic neuronal damage triggers large intracellular calcium transients that are associated both with subsequent neuronal degeneration and cell death, and alternatively with regenerative repair and outgrowth [1, 2]. Combining femtosecond laser ablation with the use of genetically encoded calcium sensitive fluorophores, we can optically measure intracellular calcium signaling within a specific target neuron of an intact adult C. elegans in response to precision laser damage [3-5]. Here we characterize the damage induced calcium signal across a variety of laser ablation experiments. This includes variations in the proximity of the damage point to the cell soma, targeting of distinct morphological structures and different neuronal types, modulation of laser power, different animal ages, and multiple surgeries to the same neuron. Results are revealing complex subcellular calcium dynamics that are precisely tuned to control cell fate. We find that in general large, extended calcium transients correlate with cell degeneration, while particularly small transients are associated with reduced regenerative outgrowth. This suggests an optimum window in which elevation of cytoplasmic calcium successfully facilitates neuronal repair and outgrowth without initiating cell death. Our results are helping to pinpoint the critical aspects of this signaling pathway that dictate neuronal survival and regeneration following traumatic damage. 1.Coleman, M., Axon degeneration mechanisms: commonality amid diversity. Nat Rev Neurosci, 2005. 6(11): p. 889-98. 2.Kamber, D., H. Erez, and M.E. Spira, Local calcium-dependent mechanisms determine whether a cut axonal end assembles a retarded endbulb or competent growth cone. Exp Neurol, 2009. 219(1): p. 112-25. 3.Yanik, M.F., et al., Neurosurgery: functional regeneration after laser axotomy. Nature, 2004. 432(7019): p. 822. 4.Gabel, C.V., et al., Distinct cellular and molecular mechanisms mediate initial axon development and adult-stage axon regeneration in C. elegans. Development, 2008. 135(6): p. 1129-36. 5.Ghosh-Roy, A., et al., Calcium and Cyclic AMP Promote Axonal Regeneration in Caenorhabditis elegans and Require DLK-1 Kinase. Journal of Neuroscience, 2010. 30(9): p. 3175-3183.

Sun, Lin et al. (2013) International Worm Meeting "UNC-68/RyR channels modulate critical sub-cellular calcium signals during normal and optogenetically enhanced neuronal regeneration."

Gabel, Christopher V., Chung, Samuel, Alkema, Mark, Shay, James, Sun, Lin, Clark, Christopher

[International Worm Meeting, 2013]

Sub-cellular calcium signaling plays an important role in a neuron's ability to recover from traumatic damage. Employing femtosecond laser surgery, time-lapse microscopy, fluorescent calcium imaging and optogenetic photo-stimulation, we can damage a single neuron, quantify its regeneration, measure sub-cellular calcium signals and dynamically manipulate cell physiology in vivo. Extending previous C. elegans studies that measured an initial (~5 min) damage induced calcium transient within an axotomized neuron, we observe a prolonged, localized calcium signal within the vicinity of the damage point for 5 h following laser axotomy of the ALM neuron. This signal is eliminated by mutation of unc-68 encoding the C. elegans homologue of the ryanodine receptor (RyR) channel, a calcium release channel in the endoplasmic reticulum membrane. In addition, the same unc-68(e540) mutants exhibit a >50% reduction in regeneration outgrowth at 5h and 24h post-lesion as well as severely disrupted regeneration guidance. Employing optogenetic techniques we periodically stimulate axotomized ALM neurons by photo-activation of the cation channel channelrhodopsin-2 (ChR2). This results in a robust, >30%, increase in regeneration outgrowth over the 24 h following laser surgery. The effect is eliminated in unc-68(e540) mutants, or by pharmacologically blocking UNC-68 with dantrolene. Calcium induced calcium release via RyR channels plays an important role in amplifying sub-cellular calcium signals within a neuronal growth cone to modulate axon guidance. In C. elegans, UNC-68 is necessary for ChR2 photo-activation of muscle. In our experiments UNC-68 calcium release may amplify the ChR2 initiated signal to stimulate additional outgrowth. Taken as a whole, our results demonstrate an important role for UNC-68/RyR calcium release in stimulating early regeneration outgrowth and point to possible therapeutic control of cellular calcium physiology to enhance neuronal regeneration in vivo.

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.