Sadler PL et al. (2000) Development "Anucleate Caenorhabditis elegans sperm can crawl, fertilize oocytes and direct anterior-posterior polarization of the 1-cell embryo."

Sadler PL, Shakes DC

[Development, 2000]

It has long been appreciated that spermiogenesis, the cellular transformation of sessile spermatids into motile spermatozoa, occurs in the absence of new DNA transcription. However, few studies have addressed whether the physical presence of a sperm nucleus is required either during spermiogenesis or for subsequent sperm functions during egg activation and early zygotic development. To determine the role of the sperm nucleus in these processes, we analyzed two C. elegans mutants whose spermatids lack DNA. Here we show that these anucleate sperm not only differentiate into mature functional spermatozoa, but they also crawl toward and fertilize oocytes. Furthermore, we show that these anucleate sperm induce both normal egg activation and anterior-posterior polarity in the 1-cell C. elegans embryo. The latter finding demonstrates for the first time that although the anterior-posterior embryonic axis in C. elegans is specified by sperm, the sperm pronucleus itself is not required. Also unaffected is the completion of oocyte meiosis, formation of an impermeable eggshell, migration of the oocyte pronucleus, and the separation and expansion of the sperm-contributed centrosomes. Our investigation of these mutants confirms that, in C. elegans, neither the sperm chromatin mass nor a sperm pronucleus is required for spermiogenesis, proper egg activation, or the induction of anterior-posterior polarity.

Zhou Q et al. (1997) J Biol Chem "Molecular cloning of human plasma membrane phospholipid scramblase. A protein mediating transbilayer movement of plasma membrane phospholipids."

Zhou Q, Wiedmer T, Luhm RA, Zhao J, Stout JG, Sims PJ

[J Biol Chem, 1997]

The rapid movement of phospholipids (PL) between plasma membrane leaflets in response to increased intracellular Ca2+ is thought to play a key role in expression of platelet procoagulant activity and in clearance of injured or apoptotic cells. We recently reported isolation of a approximately 37-kDa protein in erythrocyte membrane that mediates Ca2+-dependent movement of PL between membrane leaflets, similar to that observed upon elevation of Ca2+ in the cytosol (Basse, F., Stout, J. G., Sims, P. J., and Wiedmer, T. (1996) J. Biol. Chem. 271, 17205-17210). Based on internal peptide sequence obtained from this protein, a 1,445-base pair cDNA was cloned from a K-562 cDNA library. The deduced "PL scramblase" protein is a proline-rich, type II plasma membrane protein with a single transmembrane segment near the C terminus. Antibody against the deduced C-terminal peptide was found to precipitate the approximately 37-kDa red blood cell protein and absorb PL scramblase activity, confirming the identity of the cloned cDNA to erythrocyte PL scramblase. Ca2+-dependent PL scramblase activity was also demonstrated in recombinant protein expressed from plasmid containing the cDNA. Quantitative immunoblotting revealed an approximately 10-fold higher abundance of PL scramblase in platelet ( approximately 10(4) molecules/cell) than in erythrocyte ( approximately 10(3) molecules/cell), consistent with apparent increased PL scramblase activity of the platelet plasma membrane. PL scramblase mRNA was found in a variety of hematologic and nonhematologic cells and tissues, suggesting that this protein functions in all cells.

Sanchez AD et al. (2021) STAR Protoc "A proximity labeling protocol to probe proximity interactions in C.elegans."

Feldman JL, Sanchez AD

[STAR Protoc, 2021]

Enzyme-catalyzed proximity labeling (PL) has emerged as a critical approach for identifying protein-protein proximity interactions in cells; however, PL techniques were not historically practical in living multicellular organisms due to technical limitations. Here, we present a protocol for applying PL to living <i>C.elegans</i> using the biotin ligase mutant enzyme TurboID. We demonstrated PL in a tissue-specific and region-specific manner by focusing on non-centrosomal MTOCs (ncMTOCs) of intestinal cells. This protocol is useful for targeted <i>in vivo</i> protein network profiling. For complete details on the use and execution of this protocol, please refer to Sanchez et al. (2021).

Pertel R et al. (1974) Comp Gen Pharmacol "Histamine content of the nematode, Caenorhabditis elegans."

Pertel R, Wilson SH

[Comp Gen Pharmacol, 1974]

1. The histamine contect in axenically grown nematodes was found to be 350 ng. per g. wet weight. 2. Histamine content was determined in extracts from the free-living nematode, Caenorhabditis elegans var. Bristol (strain B1-Pl) by an isotope dilution procedure which obviated error encountered in a conventional fluorescence assay. 3. This is the first report of histamine in the Nematoda.

Shakes DC et al. (2003) Development "Developmental defects observed in hypomorphic anaphase-promoting complex mutants are linked to cell cycle abnormalities."

Sadler PL, Golden A, Shakes DC, Abdolrasulnia M, Schumacher JM

[Development, 2003]

sIn C elegans, mutants in the anaphase-promoting complex or cyclosome (APC/C) exhibit defects in germline proliferation, the formation of the vulva and male tail, and the metaphase to anaphase transition of meiosis I. Oocytes lacking APC/C activity can be fertilized but arrest in metaphase of meiosis I and are blocked from further development. To examine the cell cycle and developmental consequences of reducing but not fully depleting APC/C activity, we analyzed defects in embryos and larvae of mat1/cdc-27 mutants grown at semi-permissive temperatures. Hypomorphic embryos developed to the multicellular stage but were slow to complete meiosis I and displayed aberrant meiotic chromosome separation. More severely affected embryos skipped meiosis 11 altogether and exhibited striking defects in meiotic exit. These latter embryos failed to produce normal eggshells or establish normal asymmetries prior to the first mitotic division. In developing larvae, extended M-phase delays in late-dividing cell lineages were associated with defects in the morphogenesis of the male tail. This study reveals the importance of dosage-specific mutants in analyzing molecular functions of a ubiquitously functioning protein within different cell types and tissues, and striking correlations between specific abnormalities in cell cycle

Koyiloth M et al. (2021) Biochim Biophys Acta Biomembr "Cholesterol interaction attenuates scramblase activity of SCRM-1 in the artificial membrane."

Koyiloth M, Gummadi SN

[Biochim Biophys Acta Biomembr, 2021]

Phospholipid (PL) scramblases are single-pass transmembrane protein mediating bidirectional PL translocation. Previously in silico analysis of human PL scramblases, predicted the presence of an uncharacterized cholesterol-binding domain spanning partly in the transmembrane helix as well as in the adjacent extracellular coil. This domain was found to be universally conserved in diverse organisms like Caenorhabditis elegans. In this study, we investigated the saturable cholesterol-binding domain of SCRM-1 using fluorescence sterol binding assay, Stern-Volmer quenching, Forster resonance energy transfer, and CD spectroscopy. We observed high-affinity interaction between cholesterol and SCRM-1. Our results support a previous report, which showed that the cholesterol ordering effect reduced the scramblase activity of hPLSCR1. Considering the presence of a high-affinity binding sequence, we propose that the reduction in activity could partly be due to the cholesterol binding. To validate this, we generated a C-terminal helix (CTH) deletion construct (CTH SCRM-1) and a point mutation in the putative cholesterol-binding domain I273D SCRM-1. Deletion construct greatly reduced cholesterol affinity along with loss of scramblase activity. In contrast to this, I273D SCRM-1 retained scrambling activity in proteoliposomes containing ~30mol% cholesterol but lost sterol binding ability. These results suggest that C-terminal helix is crucial for membrane insertion and in the lipid bilayer the scrambling activity of SCRM-1 is modulated through its interaction with cholesterol.

Davis ES et al. (2002) Genetics "Multiple subunits of the Caenorhabditis elegans anaphase-promoting complex are required for chromosome segregation during meiosis I."

Wille L, Chestnut BA, Golden A, Shakes DC, Davis ES, Sadler PL

[Genetics, 2002]

Two genes, originally identified in genetic screens for Caenorhabditis elegans mutants that arrest in metaphase of meiosis 1, prove to encode subunits of the anaphase-promoting complex or cyclosome (APC/C). RNA interference studies reveal that these and other APC/C subunits are essential for the segregation of chromosomal homologs during meiosis I. Further, chromosome segregation during meiosis I requires APC/C functions in addition to the release of sister chromatid cohesion.

Pramanik A et al. (2016) J Fluoresc "A Novel Technique of Synthesis of Highly Fluorescent Carbon Nanoparticles from Broth Constituent and In-vivo Bioimaging of C. elegans."

Kumar BA, Biswas S, Rai SK, Varalakshmi P, Kumbhakar P, Kole AK, Tiwary CS, Krishnaraj RN, Pramanik A

[J Fluoresc, 2016]

Here we have demonstrated a novel single step technique of synthesis of highly fluorescent carbon nanoparticles (CNPs) from broth constituent and in vivo bioimaging of Caenorhabditis elegans (C. elegans) with the synthesized CNPs has been presented. The synthesized CNPs has been characterized by the UV-visible (UV-Vis) absorption spectroscopy, transmission electron microscopy (TEM) and Raman studies. The sp (2) cluster size of the synthesized samples has been determined from the measured Raman spectra by fitting it with the theoretical skew Lorentzian (Breit-Wigner- Fano (BWF)) line shape. The synthesised materials are showing excitation wavelength dependent tunable photoluminescence (PL) emission characteristics with a high quantum yield (QY) of 3% at a very low concentration of CNPs. A remarkable increase in the intensity of PL emission from 16% to 39% in C. elegans has also been observed when the feeding concentration of CNPs to C. elegans is increased from 0.025% to 0.1% (w/v). The non-toxicity and water solubility of the synthesized material makes it ideal candidate for bioimaging.

Shakes DC et al. (2009) PLoS Genet "Spermatogenesis-specific features of the meiotic program in Caenorhabditis elegans."

Wu JC, Noritake A, Chu DS, Sadler PL, Moore LL, LaPrade K, Shakes DC

[PLoS Genet, 2009]

In most sexually reproducing organisms, the fundamental process of meiosis is implemented concurrently with two differentiation programs that occur at different rates and generate distinct cell types, sperm and oocytes. However, little is known about how the meiotic program is influenced by such contrasting developmental programs. Here we present a detailed timeline of late meiotic prophase during spermatogenesis in Caenorhabditis elegans using cytological and molecular landmarks to interrelate changes in chromosome dynamics with germ cell cellularization, spindle formation, and cell cycle transitions. This analysis expands our understanding C. elegans spermatogenesis, as it identifies multiple spermatogenesis-specific features of the meiotic program and provides a framework for comparative studies. Post-pachytene chromatin of spermatocytes is distinct from that of oocytes in both composition and morphology. Strikingly, C. elegans spermatogenesis includes a previously undescribed karyosome stage, a common but poorly understood feature of meiosis in many organisms. We find that karyosome formation, in which chromosomes form a constricted mass within an intact nuclear envelope, follows desynapsis, involves a global down-regulation of transcription, and may support the sequential activation of multiple kinases that prepare spermatocytes for meiotic divisions. In spermatocytes, the presence of centrioles alters both the relative timing of meiotic spindle assembly and its ultimate structure. These microtubule differences are accompanied by differences in kinetochores, which connect microtubules to chromosomes. The sperm-specific features of meiosis revealed here illuminate how the underlying molecular machinery required for meiosis is differentially regulated in each sex.

Burrows AE et al. (2006) Development "The C. elegans Myt1 ortholog is required for the proper timing of oocyte maturation."

Golden A, Sadler PL, Richie CT, Sceurman BK, Kosinski ME, Schumacher JM, Burrows AE

[Development, 2006]

Maturation promoting factor (MPF), a complex of cyclin-dependent kinase 1 and cyclin B, drives oocyte maturation in all animals. Mechanisms to block MPF activation in developing oocytes must exist to prevent precocious cell cycle progression prior to oocyte maturation and fertilization. This study sought to determine the developmental consequences of precociously activating MPF in oocytes prior to fertilization. Whereas depletion of Myt1 in Xenopus oocytes causes nuclear envelope breakdown in vitro, we found that depletion of the Myt1 ortholog WEE-1.3 in C. elegans hermaphrodites causes precocious oocyte maturation in vivo. Although such oocytes are ovulated, they are fertilization incompetent. We have also observed novel phenotypes in these precociously maturing oocytes, such as chromosome coalescence, aberrant meiotic spindle organization, and the expression of a meiosis II post-fertilization marker. Furthermore, co-depletion studies of CDK-1 and WEE-1.3 demonstrate that WEE-1.3 is dispensable in the absence of CDK-1, suggesting that CDK-1 is a major target of WEE-1.3 in C. elegans oocytes.