(2019) Development "The people behind the papers - Julia Brandt, Mary Rossillo and Niels Ringstad."

[Development, 2019]

A fundamental aim in developmental biology is to understand how the various cell types of the body are specified by differential gene regulation. <i>Caenorhabditis</i><i>elegans</i> nervous system development provides a powerful system for studying this, as exemplified by a new Development paper reporting on how the BAG neurons that help the worm sense oxygen and carbon dioxide are specified. We caught up with first authors Julia Brandt and Mary Rossillo and their supervisor Niels Ringstad (Associate Professor at the Skirball Institute of Biomolecular Medicine and Department of Cell Biology at New York University) to find out more about the story.

Kreis HA et al. (1933) Trans Am Microsc Soc "Two new species of Rhabditis (R macrocerca & R clavopapillata) assoc with dogs and monkeys in experimental Strongyloides studies."

Faust EC, Kreis HA

[Trans Am Microsc Soc, 1933]

From time to time during our studies on experimental Strongyloides infections, freshly passed stools of dogs have been contaminated with species of Rhabditis. At first it was believed that the contaminations resulted from improper sterilization of the Petri dishes used in collecting the feces, but after this possibility was eliminated the worms appeared with equal consistency. Sterilization of the cages in which the animals were housed also failed to reduce the contamination. Studies were then made of the animals themselves and it was found that the worms were developing in abundance among the perianal hairs, particularly in those dogs which had loose bowel movements. To a lesser extent the hairs of the abdomen, legs and even the face were also discovered to be favorable breeding places for these rhabditids. Thorough cleansing of the dogs externally with cresol solutions reduced the contamination appreciably but did not completely eliminate

Fields SD et al. (1998) J Cell Sci "The S. cerevisiae CLU1 and D. discoideum cluA genes are functional homologues that influence mitochondrial morphology and distribution."

Fields SD, Conrad MN, Clarke M

[J Cell Sci, 1998]

The cluA gene, encoding a novel 150 kDa protein, was recently characterized in Dictyostelium discoideum; disruption of cluA impaired cytokinesis and caused mitochondria to cluster at the cell center. The genome of Saccharomyces cerevisiae contains an open reading frame (CLU1) that encodes a protein that is 27% identical, 50% similar, to this Dictyostelium protein. Deletion of CLU1 from S. cerevisiae did not affect cell viability, growth properties, sporulation efficiency, or frequency of occurrence of cells lacking functional mitochondria. However, in clu1Delta cells the mitochondrial reticulum, which is normally highly branched, was condensed to one side of the cell. Transformation of cluA- Dictyostelium mutants with the yeast CLU1 gene yielded amoebae that divided normally and had dispersed mitochondria. The mitochondria in cluA- Dictyostelium cells complemented with CLU1 were not as widely scattered as in cluA+ Dictyostelium cells, but formed loose clusters throughout the cytoplasm. These results indicate that the products of the CLU1 and cluA genes, in spite of their limited homology, are functional homologues.

Peixoto CA et al. (1995) Tissue Cell "Freeze-fracture and deep-etched view of the cuticle of Caenorhabditis elegans."

Peixoto CA, DeSouza W

[Tissue Cell, 1995]

At ultrastructural level, the Caenorhabditis elegans (C. elegans) cuticle shows the presence of well-defined layers, one of them is a membrane-like structure designated as epicuticle, always present on the outermost surface of nematodes. Freeze-fracture replicas revealed the existance of two faces of the epicuticle: a inner face containing numerous particles, and a almost smooth outer face. Deep etching replicas confirmed the existance of these two faces of the epicuticle showing in some replicas two particle populations on the outer face of L4 and adult forms of C. elegans. Also a previously unrecognized structure was noted in the cuticle of C. elegans, a matrix composed by network of globular and filamentous structures, leaving in between them spaces, which probably are occupied by water in the living adult and L4 larvae specimen. This network demonstrates either a compact nature or loose nature according to their cuticle location. Deep etching replicas of the adults nematode revealed large spaces between the cortical and basal layers which are regularly interrupted by struts connecting each other by fibers in a particular arrangement.

Cordeddu V et al. (2009) Nat Genet "Mutation of SHOC2 promotes aberrant protein N-myristoylation and causes Noonan-like syndrome with loose anagen hair."

Tartaglia M, Cordeddu V, Tenconi R, Schackwitz W, Bazzicalupo P, Iyengar R, Cardinale A, Pennacchio LA, Bartholdi D, Ma'ayan A, Rossi C, Martinelli S, Selicorni A, Flex E, Cecchetti S, Zampino G, Digilio MC, Kutsche K, Lipzen A, Zenker M, Lepri F, Anichini C, Di Schiavi E, Dallapiccola B, Fodale V, Ferrero GB, Martin J, Sarkozy A, Gelb BD, Merlo D, Mazzanti L

[Nat Genet, 2009]

N-myristoylation is a common form of co-translational protein fatty acylation resulting from the attachment of myristate to a required N-terminal glycine residue. We show that aberrantly acquired N-myristoylation of SHOC2, a leucine-rich repeat-containing protein that positively modulates RAS-MAPK signal flow, underlies a clinically distinctive condition of the neuro-cardio-facial-cutaneous disorders family. Twenty-five subjects with a relatively consistent phenotype previously termed Noonan-like syndrome with loose anagen hair (MIM607721) shared the 4A&gt;G missense change in SHOC2 (producing an S2G amino acid substitution) that introduces an N-myristoylation site, resulting in aberrant targeting of SHOC2 to the plasma membrane and impaired translocation to the nucleus upon growth factor stimulation. Expression of SHOC2(S2G) in vitro enhanced MAPK activation in a cell type-specific fashion. Induction of SHOC2(S2G) in Caenorhabditis elegans engendered protruding vulva, a neomorphic phenotype previously associated with aberrant signaling. These results document the first example of an acquired N-terminal lipid modification of a protein causing human disease.

Fiori A et al. (2012) Eukaryot Cell "The heat-induced molecular disaggregase Hsp104 of Candida albicans plays a role in biofilm formation and pathogenicity in a worm infection model."

Van Dijck P, Govaert G, Kucharikova S, Fiori A, Cammue BP, Thevissen K

[Eukaryot Cell, 2012]

The consequences of deprivation of the molecular chaperone Hsp104 in the fungal pathogen Candida albicans were investigated. Mutants lacking HSP104 became hypersusceptible to lethally high temperatures, similarly to the corresponding mutants of Saccharomyces cerevisiae, whereas normal susceptibility was restored upon reintroduction of the gene. By use of a strain whose only copy of HSP104 is an ectopic gene under the control of a tetracycline-regulated promoter, expression of Hsp104 prior to the administration of heat shock could be demonstrated to be sufficient to confer protection from the subsequent temperature increase. This result points to a key role for Hsp104 in orchestrating the cell response to elevated temperatures. Despite their not showing evident growth or morphological defects, biofilm formation by cells lacking HSP104 proved to be defective in two established in vitro models that use polystyrene and polyurethane as the substrates. Biofilms formed by the wild-type and HSP104-reconstituted strains showed patterns of intertwined hyphae in the extracellular matrix. In contrast, biofilm formed by the hsp104/hsp104 mutant showed structural defects and appeared patchy and loose. Decreased virulence of the hsp104/hsp104 mutant was observed in the Caenorhabditis elegans infection model, in which high in vivo temperature does not play a role. In agreement with the view that stress responses in fungal pathogens may have evolved to provide niche-specific adaptation to environmental conditions, these results provide an indication of a temperature-independent role for Hsp104 in support of Candida albicans virulence, in addition to its key role in governing thermotolerance.

Ward S et al. (1988) J Mol Biol "Genomic organization of major sperm protein genes and pseudogenes in the nematode Caenorhabditis elegans."

Hogan E, Albertson DG, Ward S, Klass MR, Burke DJ, Sulston JE, Ammons D, Coulson AR

[J Mol Biol, 1988]

The major sperm proteins (MSPs) are a family of closely related, small, basic proteins comprising 15% of the protein in Caenorhabditis elegans sperm. They are encoded by a multigene family of more than 50 genes, including many pseudogenes. MSP gene transcription occurs only in late primary spermatocytes. In order to study the genomic organization of transcribed MSP genes, probes specific for the 3' untranslated regions of sequenced cDNA clones were used to isolate transcribed genes from genomic libraries. These and other clones of MSP genes were located in overlapping cosmid clones by DNA fingerprinting. These cosmids were aligned with the genetic map by overlap with known genes or in-situ hybridization to chromosomes. Of 40 MSP genes identified, 37, including all those known to be transcribed, are organized into six clusters composed of 3 to 13 genes each. Within each cluster, MSP genes are not in tandem but are separated by at least several thousand bases of DNA. Pseudogenes are interspersed among functional genes. Genes with similar 3' untranslated sequences are in the same cluster. The six MSP clusters are confined to only three chromosomal loci; one on the left arm of chromosome II and two near the middle of chromosome IV. Additional sperm-specific genes are located in one cluster of MSP genes on chromosome IV. The multiplicity of MSP genes appears to be a mechanism for enhancing MSP synthesis in spermatocytes, and the loose clustering of genes could be a result of the mechanism of gene duplication or could play a role in regulation.

Li L et al. (2018) J Neurosci "SNT-1 functions as the Ca2+ sensor for tonic and evoked neurotransmitter release in C. elegans."

Liu H, Li L, Chandra M, Hu Z, Collins BM, Wang W

[J Neurosci, 2018]

Synaptotagmin-1 (Syt1) binds Ca²⁺ through its tandem C2 domains (C2A and C2B) and triggers Ca²⁺-dependent neurotransmitter release. Here we show that <i>snt-1</i>, the homolog of mammalian Syt1, functions as the Ca²⁺ sensor for both tonic and evoked neurotransmitter release at the <i>C. elegans</i> neuromuscular junction. Mutations that disrupt Ca²⁺ binding in double C2 domains of SNT-1 significantly impaired tonic release, whereas disrupting Ca²⁺ binding in a single C2 domain had no effect, indicating that the Ca²⁺ binding of the two C2 domains is functionally redundant for tonic release. Stimulus-evoked release was significantly reduced in <i>snt-1</i> mutants, with prolonged release latency as well as faster rise and decay kinetics. Unlike tonic release, evoked release was triggered by Ca²⁺ binding solely to the C2B domain. Moreover, we showed that SNT-1 plays an essential role in the priming process in different subpopulations of synaptic vesicles with tight or loose coupling to Ca²⁺ entry.<b>SIGNIFICANCE STATEMENT</b>We showed that SNT-1 in <i>C. elegans</i> regulates evoked neurotransmitter release through Ca²⁺ binding to its C2B domain, a similar way to Syt1 in the mouse CNS and the fly NMJ. However, the largely decreased tonic release in <i>snt-1</i> mutants argues SNT-1 has a clamping function. Indeed, Ca²⁺-binding mutations in the C2 domains in SNT-1 significantly reduced the frequency of the miniature excitatory postsynaptic current (mEPSC), indicating that SNT-1 also acts as a Ca²⁺ sensor for tonic release. Therefore, revealing the differential mechanisms between invertebrates and vertebrates will provide significant insights into our understanding how synaptic vesicle fusion is regulated.

Gill HK et al. (2016) PLoS Genet "Integrity of Narrow Epithelial Tubes in the C. elegans Excretory System Requires a Transient Luminal Matrix."

Forman-Rubinsky R, Gill HK, Ayala-Figueroa J, Cohen JD, Bickard K, Parry JM, Poggioli C, Hall DH, Sundaram MV, Pu P

[PLoS Genet, 2016]

Most epithelial cells secrete a glycoprotein-rich apical extracellular matrix that can have diverse but still poorly understood roles in development and physiology. Zona Pellucida (ZP) domain glycoproteins are common constituents of these matrices, and their loss in humans is associated with a number of diseases. Understanding of the functions, organization and regulation of apical matrices has been hampered by difficulties in imaging them both in vivo and ex vivo. We identified the PAN-Apple, mucin and ZP domain glycoprotein LET-653 as an early and transient apical matrix component that shapes developing epithelia in C. elegans. LET-653 has modest effects on shaping of the vulva and epidermis, but is essential to prevent lumen fragmentation in the very narrow, unicellular excretory duct tube. We were able to image the transient LET-653 matrix by both live confocal imaging and transmission electron microscopy. Structure/function and fluorescence recovery after photobleaching studies revealed that LET-653 exists in two separate luminal matrix pools, a loose fibrillar matrix in the central core of the lumen, to which it binds dynamically via its PAN domains, and an apical-membrane-associated matrix, to which it binds stably via its ZP domain. The PAN domains are both necessary and sufficient to confer a cyclic pattern of duct lumen localization that precedes each molt, while the ZP domain is required for lumen integrity. Ectopic expression of full-length LET-653, but not the PAN domains alone, could expand lumen diameter in the developing gut tube, where LET-653 is not normally expressed. Together, these data support a model in which the PAN domains regulate the ability of the LET-653 ZP domain to interact with other factors at the apical membrane, and this ZP domain interaction promotes expansion and maintenance of lumen diameter. These data identify a transient apical matrix component present prior to cuticle secretion in C. elegans, demonstrate critical roles for this matrix component in supporting lumen integrity within narrow bore tubes such as those found in the mammalian microvasculature, and reveal functional importance of the evolutionarily conserved ZP domain in this tube protecting activity.