C. Elegans Research: Update #2

(Originally written 6/11/18)

            I have finished the wet lab portion of my project, and I learned so much while working on campus!

            In the first week of my research, I learned how to do some new lab chores and techniques. When I arrived, I had to first tend to my worms! Because the mutation I am working with is temperature sensitive, I had to shift young worms up to a 25 C incubator in order to make the spe-26 mutant worms sterile. Each day, I transferred worms that had laid embryos to new plates, so that when the embryos hatched, all worms on a single plate would be the same age. I put half of my plates in the 25 C incubator to serve as my experimental worms, and kept the other half at 16 C so that they would stay fertile and would continue to lay embryos for me to work with.

            In my first post, I wrote that I would be visualizing actin and tubulin dynamics in spermatogenesis, in order to study the function of SPE-26. In order to visualize cell divisions, I first had to dissect the worms, then used immunocytology to stain specific cell parts.

            While on campus, I dissected and fixed a lot of worms! In order to dissect C. elegans, I transferred about 7 worms to a glass slide with a buffer solution, and used a small needle to cut each worm near the gonad. When a worm is cut, the gonad separates from the worm’s body, so it is easy to view the entire germline. Once I cut all of the worms on a slide, I preserved the slide in either methanol to dehydrate the specimen, or paraformaldehyde to generate chemical cross-links, and prepare it for staining. For each study I completed, I prepared 8-16 slides, including both wild-type and mutant worms.

           After dissecting worms, I used immunocytology to stain cell parts of interest. In immunocytology, antibodies tagged with fluorescent markers bind to specific proteins in cells. Because each antibody is specific to a single molecule, applying an antibody solution to the specimen will only stain the molecule of interest. In my project, I am interested in studying actin and tubulin dynamics in spermatogenesis, so I used antibodies that bound specifically to actin, tubulin, and DNA. Once I stained these molecules with fluorescent antibodies, I was able to view my slides with an epifluorescent microscope, which allowed me to view and photograph the cells in each worm’s germline.

            During my time in the lab, I completed one immunoprep using DNA/tubulin staining, two preps using DNA/actin staining, and one prep using DNA/ERM-1 staining. ERM-1 is an actin-binding protein that connects actin to the cellular membrane; I want to analyze the dynamics of this protein to gather more insight into how actin is partitioned during spermatogenesis. Each prep yielded at least 20 photographs of C. elegans gonads, which I will study more carefully using photoshop once I return home. After compiling my photographs, I will be able to compare spermatogenesis in the control and mutant worms. Upon some basic analysis, my findings have been consistent with previous data that spe-26 mutants are unable to produce mature spermatids, and fail to properly polarize and form RBs (Winter et al., 2017). In wild-type male worms, spermatids form a bi-polar budding figure as they detach from the RBs; thus, a single spermatid pinches off from either end of the RB. In spe-26 worms, it seems that the spermatids fail to polarize, and the developing spermatids never fully divide. This produces a large cell with multiple nuclei. (See below for a comparison of bipolar and unipolar budding figures.) I will need to analyze my data more thoroughly to see the extent to which mutant cells attempt to divide before arresting development, and to see exactly what goes wrong with each cell part during development.

Screen Shot 2018-07-31 at 9.57.50 PM

Figure 1. A comparison of normal spermatid budding (left) and spe-26 mutant unipolar budding (right). In normal budding, the mature spermatids bud from either side of the RB, which contains actin, tubulin, and other cellular waste. In arrested spermatid development caused by a spe-26 mutation, the DNA polarizes to one side, and fails to bud from the RB.

 

References:

Winter, Ethan S. et al. (2017). Cytoskeletal variations in an asymmetric cell division support

diversity in nematode sperm size and sex ratios. Development, (144), 3253-3263.

 

Comments

  1. nmcaputo says:

    Hey Kayleigh, I am so impressed by not only your research design, but also the many lab techniques you utilized in your project! It’s incredible that you are so comfortable and competent in the laboratory this early on in your undergraduate career.
    I’m fascinated by the fact that you were able to control the fertility of the C. elegans specimens simply by controlling the temperature. I knew that C. elegans are a popular research organism because of their transparent skin and short life cycle, but this temperature-sensitive characteristic only contributes to their usefulness as a model organism. I also had a question for you: how long did the immunocytology staining take, and how difficult was it to complete this process? Is it similar to the process used to stain cells in order to see them more clearly under a microscope, or is it much more involved? I look forward to seeing your completed project!

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