Presumptive TRP channel CED-11 promotes cell volume decrease and facilitates degradation of apoptotic cells in

Apoptotic cells undergo a series of morphological changes. These changes are dependent on caspase cleavage of downstream targets, but which targets are signifi cant and how they facilitate the death process are not well understood. In Caenorhabditis elegans an increase in the refractility of the dying cell is a hallmark morphological change that is caspase dependent. We identify a presumptive transient receptor potential (TRP) cation channel, CED-11, that acts in the dying cell to promote the increase in apoptotic cell refractility. CED-11 is required for multiple other morphological changes during apoptosis, including an increase in electron density as visualized by electron microscopy and a decrease in cell volume. In ced-11 mutants, the degradation of apoptotic cells is delayed. Mutation of ced-11 does not cause an increase in cell survival but can enhance cell survival in other cell-death mutants, indicating that ced-11 facilitates the death process. In short, ced-11 acts downstream of caspase activation to promote the shrinkage, death, and degradation of apoptotic cells. Keywords: TRP channel; apoptosis; C. elegans; cell volume; apoptotic volume decreaseNational Institutes of Health (U.S.) (Grant T32GM007287

Distinct Neural Circuits Control Rhythm Inhibition and Spitting by the Myogenic Pharynx of C. elegans

Neural circuits have long been known to modulate myogenic muscles such as the heart, yet a mechanistic understanding at the cellular and molecular levels remains limited. We studied how light inhibits pumping of the Caenorhabditis elegans pharynx, a myogenic muscular pump for feeding, and found three neural circuits that alter pumping. First, light inhibits pumping via the I2 neuron monosynaptic circuit. Our electron microscopic reconstruction of the anterior pharynx revealed evidence for synapses from I2 onto muscle that were missing from the published connectome, and we show that these “missed synapses” are likely functional. Second, light inhibits pumping through the RIP-I1-MC neuron polysynaptic circuit, in which an inhibitory signal is likely transmitted from outside the pharynx into the pharynx in a manner analogous to how the mammalian autonomic nervous system controls the heart. Third, light causes a novel pharyngeal behavior, reversal of flow or “spitting,” which is induced by the M1 neuron. These three neural circuits show that neurons can control a myogenic muscle organ not only by changing the contraction rate but also by altering the functional consequences of the contraction itself, transforming swallowing into spitting. Our observations also illustrate why connectome builders and users should be cognizant that functional synaptic connections might exist despite the absence of a declared synapse in the connectome.United States. National Institutes of Health (GM24663

Mutations in Nonessential eIF3k and eIF3l Genes Confer Lifespan Extension and Enhanced Resistance to ER Stress in Caenorhabditis elegans

The translation initiation factor eIF3 is a multi-subunit protein complex that coordinates the assembly of the 43S pre-initiation complex in eukaryotes. Prior studies have demonstrated that not all subunits of eIF3 are essential for the initiation of translation, suggesting that some subunits may serve regulatory roles. Here, we show that loss-of-function mutations in the genes encoding the conserved eIF3k and eIF3l subunits of the translation initiation complex eIF3 result in a 40% extension in lifespan and enhanced resistance to endoplasmic reticulum (ER) stress in Caenorhabditis elegans. In contrast to previously described mutations in genes encoding translation initiation components that confer lifespan extension in C. elegans, loss-of-function mutations in eif-3.K or eif-3.L are viable, and mutants show normal rates of growth and development, and have wild-type levels of bulk protein synthesis. Lifespan extension resulting from EIF-3.K or EIF-3.L deficiency is suppressed by a mutation in the Forkhead family transcription factor DAF-16. Mutations in eif-3.K or eif-3.L also confer enhanced resistance to ER stress, independent of IRE-1-XBP-1, ATF-6, and PEK-1, and independent of DAF-16. Our data suggest a pivotal functional role for conserved eIF3k and eIF3l accessory subunits of eIF3 in the regulation of cellular and organismal responses to ER stress and agingNational Institutes of Health (U.S.) (Grant R01-GM084477)National Institutes of Health (U.S.) (Pre-Doctoral Training Grant T32GM007287

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

A <i>Caenorhabditis elegans</i> protein with a PRDM9-like SET domain localizes to chromatin-associated foci and promotes spermatocyte gene expression, sperm production and fertility

<div><p>To better understand the tissue-specific regulation of chromatin state in cell-fate determination and animal development, we defined the tissue-specific expression of all 36 <i>C</i>. <i>elegans</i> presumptive lysine methyltransferase (KMT) genes using single-molecule fluorescence <i>in situ</i> hybridization (smFISH). Most KMTs were expressed in only one or two tissues. The germline was the tissue with the broadest KMT expression. We found that the germline-expressed <i>C</i>. <i>elegans</i> protein SET-17, which has a SET domain similar to that of the PRDM9 and PRDM7 SET-domain proteins, promotes fertility by regulating gene expression in primary spermatocytes. SET-17 drives the transcription of spermatocyte-specific genes from four genomic clusters to promote spermatid development. SET-17 is concentrated in stable chromatin-associated nuclear foci at actively transcribed <i>msp</i> (major sperm protein) gene clusters, which we term <i>msp</i> locus bodies. Our results reveal the function of a PRDM9/7-family SET-domain protein in spermatocyte transcription. We propose that the spatial intranuclear organization of chromatin factors might be a conserved mechanism in tissue-specific control of transcription.</p></div

<i>set-17</i> functions in the germline to promote sperm-production and fertility.

<p>A) Broodsizes of mutants defective in KMT genes expressed in the germline. Progeny number was determined for single adult hermaphrodites over three days and progeny were scored as adults after 3–6 days. Grey shaded area indicates the range of the wild-type 95% confidence interval. n > 10 for all genotypes. <i>set-23</i>, <i>set-24</i> and <i>set-27</i> mutants were not available, and instead these genes were examined using RNAi treated wild-type animals; fertility was not affected. B) Broodsize of wild-type, <i>set-17</i>, <i>set-17; P</i>_{<i>set-17</i>}::<i>set-17(+)</i> (expressing wild-type <i>set-17</i> from its endogenous promoter) and <i>set-17; P</i>_<i>mex-5</i>::<i>set-17(+)</i> (expressing wild-type <i>set-17</i> from the <i>mex-5</i> germline-specific promoter) hermaphrodites. n > 20; *** P < 0.0001, ** P < 0.0012, t-test. C) Progeny of mating single males (wild-type or <i>set-17</i> mutant) and single females (wild-type or <i>set-17</i> mutant). Strains carried the <i>fog-2(q71)</i> mutation, which feminizes hermaphrodites by suppressing hermaphrodite but not male sperm production and ensured that all progeny were cross progeny. After 24 hr the male was removed, and the female allowed to lay progeny until completion. Females were placed on fresh plates every 24 hr over 4 days. Cross progeny were scored as number of adult female progeny 3–7 days after mating; only females were scored because adult males burrow, crawl off plates and clump together, making their quantification unreliable. n > 15. *** P < 0.0001, t-test. D) Spermatid counts in individual spermathecas of the indicated genotypes. Spermatid counts were determined by imaging DAPI-stained spermatid nuclei in hermaphrodites fixed in 4% formaldehyde 12 hr post-L4 around the time of first fertilization. n: wild-type = 26, <i>set-17(-)</i> = 32, <i>set-17(-);</i> P_{<i>set-17</i>}::<i>set-17(+)</i> = 33, <i>set-17(-);</i> P_<i>mex-5</i>::<i>set-17(+)</i> = 15. *** P < 0.0001, t-test.</p

SET-17 promotes transcription at spermatogenic gene clusters.

<p>A) Endogenous <i>msp</i> mRNA expression determined by smFISH using a probe-set that recognizes transcripts from 28 <i>msp</i> genes in wild-type or <i>set-17</i> sperm-producing germlines of L4 hermaphrodites. White lines delineate the gonad. Scale bar, 10 μm. B) Levels of endogenous <i>msp</i> mRNA as measured by smFISH in mature primary spermatocytes in wild-type and <i>set-17</i> L4 hermaphrodites. n > 10, P < 0.001, t-test. C) Left, endogenous <i>msp</i> RNA transcription sites (TSs) in the nuclei of wild-type and <i>set-17</i> primary spermatocytes in L4 hermaphrodites; scale bar, 5 μm. Right, percent of primary spermatocyte nuclei with at least one <i>msp</i> TS (positive). Wild type n = 177, <i>set-17</i> n = 199. *** P < 0.0001, t-test. D) Frequency distribution of TSs in primary spermatocyte nuclei of wild-type or <i>set-17(-)</i> sperm-producing L4 hermaphrodites. Avg. TSs per nucleus: wild type, 1.75, n = 141; <i>set-17</i>, 0.82, n = 153; P < 0.0001, KS or MW test. E) Probability that a single <i>msp</i> cluster is transcribed in a wild-type or <i>set-17</i> L4 hermaphrodite. Calculated from the TS distributions for wild-type and <i>set-17</i> animals shown in (D). P < 0.0001, t-test. F) Co-localization of endogenous <i>msp</i> RNA TSs (as visualized by smFISH) and SET-17::GFP (as visualized by GFP protein fluorescence) in the nuclei of primary spermatocytes in the male germline. White circles, nuclear circumference; scale bar, 1 μm. G) Individual frames from an <i>in vivo</i> FRAP time series study of two primary spermatocytes and a hypodermal nucleus. White lines, nuclear circumference; scale bar, 1 μm; red circle, focal area of laser bleaching and region of measurement. H) Normalized fluorescence intensity measurement in the area of bleaching for the three experiments shown in (G). t = 0, pre-bleaching intensity. Recovery of SET-17::GFP is 10-fold slower in spermatocyte foci than in hypodermal nuclei. I) Normalized FRAP data for 16 experiments examining spermatocyte foci. Black dotted line indicates average half-maximal recovery time, 61 s, calculated from exponential fits for each experiment. J) Average half-maximal recovery times from FRAP studies of SET-17::GFP in spermatocyte foci (Foci t_1/2) or hypodermal nuclei (Hyp t_1/2). Error bars: SEM; P < 0.05, t-test.</p

L1 tissue-specific expression patterns of all <i>C</i>. <i>elegans</i> KMT genes.

<p>Most KMT gene were expressed with tissue-specificity. Expression in a tissue was defined as more than one transcript detected in multiple cells in all animals. Mam Orth, mammalian ortholog of the <i>C</i>. <i>elegans</i> protein; SET target, experimentally shown to catalyze histone lysine methylation at these targets.</p

ELT-1 acts with SET-17 to promote spermatogenic gene cluster expression and fertility.

<p>A) Overlays among the sets of the 44 predicted spermatogenic <i>elt-1</i> targets (from data of del Castilles-Olivera et al., 2009), the 60 spermatogenic <i>set-17</i> misregulated genes (from our <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007295#pgen.1007295.g006" target="_blank">Fig 6A</a>) and the 28 <i>msp</i> genes. B) Average log₂-fold change in expression from RNAseq studies compared to the wild type for the 44 predicted <i>elt-1</i> target genes in <i>set-17</i> (purple), <i>set-17; P</i>_{<i>set-17</i>}::<i>set-17(+)</i> (orange) and <i>set-17; P</i>_<i>mex-5</i>::<i>set-17(+)</i> (yellow) mutant whole L4 hermaphrodite. **** P < 0.0001, MW or KS-test. C) Endogenous <i>msp</i> mRNA detection by smFISH in primary spermatocytes of <i>elt-1</i> partial loss-of-function (rf) L4 hermaphrodites. Scale bar, 10 μm. D) Endogenous <i>msp</i> mRNA levels as visualized by smFISH in mature primary spermatocytes in wild-type (black), <i>elt-1</i> (purple), <i>set-17</i> (red) and <i>set-17; elt-1</i> (magenta) L4 hermaphrodites. n > 15. E) Number of progeny of wild-type (black), <i>elt-1</i> (purple), <i>set-17</i> (red) and <i>set-17; elt-1</i> mutants (magenta). n > 20. F) Fertility correlates with endogenous <i>msp</i> mRNA levels as visualized by smFISH (data from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007295#pgen.1007295.g007" target="_blank">Fig 7C and 7D</a>). Pearson-correlation r = 0.9981, P < 0.002 (2-tailed). G) Model of SET-17 foci composition and function in <i>msp</i> gene expression in primary spermatocyte nuclei at spermatogenic gene clusters. See text for details.</p