Shakes, Diane et al. (2009) International Worm Meeting "EMB-1 is a novel protein involved in the metaphase-to-anaphase transition."

Golden, Andy, Shakes, Diane

[International Worm Meeting, 2009]

The Anaphase Promoting Complex (APC) is a multi-subunit E3 ubiquitin ligase that promotes the metaphase-to-anaphase transition during meiotic and mitotic divisions. Temperature-sensitive (ts) mutants in mat-1, mat-2, mat-3, emb-27, and emb-30 arrest as 1-cell embryos, stuck in metaphase of meiosis I. These five genes code for five subunits of the APC. The ts alleles of emb-1 have grabbed our attention because their arrest phenotype is indistinguishable from the APC mutants. Furthermore, genetic doubles constructed between emb-1(hc62) and the APC mutants cannot be maintained at the permissive temperature, a common feature of any APC double mutant. Additionally, suppressors that suppress the APC mutants (Stein et al., 2007) also suppress emb-1. What is EMB-1 you may ask? We mapped emb-1 to a tiny interval on LG III and used RNAi to phenocopy the 1-cell arrest phenotype. Rescue and sequencing confirmed that emb-1 codes for a novel protein with no known homologies outside of Caenorhabditis species. Localization studies are underway. We propose that EMB-1 is a novel subunit or regulator of the APC in C. elegans.

Levitan DJ et al. (1994) Worm Breeder's Gazette "sum-1 Encodes a Protein Containing Seven Transmembrane Domains."

Levitan DJ, Greenwald IS

[Worm Breeder's Gazette, 1994]

sum-l encodes a protein containing seven transmembrane domains Diane Levitan and Iva Greenwald, HHMI, Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032

Boyd L et al. (1994) Worm Breeder's Gazette "Asymmetric PAR-2 at First Cleavage"

Boyd L, Kemphues KJ, Levitan DJ

[Worm Breeder's Gazette, 1994]

Asymmetric PAR-2 at First Cleavage Lynn Boyd1, Diane Levitan2, and Ken Kemphues1, 1)Section of Genetics and Development, Cornell University; 2) Department of Cellular and Developmental Biology, Harvard University (current address: Columbia University)

Winter, Ethan S. et al. (2013) International Worm Meeting "Investigating the cellular mechanisms of skewed sex ratios in non-C. elegans nematodes."

Winter, Ethan S., Shakes, Diane C.

[International Worm Meeting, 2013]

During the post-meiotic phase of spermatogenesis, various proteins and cellular components are either retained by the sperm or discarded in a cytoplasmic "waste bag" (residual body). In nematodes, this spermatid-residual body partitioning event occurs unusually early; either during or immediately after anaphase II. In addition, since nematode sperm are non-flagellated, one of the discarded components is tubulin, which is essential for meiotic chromosome segregation but (except for the centriole) superfluous for the subsequent function of either the spermatids or crawling spermatozoa. Instead, the motility of nematode spermatozoa is driven by the major sperm protein (MSP) which forms dynamic filaments within the treadmilling pseudopod. Our lab has recently discovered that a dramatic sex skew in the non-C. elegans species, Rhabditis sp. SB347 (Felix, 2004) can be explained by an asymmetric partitioning of cellular components during anaphase II of spermatogenesis (Shakes et al, 2011) To investigate the evolution of this trait and rule out that it is merely an isolated curiosity of R. sp. SB347, we are analyzing spermatogenesis in other members of this clade. Here we report similar partitioning patterns during spermatogenesis in three species R. sp.SB372, R. sp. JU1782, and R. sp. JU1809. As in the dividing secondary spermatocytes of SB347, MSP is partitioned exclusively to the X-bearing sperm. In contrast, tubulin and other non-essential components are partitioned exclusively to the nullo-X sperm; true residual bodies are never observed. We also find that the two chromatin masses exhibit differential compaction. Thus, these meiotic asymmetries serve as a common mechanism for generating skewed sex ratios within this clade and potentially throughout the phylum. Ultimately, these ongoing studies may provide new insights regarding not only the evolution of reproductive modes and sexual plasticity but also the cellular and biochemical mechanisms of cell polarization.

Morrison, Kayleigh N. et al. (2021) MicroPubl Biol

Morrison, Kayleigh N., Ragle, James Matthew, Shakes, Diane C., Uyehara, Christopher M., Ward, Jordan D.

[MicroPubl Biol, 2021]

During the process of cell differentiation, specific cytoskeletal proteins can sequentially assemble into a wide variety of diverse molecular superstructures. Nematode spermatogenesis provides a powerful system for studying these transitions since sperm-specific transcription ceases prior to the meiotic divisions and translation ceases shortly thereafter (Chu and Shakes, 2013). Therefore, structural transitions that follow the meiotic divisions must be carried out by the remodeling of already synthesized proteins. The Major Sperm Protein (MSP) is a nematode-specific cytoskeletal element whose polymerization dynamics drive the pseudopod-based motility of the activated sperm (Roberts, 2005). In C. elegans, MSP additionally functions as the extracellular signaling molecule for triggering both ovulation and oocyte maturation (Miller et al., 2003). MSP is highly abundant in sperm, where it reaches 10-15% of total and 40% of soluble cellular protein (Roberts 2005). Within developing spermatocytes, MSP is packaged into fibrous bodymembranous organelle (FB-MO) complexes (Fig. 1A, Roberts et al., 1986). By assembling into paracrystalline FBs, MSP is both sequestered away from the critical meiotic processes of chromosome segregation and cytokinesis while also being packaged for efficient segregation into spermatids during the post-meiotic partitioning process (Chu and Shakes 2013, Nishimura and LHernault, 2010, Price et al., 2021). Following the meiotic divisions and sperm individualization, FBs disassemble, and MSP disperses as dimers throughout the spermatid cytoplasm (Fig. 1A). When sperm activate to form motile spermatozoa, MSP polymerization within the pseudopod drives the motility of the crawling sperm (Chu and Shakes, 2013). Thus, MSP exists in at least three distinct molecular states: 1) in highly organized paracrystalline FBs within developing spermatocytes 2) as unpolymerized dimers within spermatids, and 3) in dynamically polymerizing filaments and fibers within crawling spermatozoa.

Sadler, Penny L. et al. (2011) International Worm Meeting "Using C. elegans to teach transmission genetics in a large introductory biology lab."

Sadler, Penny L., Shakes, Diane C.

[International Worm Meeting, 2011]

Transmission genetics is a core topic in most introductory biology courses and yet many students rarely think about genetics beyond Mendel's classical experiments with peas. In our introductory biology laboratory course at the College of William and Mary, we expand students' notions of transmission genetics by using C. elegans as the model system to determine whether an "unknown" Unc mutation is linked or unlinked to a known Dpy marker. In a course that serves not only biology majors but also neuroscience majors, pre-meds, and non-majors, the students see direct relevance in studying mutations with direct links to human neurological conditions. Our genetics module is a four-week session that culminates in a writing workshop designed to aid students in writing their first formal lab report. The first week serves as an introduction to best practices in using a dissecting scope, sexing the worms, distinguishing single mutant morphological phenotypes, and setting up a Po cross with 1mm long males and hermaphrodites. During the second week, the students score their P0 cross, set up their F1 crosses, and practice distinguishing wild type, single mutants and double mutant hermaphrodites. During the third week, the students score their F2 progeny, analyze their data, and test their data set against a null hypothesis that the two genes are unlinked using Chi-square. The format of the lab report is introduced and discussed as a class. During the fourth week, student teams meet with their lab instructor to go over a rough draft of their lab report. This one-on-two time dedicated to improving the content of the report allows for correction of major misconceptions and errors in formatting the report. The fourth lab week is also dedicated to a self-guided online Wormbase bioinformatics exercise which is designed to help students formulate their ideas for the future directions sections of their lab report. The poster will also present student learning objectives and assessment, and discuss the logistics of preparing the materials that enable over 400 freshmen carry out a series of genetic crosses during a four week lab module under the guidance of masters level graduate teaching assistants and a single, full-time lab coordinator.

Singaravelu, Gunasekaran et al. (2009) International Worm Meeting "Isolation of Caenorhabditis elegans mutants defective in sperm function."

Shakes, Diane, Singaravelu, Gunasekaran, Singson, Andrew

[International Worm Meeting, 2009]

The functional components required during fertilization can be conveniently studied in Caenorhabditis elegans. Here we describe a screening strategy for isolation of C. elegans mutants defective in sperm function (Spe) or egg function (Egg). We mutagenized the hermaphrodite population of the genotype sem-2(n1343) Is[Pelt-7::gfp + rol-6 (su 1006)] I using Ethyl methyl sulfonate (EMS) and screened for the self sterile mutants that could be rescued by allowing them to mate with wild type males. The sem-2(n1343) genetic background prevents the worm from laying eggs and the progeny are hatched inside the body. The transgene, Pelt-7::gfp, drives the expression of gfp under elt-7 promoter, which is active in intestine. Thus, amid the mutagenized population, sterile mutants can be readily identified by the absence of fluorescing eggs and/or larvae in their uterus. In our most recent screen, we isolated twenty-four non-conditional sterile Spe mutants and eleven temperature sensitive Spe mutants. Our preliminary characterization suggests that various aspects of C. elegans reproductive biology such as sperm development, differentiation and/or functions are compromised in these mutants. A slightly modified strategy can be used to identify mutants that cause defective oogenesis or oocyte function. We are currently mapping and further characterizing these mutants with the aim of unraveling the genetic determinants behind the normal, reproductively competent sperm.

Peterson, Jackson et al. (2013) International Worm Meeting "Localization dynamics of SPE-6, a sperm-specific CK1 in C. elegans."

Waller, Brianna, Shakes, Diane, Peterson, Jackson

[International Worm Meeting, 2013]

During spermatogenesis, diploid germ cells develop into small, haploid spermatozoa which only acquire full motility during the final step of sperm activation. In C. elegans, sperm activation occurs in response to an external signal which converts sessile round spermatids into bipolar, crawling spermatozoa. Notably, this final conversion occurs in the absence of either transcription or translation, suggesting that these changes are driven solely by post-translational modifications. In fact, since C. elegans spermatocytes cease transcription prior to the meiotic divisions and cease translation immediately after the meiotic divisions, many of the key developmental changes occur in the absence of transcription and translation. Therefore, key motility proteins such as the major sperm protein (MSP) must be translated early in spermatogenesis and sequestered within fibrous body membranous organelles (FB-MOs). Here we describe the dynamic localization of a sperm-specific casein kinase 1 (SPE-6). In wild type spermatocytes, SPE-6 is present in both the cytoplasm and in association with FB-MOs. During the post-meiotic budding division, SPE-6 segregates to the spermatids and away from residual bodies, eventually localizing to the sperm chromatin. Then during sperm activation, SPE-6 re-localizes to the pseudopod. In several non-null alleles of spe-6, sperm activation occurs in the absence of an activation signal. Our analysis of spe-6(hc163) reveals that this precocious activation phenotype is accompanied by dramatic alterations in SPE-6 localization patterns. In developing spermatocytes, the mutant protein is mostly cytosolic. During the budding division, it segregates aberrantly to the residual body; any SPE-6 that does end up in spermatids fails to localize to the chromatin. We present a model of how this aberrant pattern of protein localization may explain the precocious activation phenotype and provide clues regarding the function of SPE-6 in sperm activation.

Messina, Kari et al. (2011) International Worm Meeting "Novel Protein SPE-7 is Required for Meiotic Chromosome Segregation and Fibrous Body Assembly."

Shakes, Diane C., Presler, Marc, Messina, Kari

[International Worm Meeting, 2011]

The development of functional spermatozoon from uncommitted germ cells requires the progression of two distinct yet presumably interacting cellular programs: the meiotic and cell differentiation programs of spermatogenesis. One spermatogenesis-defective factor required for the normal progression of both of these programs is SPE-7. spe-7 mutant spermatocytes progress through the early phases of meiosis, detach from the rachis, and set up a metaphase I spindle. However meiotic chromosome segregation is aberrant and the spermatocytes never undergo cytokinesis. Ultimately, the spe-7 spermatocytes arrest with cortically localized actin and microtubules that reassemble into a network-like pattern. Surprisingly, we found that in developing wildtype spermatocytes, SPE-7 localizes to fibrous bodies, the paracrystaline storage form of the major sperm protein, MSP. During the meiotic divisions, the localization of SPE-7 is somewhat dynamic but it ultimately segregates with the fibrous bodies into the budding spermatids before disappearing altogether. This localization pattern is apparently functional as a reexamination of spe-7 spermatocytes revealed that they express MSP but are defective for fibrous body assembly. spe-7 encodes a novel protein with close homologs limited to other Caenorhabditis species. Both germline microarrays and western blots indicate that SPE-7 is expressed specifically during spermatogenesis, and it is one of the many genes regulated by the sperm-specific transcription factor, SPE-44 (see abstract by Kulkarni et al.). We are currently pursuing additional genetic and biochemical studies designed to understand the dynamic localization of SPE-7 as well as its interactions with other known spe genes.

Golden, Andy et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "EMB-1, SUCH-1, and GFI-3, three Anaphase-Promoting Complex subunits, are required for the meiotic divisions of fertilized embryos"

Stein, Kathryn, Nesmith, Jessica, Golden, Andy, Shakes, Diane

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

Chromosome segregation during meiosis and mitotis requires the activity of the Anaphase-Promoting Complex (APC), a multi-subunit E3 ubiquitin ligase that ubiquitylates target proteins, which are then degraded by the 26S proteasome. RNAi and mutational analysis has revealed that most APC subunits in C. elegans are required for the meiotic divisions in fertilized embryos. Depletion of any of these subunits results in embryos arrested in metaphase of meiosis I. Temperature-sensitive alleles of emb-1 share this arrest phenotype at non-permissive temperatures. Alleles of emb-1 also enhance other APC mutants at permissive temperatures and are suppressed by mutants that suppress other APC mutan ts. We mapped and cloned emb-1; it encodes an 81 amino acid protein with no known function. The Oegema lab has demonstrated that EMB-1 co-purifies with APC subunits. Together, these data suggest that EMB-1 is a novel regulator or subunit of the APC.