McNally, Francis et al. (2021) International Worm Meeting "Role of Cohesin in Chromosome-Dependent Meiotic Spindle Assembly"

McNally, Karen, McNally, Francis

[International Worm Meeting, 2021]

Accurate chromosome segregation during mitosis or meiosis requires a bipolar array of microtubules with microtubule plus ends oriented toward chromosomes and microtubule minus ends oriented toward the poles. Mutants that assemble spindles which contain more than or fewer than two poles typically produce aneuploid progeny. During mitosis and male meiosis, bipolar spindle formation is initiated by the duplication of the centrosomes. Because female meiotic spindles in C. elegans and vertebrates do not have centrosomes, we asked whether the discrete bipolar structure of bivalents is instead required for the initiation of bipolar spindle assembly. To address this question, we conducted time-lapse imaging of spindle assembly using GFP::tubulin or the spindle pole markers GFP::ASPM-1 or GFP::LIN-5 in cohesin mutants that have single chromatids during segregation stages of meiosis. Severson and Meyer (eLife.03467) previously reported that rec-8, spo-11 double mutants and rec-8, spo-11, coh-3, coh-4 quadruple mutants both have single chromatids at metaphase I and metaphase II. Our results revealed that bipolar spindles assemble around rec-8, spo-11 metaphase I single chromatids but not around rec-8, spo-11, coh-3, coh-4 metaphase I single chromatids nor any of the metaphase II single chromatids. These results suggest that chromosome bipolarity is not required for bipolar spindle assembly and further suggest that non-cohesive cohesin can promote bipolar spindle assembly. To address how non-cohesive cohesin might promote bipolar spindle assembly, we monitored chromosome-associated AIR-2. AIR-2 normally concentrates in a ring between homologous chromosomes at metaphase I and between sister chromatids at metaphase II. We found intense labeling of AIR-2 on the assembly-competent metaphase I single chromatids of rec-8, spo-11 mutants but minimal GFP::AIR-2 was associated with any of the assembly-incompetent single chromatids. We are currently asking whether AIR-2 is the only spindle assembly factor recruited by cohesin and trying to identify the downstream effectors of AIR-2.

Francis McNally et al. (2007) International Worm Meeting "Restriction of meiotic spindle length by microtubule severing."

Karen McNally, Francis McNally

[International Worm Meeting, 2007]

In animals, female meiotic spindles mediate the expulsion of chromosomes into polar bodies to generate a haploid egg. Time-lapse imaging of fluorescent protein fusions has revealed that wild-type C. elegans meiotic spindles undergo discrete length changes during each division. Meiosis I spindles, for example, maintain a constant metaphase length of 7.7 um for several minutes. Meiosis I spindles then shorten until they reach 58% of their starting length. During this initial shortening phase, the average fluorescence intensity of GFP-tubulin doubles (suggesting an inward sliding mechanism) and the spindles remain parallel to the cortex. After this initial shortening phase, spindles rotate perpendicular to the cortex, then initiate anaphase chromosome separation as they proceed through a second phase of spindle shortening. During the second shortening phase, the average fluorescence intensity of GFP tubulin decreases at poles while increasing in the midzone, suggesting a redistribution of microtubules within the spindle. At the end of the second shortening phase, MI spindles are 2.8 um long (38% of starting length). MEI-1 and MEI-2 are the C. elegans orthologs of the 2 subunits of a conserved microtubule-severing ATPase called katanin. MEI-1 and MEI-2 are localized on meiotic spindles and purified complexes of MEI-1 and MEI-2 sever microtubules in vitro, indicating that MEI-1/MEI-2 may regulate microtubule length within the meiotic spindle. We have analyzed a partial loss of function allele of mei-2 called ct98. mei-2(ct98) meiosis I spindles exhibit 4 defects. First, mei-2(ct98) MI spindles initially assembly with a longer than wild-type metaphase length of 9.6 um. Second, they do not rotate. Third, they do not undergo the second phase of spindle shortening although they undergo the first phase of shortening with wild-type velocity. Fourth, microtubules do not redistribute from the poles to the midzone. As a result of these defects, mei-2(ct98) spindles reach a final minimum length of 6.2 um (65% of starting length) and induce formation of larger than wild-type polar bodies. These results are consistent with a model in which microtubule severing reduces the average microtubule length during spindle assembly and during the second phase of spindle shortening. We are currently analyzing a collection of mei-1 misense alleles provided by Paul Mains to establish a relationship between in vitro microtubule-severing activity and in vivo spindle length control in order to address the perplexing question of why null alleles of mei-1 or mei-2 prevent assembly of bipolar meiotic spindles rather than causing assembly of extremely long meiotic spindles.

Bailey, Cynthia et al. (2021) International Worm Meeting "The role of ATX-2 and VPR-1 in sperm positioning within the C. elegans meiotic embryo"

McNally , Francis, Bailey, Cynthia

[International Worm Meeting, 2021]

Fertilization occurs during female meiosis in most animals, which raises the question of what prevents the sperm body (DNA, centrioles, and organelles) from interacting with the meiotic spindle. In C. elegans, the meiotic spindle and sperm body are maintained in opposite thirds of the ellipsoid zygote despite vigorous cytoplasmic streaming. In a previous study (Panzica et al. 2017. J Cell Biol 216: 2273), the sperm body was relatively stationary in control cells but moved long distances with the yolk granules when actin was depolymerized, resulting in sperm DNA within 2 microm of the meiotic spindle. This result led to the idea that the sperm body is anchored at the site of fertilization by cortical actin while maternal organelles move freely with cytoplasmic streaming. Simultaneous live imaging of paternal mitochondria, maternal ER and maternal yolk granules, however, revealed episodes with all these organelles moving together. In agreement with Kimura (2020. Mol Biol Cell 31:1765) we observed long range excursions of the sperm contents even in control embryos. These movements, however, were mostly in the short axis of the embryo. Long-distance excursions of the sperm contents toward the meiotic spindle were infrequent and followed by a return to the original spindle distal position. We are currently exploring the possibility that contacts between the maternal ER, paternal mitochondria, and cortical F-actin may play a role in restricting the movement of the sperm body to the future posterior end of the embryo. To begin, we have first defined the cell-cycle changes in ER structure by filming meiotic embryos within mKate::tubulin GFP::SP12 hermaphrodites and the cell-cycle changes in cytoplasmic streaming by filming embryos within GFP::SP12 hermaphrodites mated with mCherry::MEV-1 males. We are now using this baseline to analyze ER structure and movement of ER and paternal mitochondria in embryos depleted of maternal ATX-2, VPR-1, HSP-4, PFN-1 and other candidates predicted to disrupt sheet-like ER.

Gong, Ting et al. (2021) International Worm Meeting "C. elegans maximize the number of euploid progeny from zim-2 parents with crossover failure on chromosome V."

Gong, Ting, McNally, Francis, Tran, Thuy-Linh

[International Worm Meeting, 2021]

Gametes with incorrect chromosome number can lead to embryonic lethality and developmental deficiency if inherited. Parental trisomy or crossover failure are two conditions expected to yield random meiotic segregation and 25% trisomy among offspring. We previously demonstrated that parental trisomy of the X can be corrected among progeny by preferentially placing the extra chromosome in the first polar body (Cortes, eLife.06056). If 100% efficient, such a univalent elimination mechanism would generate 100% lethal monosomy among progeny of parents with crossover failure. However, high viability rates have been reported among progeny of him-8 mutants (crossover failure on X) (Hodgkin, Genetics 91:67), zim-2 mutants (crossover failure on V) and zim-1 mutants (crossover failure on II and III) (Jaramillo-Lambert, Curr Biol 20:2078), suggesting the possible existence of a distributive segregation mechanism that is more efficient during spermatogenesis than during oogenesis. Here we directly measured the frequency of monosomy, disomy and trisomy among the progeny of zim-1 mutants and zim-2 mutants mated to wild type using PCR polymorphisms that differ between three strain backgrounds. Zim-1 hermaphrodites mated to wild-type males produced 72% disomy II progeny (n=33) and 57% disomy III progeny (n=54). This difference from the 50% expected from random segregation was not significant. Zim-2 hermaphrodites mated with wild-type males produced 73% disomy V progeny (n=65) which was not significantly different than random segregation when corrected for 24% (n=42) crossovers on V observed by counting diakinesis bivalents in oocytes. In striking contrast, zim-2 males mated to wild-type hermaphrodites produced 95% disomy V progeny (n=94) which is significantly higher than random segregation. We are currently testing whether this is due to a high frequency of crossovers on V in zim-2 males, more efficient fertilization by euploid sperm, or a genuine distributive segregation system during male meiosis. A side benefit of our approach is that we can determine the phenotypes of most single chromosome aneuploidies. We have thus far observed monosomies of II, III, or V only in dead embryos and have observed trisomies of II, III, or V only among hatched larvae.

Vargas, Elizabeth et al. (2017) International Worm Meeting "Trisomy correction occurs during both meiosis I and meiosis II."

Korf, Ian, Friedman, Jacob, McNally, Francis, McNally, Karen, Vargas, Elizabeth, Wang, David

[International Worm Meeting, 2017]

The vast majority of eukaryotes have exactly two copies of each chromosome and reproduce sexually through the process of meiosis. Accurate chromosome segregation requires the physical attachment of exactly two homologous chromosomes by a crossover so that each homologous chromosome can form attachments to opposite poles of a bipolar spindle. Thus a third copy of a chromosome might be expected to segregate randomly, resulting in 50% of progeny inheriting the extra chromosome. Hodgkin et al. (Genetics 91:67) reported that C. elegans with a third copy of the X chromosome have far fewer than 50% trisomic progeny. Cortes et al. (Elife 4:e06056) found that the preferential elimination of the extra X occurs during anaphase I of female meiosis. To test whether extra copies of all C. elegans chromosomes are preferentially eliminated, we scored the inheritance of three different DNA polymorphisms for each autosome among the progeny of triploid hermaphrodites mated with diploid males. For all 5 autosomes, the frequency of trisomic offspring was significantly less than expected from random segregation. Preferential elimination of the extra copy of chromosome V was confirmed by FISH. We also generated a trisomy IV worm strain and when trisomy IV hermaphrodites were mated to diploid males, the frequency of triplo IV progeny was again much lower than expected from random segregation. Cortes et al. (2015) found that univalent X chromosomes do not lose cohesion during anaphase I and thus lag before being preferentially captured by the polar body. Counting of mCherry:histone-labeled chromosomes in triploids revealed that chromosome number decreased between metaphase I and metaphase II and decreased again between metaphase II and diakinesis in the adult progeny. Data from live imaging of anaphase chromosome segregation and REC-8 staining of fixed meiotic spindles support a model in which some univalents remain intact during anaphase I whereas other univalents segregate apart at anaphase I. The resulting single chromatids then lag during anaphase II and are preferentially captured by the second polar body. These data demonstrate that asymmetric female meiotic divisions can correct triploidy and trisomy in C. elegans.

Juanico, Iris Y et al. (2021) MicroPubl Biol

Gong, Ting, McNally, Francis J, Juanico, Iris Y, Meyer, Christina M, McCarthy, John E

[MicroPubl Biol, 2021]

RMD-1 was discovered as a cytoplasmic regulator of microtubule dynamics in C. elegans and several highly homologous proteins were inferred from the C. elegans and human genome sequences (Oishi et al., 2007). One of the human homologs, RMD-3/PTPIP51, has an N-terminal extension relative to other RMDs that targets it to the mitochondrial outer membrane and which binds the endoplasmic reticulum (ER) protein VAPB (De Vos et al., 2012). This interaction mediates tethering of mitochondria to the ER in human cells (Stoica et al., 2014). None of the C. elegans RMDs have sequence homology with the N-terminal extension found on human RMD-3/PTPIP51, however, C. elegans RMD-2 has an N-terminal extension relative to the other RMDs and this extension has potential to form a transmembrane helix as determined by TMpred release 25 (https://embnet.vital-it.ch/software/TMPRED_form.html). RMD-2 is present in sperm whereas RMD-3 and RMD-6 are highly enriched in sperm (Ma et al., 2014). Paternal mitochondria are eventually degraded in the embryo (Sato and Sato, 2011). However, they are maintained, along with other sperm contents, at the opposite end of the zygote from the female meiotic spindle during meiosis (Fig. 1). We hypothesized that RMD-2, 3 and 6 on paternal mitochondria bind to the VAPB homolog, VPR-1, on maternal ER at fertilization to tether the sperm contents at the future posterior end of the zygote.

Johnson, Jacque-Lynne F. et al. (2009) International Worm Meeting "Levels of the ubiquitin ligase substrate adaptor MEL-26 are inversely correlated with MEI-1/katanin microtubule severing activity during both meiosis and mitosis."

Mains, Paul E., McNally, Karen, Raharjo, Eko, Lu, Chenggang, Johnson, Jacque-Lynne F., McNally, Francis J.

[International Worm Meeting, 2009]

The MEI-1/MEI-2 katanin microtubule-severing complex is required for meiotic spindle formation in C. elegans. While essential for meiosis, MEI-1/MEI-2 must be quickly downregulated to prevent interference with formation of the first mitotic spindle. Post-meiotic inactivation of MEI-1 is accomplished by two independent protein degradation pathways. One pathway requires MEL-26 as a substrate-specific adaptor for the CUL-3 based E3 ubiquitin ligase, while a parallel pathway requires phosphorylation of MEI-1 by MBK-2 kinase. MBK-2 mediated degradation of MEI-1 appears to be coupled with meiotic exit after which MEL-26 completes the degradation process. A phosphorylation system involving Protein Phosphatase 4 also acts independently of these degradation systems to regulate meiotic and mitotic MEI-1 activity (Han et al, 2009). The parallel MEL-26 and MBK-2 systems explain how MEI-1 microtubule-severing activity is degraded during mitosis, but how is MEI-1 degradation delayed until its essential meiotic function is complete. Stitzel et al (2006) showed that MBK-2 activity is held in check by EGG-3 until meiosis is completed. Our work reveals that the MEL-26 branch of the MEI-1 degradation pathway is regulated in part by MEL-26 protein accumulation. Affinity-purified antibodies revealed that MEL-26 is present at low (but nonzero) levels until meiosis is complete, after which MEL-26 levels increase substantially, paralleling the increase in post-meiotic MEI-1 degradation. During meiosis, MEL-26 levels are kept low by another type of ubiquitin ligase containing CUL-2, and by an upstream activator of ubiquitin ligase activity, RFL-1. Thus, a cascade of proteolysis, triggered at fertilization, results in the correct timing of MEI-1 activity. However, premature meiotic activity of either MEL-26 or MBK-2 (independently or together) is not sufficient to block spindle formation, indicating that additional factors are rate limiting for the downregulation of MEI-1. While it was initially thought that MEL-26 did not play a role during meiosis, we find the low levels of meiotic MEL-26 have a subtle function, acting to moderate MEI-1 activity in the meiotic spindle. In addition, our results demonstrate that while RFL-1 has additional essential targets, MEI-1 is the only essential target for MEL-26 and possibly for the E3 ubiquitin ligase CUL-3.

Lee M-H et al. (1997) International C. elegans Meeting "TOWARD THE IDENTIFICATION OF IN VIVO RNA TARGETS OF GLD-1."

Lee M-H, Schedl TB

[International C. elegans Meeting, 1997]

gld-1 is a female germ cell specific tumor suppressor gene that is essential for normal oocyte differentiation and meiotic prophase progression (Francis et al., 1995a,b). GLD-1 is a member of a small family of proteins, including the mouse/human Sam68 and Quaking proteins, that are highly related over an ~ 200 amino acid region which contains a 70 to 100 amino acid RNA binding domain called the KH motif (Jones and Schedl, 1995). Extensive mutational analysis of gld-1 has demonstrated the importance of conserved sequences for its in vivo function. GLD-1 is a cytoplasmic protein and therefore is likely to control mRNA translation or RNA stability in the cytoplasm (Jones et al., 1996). Currently, no obvious RNA targets have been identified from genetic analysis. We have employed a biochemical approach to identify in vivo RNA targets of GLD-1. The strategy is based on the ability of anti GLD-1 antibodies to immunoprecipitate (IP) GLD-1 from a worm lysate and the strong likelihood that GLD-1 present in the lysate is functional and bound to RNAs. GLD-1 was IPed with affinity purified polyclonal antibodies from a cytosol extract of adult hermaphrodites or with rabbit IgG as a control. RNAs co-IP with GLD-1, as well as with rabbit IgG, were converted into cDNAs. Non-specifically trapped RNAs from the GLD-1 IP were eliminated by subtracting cDNAs from the GLD-1 IP with cDNAs from the control IP. The difference product after 4 rounds of subtraction (DP4) was used to screen a Lambda ZAP II cDNA library constructed with the cDNAs from the GLD-1 IP. Duplicate filters were also screened with a probe made from control IP cDNA. Clones that are positive only with the DP4 probe are currently being characterized. Francis et al., 1995a Genetics 139, 579-606; Francis et al., 1995b Genetics 139, 607-630; Jones and Schedl, 1995 Genes Dev. 9, 1491-1504; Jones et al., 1996 Dev. Biol. 180, 165-183. This work was supported by NIH Grant HD25614.

J Rogers et al. (1999) International C. elegans Meeting "Feeding For Phenotypes"

J Hubbard, J Rogers

[International C. elegans Meeting, 1999]

We were intrigued by the recent results of Timmons and Fire (1998) suggesting that RNAi-induced phenotypes could be observed by simply feeding worms with bacteria that are producing the dsRNA of interest. Given the potency of RNAi in the germ line, we wondered if this technique could be used to produce germline-defective phenotypes of interest to our lab. In addition, we sought to apply the technique to identify genes involved in germline function that may otherwise cause a loss-of-function embryonic lethal phenotype. To test this possibility, we collected several cDNAs from genes that produce loss-of-function germline-defective phenotypes (some of which also cause embryonic or larval lethatity) and introduced them into the "double T7" vector described by Timmons and Fire. We are testing these constructs by feeding synchronous populations of L1 larvae with bacteria that express dsRNA and scoring them in the adult for germline defects. Preliminary data suggests that germline phenotypes can be obtained in this manner, but that they may be restricted to events that occur relatively late in germline development. For example, gld-1 dsRNA fed to L1 larvae produced an abnormal oocyte phenotype characteristic of partial loss-of-function alleles of gld-1 (class E and F; Francis et al., 1995) at 60% penetrance. Progeny of unaffected adults were then grown on RNAi-producing bacteria. In these animals we observed the more severe tumorous phenotype in which progression through meiotic prophase is altered and oogenesis does not occur (Francis et al., 1995).

Frederic Horndli et al. (2008) C.elegans Neuronal Development Meeting "Identification of ACR-16 Specific Trafficking and Targeting Components"

Andres Maricq, Frederic Horndli, Michael Jensen

[C.elegans Neuronal Development Meeting, 2008]

Three types of receptors are present at the neuromuscular junction in C. elegans: the levamisole acetylcholine receptor (AChR) complex LevR (UNC-29, UNC-38, UNC-63, LEV-1 and LEV-8), the nicotinic AChR ACR-16 and the GABA receptor UNC-49. Both ACR-16 and the LevR complex respond to cholinergic inputs, with ACR-16 contributing to 70% of the EPSP (Francis M.M.., et al., 2005). Although a mechanism for stabilization and clustering of ACR-16 has been described (Francis MM., et al., 2005), very little is known, about how AChR receptors are transported and inserted at the NMJ. Using RNAi targeting of known transport motors and cargo adaptor proteins, in combination with either unc-29 (x29) or acr-16 (ok789) deletion backgrounds we have successfully identified the kinesin-1 transport complex (composed of unc-116, klc-2, unc-16 and unc-14 (Byrd DT., et aI., 2001, Sakamoto R., et al., 2004)) as an ACR-16 specific transport complex. Indeed, RNAi and deletion mutants for all elements of this complex in combination with unc-29 (x29), exhibit almost complete paralysis as well as ACR-16 GFP mislocalization. In addition, the same RNAi and deletion mutants for the elements of the kinesin-1 complex do not affect acr-16(ok789) movement nor UNC-29 GFP and UNC-49 GFP localization. Encouraged by these results we have started a whole genome RNAi screening approach using the unc-29(x29) and acr-16(ok789) background to discover more elements related to ACR-16 and UNC-29 trafficking, targeting, insertion and stabilization.