LIN-44/Wnt Directs Dendrite Outgrowth through LIN-17/Frizzled in C. elegans Neurons

Nervous system function requires proper development of two functional and morphological domains of neurons, axons and dendrites. Although both these domains are equally important for signal transmission, our understanding of dendrite development remains relatively poor. Here, we show that in C. elegans the Wnt ligand, LIN-44, and its Frizzled receptor, LIN-17, regulate dendrite development of the PQR oxygen sensory neuron. In lin-44 and lin-17 mutants, PQR dendrites fail to form, display stunted growth, or are misrouted. Manipulation of temporal and spatial expression of LIN-44, combined with cell-ablation experiments, indicates that this molecule is patterned during embryogenesis and acts as an attractive cue to define the site from which the dendrite emerges. Genetic interaction between lin-44 and lin-17 suggests that the LIN-44 signal is transmitted through the LIN-17 receptor, which acts cell autonomously in PQR. Furthermore, we provide evidence that LIN-17 interacts with another Wnt molecule, EGL-20, and functions in parallel to MIG-1/Frizzled in this process. Taken together, our results reveal a crucial role for Wnt and Frizzled molecules in regulating dendrite development in vivo

SARS-CoV-2 B.1.617.2 Delta variant replication and immune evasion

Abstract: The B.1.617.2 (Delta) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in the state of Maharashtra in late 2020 and spread throughout India, outcompeting pre-existing lineages including B.1.617.1 (Kappa) and B.1.1.7 (Alpha)1. In vitro, B.1.617.2 is sixfold less sensitive to serum neutralizing antibodies from recovered individuals, and eightfold less sensitive to vaccine-elicited antibodies, compared with wild-type Wuhan-1 bearing D614G. Serum neutralizing titres against B.1.617.2 were lower in ChAdOx1 vaccinees than in BNT162b2 vaccinees. B.1.617.2 spike pseudotyped viruses exhibited compromised sensitivity to monoclonal antibodies to the receptor-binding domain and the amino-terminal domain. B.1.617.2 demonstrated higher replication efficiency than B.1.1.7 in both airway organoid and human airway epithelial systems, associated with B.1.617.2 spike being in a predominantly cleaved state compared with B.1.1.7 spike. The B.1.617.2 spike protein was able to mediate highly efficient syncytium formation that was less sensitive to inhibition by neutralizing antibody, compared with that of wild-type spike. We also observed that B.1.617.2 had higher replication and spike-mediated entry than B.1.617.1, potentially explaining the B.1.617.2 dominance. In an analysis of more than 130 SARS-CoV-2-infected health care workers across three centres in India during a period of mixed lineage circulation, we observed reduced ChAdOx1 vaccine effectiveness against B.1.617.2 relative to non-B.1.617.2, with the caveat of possible residual confounding. Compromised vaccine efficacy against the highly fit and immune-evasive B.1.617.2 Delta variant warrants continued infection control measures in the post-vaccination era

Meiotic recombination modulates the structure and dynamics of the synaptonemal complex during <i>C</i>. <i>elegans</i> meiosis

<div><p>During meiotic prophase, a structure called the synaptonemal complex (SC) assembles at the interface between aligned pairs of homologous chromosomes, and crossover recombination events occur between their DNA molecules. Here we investigate the inter-relationships between these two hallmark features of the meiotic program in the nematode <i>C</i>. <i>elegans</i>, revealing dynamic properties of the SC that are modulated by recombination. We demonstrate that the SC incorporates new subunits and switches from a more highly dynamic/labile state to a more stable state as germ cells progress through the pachytene stage of meiotic prophase. We further show that the more dynamic state of the SC is prolonged in mutants where meiotic recombination is impaired. Moreover, in meiotic mutants where recombination intermediates are present in limiting numbers, SC central region subunits become preferentially stabilized on the subset of chromosome pairs that harbor a site where pro-crossover factors COSA-1 and MutSγ are concentrated. Polo-like kinase PLK-2 becomes preferentially localized to the SCs of chromosome pairs harboring recombination sites prior to the enrichment of SC central region proteins on such chromosomes, and PLK-2 is required for this enrichment to occur. Further, late pachytene nuclei in a <i>plk-2</i> mutant exhibit the more highly dynamic SC state. Together our data demonstrate that crossover recombination events elicit chromosome-autonomous stabilizing effects on the SC and implicate PLK-2 in this process. We discuss how this recombination-triggered modulation of SC state might contribute to regulatory mechanisms that operate during meiosis to ensure the formation of crossovers while at the same time limiting their numbers.</p></div

The SC switches from a more dynamic to a less dynamic state during pachytene progression.

<p><b>A.</b> Confocal images of GFP::SYP-3 fluorescence from a FRAP experiment assessing fluorescence recovery in nuclei from the mid-pachytene region of a wild-type worm, following a Z-stack bleach protocol conducted using a Leica SP2 confocal microscope. Arrows indicate partially bleached nuclei and circle indicates a fully bleached nucleus; images are sum projections of image stacks encompassing whole nuclei. Scale bar is 5μm. <b>B.</b> Panels showing partial projection images depicting the upper half of a nucleus from A, illustrating clear recovery of fluorescence for an SC that was fully contained within the bleached portion of the nucleus. <b>C.</b> Quantitation of WT FRAP experiments performed using the confocal microscope (as depicted in A). Each curve in the graph plots the recovery of a single partially bleached nucleus, with the color indicating the zone (early, mid or late pachytene) of the scored nucleus, as depicted in the cartoon gonad scheme above the graph. Extent of recovery represents the ratio of the fluorescence (at a given post-bleach time point) in an ROI from the bleached portion of the nucleus to the pre-bleach fluorescence in that ROI, normalized to the total fluorescence in that nucleus. <b>D.</b> Panels of images of EmGFP::SYP-3 fluorescence in representative early, mid and late pachytene nuclei from FRAP experiments performed using the Deltavision OMX wide-field microscope. Photobleaching was focused at a single plane (in contrast to a stepwise Z-stack bleach), using small ROIs (1.2–3μm²) to bleach limited SC segments; green rectangles represent bleached regions. Images are partial projections showing the half of the nucleus containing the bleached SC segments. <b>E.</b> Quantitation of fluorescence recovery in early, mid and late pachytene nuclei for experiments of the type depicted in D (where strong recovery is defined as apparent evening-out of fluorescence between bleached and unbleached SC segments; see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006670#sec013" target="_blank">Materials and methods</a> for scoring methodology). *p = 0.0049; ***p < 0.0001; two-tailed Fisher exact test. <b>F.</b> Panel of EmGFP::SYP-3 images showing FRAP of an SC that had been bleached along its entire length (arrow), indicating that recovery involves redistribution of subunits among different SCs within a nucleus. <b>G.</b> GFP::SYP-3 images of a field containing a mixture of unbleached reference nuclei (green arrowheads) and nuclei in which a substantial portion of the SCs were bleached (yellow arrows). Fluorescence of the unbleached SC segments diminishes relative to unbleached reference nuclei, reflecting redistribution of subunits among the SCs within each bleached nucleus.</p

Localization of PLK-2 with meiotic chromosomes is highly dynamic and is influenced by recombination.

<p><b>Top:</b> Dynamic localization of PLK-2 during wild-type meiosis. Representative images of spread nuclei from each indicated stage (zygotene, early pachytene, late pachytene, diplotene) in wild-type worms expressing HA-tagged PLK-2. As previously reported [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006670#pgen.1006670.ref042" target="_blank">42</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006670#pgen.1006670.ref043" target="_blank">43</a>], PLK-2 localizes to the pairing centers at the nuclear envelope during zygotene, while homolog pairing is being established and SCs are assembling, In early pachytene, PLK-2 becomes localized along synapsed bivalents that are engaged in recombination (<i>i</i>.<i>e</i>. displaying at least one MSH-5 focus). Prior to the early-to-late pachytene transition, PLK-2 localizes along the whole length of a bivalent. After the early-to-late pachytene transition, PLK-2 starts to retract, eventually becoming restricted to one side of the nascent CO site (marked by MSH-5); this restriction of PLK-2 to the “short arm” of the bivalent can be detected prior to detection of a similar retraction of SYP-1 that becomes visible by the end of pachytene (see late pachytene panels). By diplotene, both PLK-2 and SYP-1 are concentrated around chiasmata and on the short arm. <b>Bottom:</b> Recombination-triggered relocalization of PLK-2 is detected prior to SYP-1 enrichment on chromosomes undergoing recombination. Representative images of spread nuclei from each indicated stage (early pachytene, mid pachytene and late pachytene) from a <i>dsb-2</i> mutant expressing PLK-2::HA. During early pachytene, PLK-2 is first observed to be enriched on SCs near recombination sites marked by a MSH-5 focus and then becomes concentrated along the whole length of the recombining bivalent. Full-length localization of PLK-2 along a bivalent with a MSH-5 focus is detected prior to similar enrichment of SYP-1, which is first detected in mid pachytene. In nuclei lacking recombination events, PLK-2 is distributed uniformly among the bivalents (not depicted), as observed for SYP-1. Note: PLK-2 localizes not only to nuclei, but also to other extra-nuclear structures in the gonad, such as the rachis walls; consequently, IF signals from these other structures will tend to obscure the nuclear PLK-2 signals in maximum intensity projections of fields of nuclei. Thus, to facilitate visualization of subnuclear localization of PLK-2, representative spread nuclei were individually cropped from the edges of whole gonad spreads. In addition, the MSH-5 signal was overlaid as a screen on top of the other channels in order to facilitate visualization of the locations of MSH-5 foci in the projected images. Scale bar is 5μm.</p

When DSBs are limiting, SYP-1 is preferentially enriched on bivalents with a site where pro-crossover factors COSA-1 and MutSγ are concentrated.

<p><b>A</b> and <b>B</b>. Representative fields of whole-mount late pachytene nuclei in recombination initiation mutants <i>spo-11(me44)</i> and <i>dsb-2(me96)</i> immunostained for HTP-3, SYP-1 and GFP::COSA-1. Images are shown in two different color schemes to emphasize the correspondence between the presence of a GFP::COSA-1 focus and enrichment of SYP-1 on a chromosome pair. Scale bar represents 2μm. <b>C.</b> Representative field of spread nuclei at the early-to-late pachytene transition from the gonad of a <i>spo-11(me44)</i> mutant worm grown at 25°C and immunostained for HTP-3, SYP-1, MSH-5 and HIM-8. HIM-8 was used identify the X chromosomes, as it marks the pairing center locus located near the left end of X. In the nuclei containing a MSH-5 focus, SYP-1 is enriched on the chromosome pair harboring the focus. The SYP-1-enriched chromosome with a recombination focus can either be the X chromosome (middle right) or an autosome (upper left). In the remaining nuclei, no chromosome displays a recombination focus and SYP-1 is distributed equally among all chromosomes. (For this experiment, worms were raised at 20°C until the late L4 stage, then shifted to 25°C for 24 h prior to dissection; under these conditions, the incidence of nuclei with a SYP-1-enriched chromosome harboring a recombination focus is higher than at 20°C.) Scale bar is 5μm. <b>D</b>. Isolated late pachytene nuclear spreads from the <i>dsb-2(me96)</i> mutant, illustrating even distribution of SYP-1 among all chromosomes in a nucleus lacking a GFP::COSA-1 focus (left) and enrichment of SYP-1 on the single chromosome harboring a COSA-1 focus in the middle and right nuclei.</p

Levels of SC central region components SYP-1 and SYP-3 increase during pachytene progression.

<p><b>A.</b> Immuno-detection of wild-type distribution of SC components in a fixed whole-mount gonad (top) and higher magnification images of representative fields of nuclei in early (bottom left) and late pachytene (bottom right) stages. Chromosome axis protein HTP-3 is shown in red; SC central region protein SYP-1 is shown in green. <b>B.</b> Detection of SC central region component SYP-3 fused to GFP (EmGFP::SYP-3) in the gonad of a live worm (top). The graph (middle) represents the evolution of the average GFP::SYP-3 fluorescence intensity in a nucleus as a function of its position in the gonad (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006670#sec013" target="_blank">Materials and methods</a> for details). The increase in GFP::SYP-3 signal during pachytene progression is illustrated in higher magnification fields of early and late pachytene nuclei (bottom). For both panels, images are maximum intensity projections of 3D data stacks encompassing whole nuclei. Scale bars represent 10 μm (whole gonad) and 2 μm (fields of nuclei).</p

PLK-2 is required both for normal late pachytene SC dynamics and for preferential enrichment of SYP-1 on COSA-1-marked chromosomes when DSBs are limiting.

<p><b>A.</b> Panels of images of EmGFP::SYP-3 fluorescence in representative late pachytene nuclei from FRAP experiments performed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006670#pgen.1006670.g002" target="_blank">Fig 2D</a>, illustrating the strong fluorescence recovery that was consistently observed in the <i>plk-2</i> mutant. <b>B.</b> Quantitation of fluorescence recovery in late pachytene nuclei from wild-type and <i>plk-2</i> mutant worms (as in Figs <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006670#pgen.1006670.g002" target="_blank">2E</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006670#pgen.1006670.g003" target="_blank">3C</a>). ***p < 0.0001; two-tailed Fisher exact test. <b>C.</b> Immunofluorescence images of late pachytene nuclei from the <i>plk-2(ok1336)</i> single mutant and <i>plk-2(ok1336); dsb-2(me96)</i> double mutant; images are maximum intensity projections of 3D data stacks encompassing whole nuclei treated as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006670#pgen.1006670.g005" target="_blank">Fig 5A and 5B</a>. Scale bar represents 2μm. In contrast to the <i>dsb-2</i> single mutant (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006670#pgen.1006670.g005" target="_blank">Fig 5B</a>), SYP-1 signals are <u>not</u> specifically enriched on the chromosomes with a COSA-1 focus in the <i>plk-2; dsb-2</i> double mutant. (Note that there are asynapsed chromosome segments present in some nuclei, reflecting partially impaired synapsis in the <i>plk-2</i> mutant background.)</p

A subset of late pachytene nuclei in DSB-defective mutants exhibit an uneven distribution of SYP-1 among their SCs.

<p><b>A.</b> Immunodetection of SC components HTP-3 (red) and SYP-1 (green) in a fixed whole-mount <i>spo-11(me44)</i> mutant gonad. In late pachytene, some nuclei, indicated by an arrowhead, display an uneven distribution of the central region component SYP-1. <b>B.</b> Representative fields of late pachytene nuclei observed in wild-type, in recombination initiation mutants <i>spo-11(me44)</i>, <i>spo-11(ok79)</i> and <i>dsb-1(tm5034)</i>, in the <i>msh-5(me23)</i>, <i>cosa-1(tm3298)</i> mutants (which are proficient for initiation of recombination but defective in conversion of processed DSBs into COs), and in the <i>cosa-1(tm3298); spo-11(me44)</i> double mutant, stained for HTP-3 (red) and SYP-1 (green). Dashed circles indicate nuclei in the mutants with uneven SYP-1 distribution. For both panels A and B, images are maximum intensity projections of 3D data stacks encompassing whole nuclei. Scale bars represent 10μm (whole gonad, panel A) and 2μm (fields of nuclei, panel B). <b>C.</b> Quantification of the fraction of nuclei with uneven SYP-1 distribution in the last half of pachytene for each indicated genotype. Asterisks indicates highly significant differences (p < 0.0001; chi-square test) when single mutants are compared to wild-type or when <i>spo-11(me44)</i> is compared to <i>cosa-1(tm3298); spo-11(me44)</i>.</p

DNA helicase HIM-6/BLM both promotes MutSγ-dependent crossovers and antagonizes MutSγ-independent interhomolog associations during caenorhabditis elegans meiosis.

Meiotic recombination is initiated by the programmed induction of double-strand DNA breaks (DSBs), lesions that pose a potential threat to the genome. A subset of the DSBs induced during meiotic prophase become designated to be repaired by a pathway that specifically yields interhomolog crossovers (COs), which mature into chiasmata that temporarily connect the homologs to ensure their proper segregation at meiosis I. The remaining DSBs must be repaired by other mechanisms to restore genomic integrity prior to the meiotic divisions. Here we show that HIM-6, the Caenorhabditis elegans ortholog of the RecQ family DNA helicase BLM, functions in both of these processes. We show that him-6 mutants are competent to load the MutSγ complex at multiple potential CO sites, to generate intermediates that fulfill the requirements of monitoring mechanisms that enable meiotic progression, and to accomplish and robustly regulate CO designation. However, recombination events at a subset of CO-designated sites fail to mature into COs and chiasmata, indicating a pro-CO role for HIM-6/BLM that manifests itself late in the CO pathway. Moreover, we find that in addition to promoting COs, HIM-6 plays a role in eliminating and/or preventing the formation of persistent MutSγ-independent associations between homologous chromosomes. We propose that HIM-6/BLM enforces biased outcomes of recombination events to ensure that both (a) CO-designated recombination intermediates are reliably resolved as COs and (b) other recombination intermediates reliably mature into noncrossovers in a timely manner