C. elegans Research: Update 3/Final Post!

Although I finished my work on campus a few months ago, I had a lot more analysis to do once I returned home! In order to finish my project and come up with some conclusions, I used photoshop to analyze the images I captured with the epifluorescent microscope. To study these photographs, I viewed the different layers in each image; this allowed me to see different cell parts, such as the DNA and tubulin, individually and as part of the entire picture. Because I completed three different types of immunopreps: DNA/tubulin staining, DNA/actin staining, and DNA/ERM-1 staining (see post #2 for an explanation), I had to analyze each type of prep separately to understand the dynamics of tubulin, actin, and ERM-1 in the mutant C. elegans strain that I was working with in lab. Studying the images I obtained from each prep helped me to understand what went wrong with each of the three proteins (listed above) during the process of spermatogenesis.

          The first prep that I studied was the DNA/actin staining. In wild-type C. elegans, actin appears as a “honeycomb” pattern surrounding spermatocytes in early development, which thickens into a ring surrounding the spermatocytes as they near division. In Metaphase I, the actin remains in a uniform ring, and in Anaphase I, the actin forms a band in the center of the spermatocyte as it divides. In the second stage of meiotic divisions, Metaphase and Anaphase II, actin migrates from the perimeter of the spermatocytes into the center of the dividing cell, where it accumulates to be discarded from the cell. At this point, the actin is in the residual body (see previous post), and the two clusters of DNA on either side of it will pinch off and become spermatids.

          In hc139 (mutant) C. elegans, this exact pattern is not observed. Early development shows normal actin patterns, but once meiosis II is reached, actin dynamics are flawed. Instead of leaving the perimeter of the cell and entering the residual body, the actin remains somewhat diffused in the cell, and is partially sequestered to one side of the cell. Because SPE-26 was previously believed to be an actin-binding protein, it makes sense that the actin patterns in mutant spermatogenesis are abnormal in the latter half of spermatogenesis, when actin is needed to separate and polarize the dividing spermatocyte. Additionally, results from this immunoprep suggest that SPE-26 is necessary for creating bipolarity in spermatocytes.

          Secondly, I studied the DNA/ERM-1 staining. As mentioned in my prior blog post, ERM-1 is a protein that links actin to the cell membrane; thus, I expected it to show similar patterns to the actin staining. There is little data available about ERM-1 dynamics in spermatogenesis, but based on my prep on wild-type worms, I was able to deduce a general pattern. ERM-1 staining shows a honeycomb pattern in early development, as actin does, and is localized to the perimeter of the cell in early divisions. Like actin, ERM-1 also forms a band in the center of the dividing cell as it progresses to Anaphase I, but the band is more faint because it is not associated with the cell membrane. It was more difficult to detect an ERM-1 band in meiosis II, possibly  because the protein breaks down once it becomes detached from the cell membrane. In both mutant and wild-type C. elegans, faint ERM-1 rings were observed around mature spermatids, which is unlike actin patterns; actin is fully discarded in residual bodies.

          Analysis of ERM-1 patterns in mutant spermatogenesis was not as useful as the actin prep, but I saw similar results from both preps. Meiosis I proceeded fairly normally, but meiosis II showed some errors. Like in the actin prep, the ERM-1 prep showed that spermatocytes failed to produce residual bodies or bipolar budding figures. Additionally, there were few to no spermatids produced by each worm, showing that the spe-26 mutation leads to sterility. The ERM-1 staining was not as useful as the actin staining because the images were not as clear as I wanted them to be, and because the pattern I observed in the dividing cells was not as specific as in the actin staining, but it was still beneficial to have another variable to study as part of my project.

          Finally, I studied the DNA/tubulin prep. The Shakes lab has already completed a lot of work on tubulin dynamics in spe-26 C. elegans, but I also studied tubulin dynamics in order to add more depth to my project and to confirm previous results. If you have studied meiosis in a biology class, you likely already understand tubulin dynamics in meiosis; tubulin is what makes up the “spindle fibers” that you learn about in class. In wild-type spermatogenesis, in early development, tubulin is diffused throughout the cell, then condenses into spindles around nuclear envelope as the cell prepares for division. In Metaphase I, the spindles migrate to the poles of the cell, and attach to the chromosomes, which have lined up in the center of the cell. In Anaphase I, the spindles pull apart the chromosomes, and move to opposite ends of the cell. Once the chromosomes are separated, a cytoplasmic bridge links the two sets of chromosomes, which undergo another round of division. In Metaphase II, the two sets of chromosomes line up again, and the spindles attach to them. Afterwards, in Anaphase II, the spindles pull the two separate sets of chromosomes apart, and four mature spermatids are formed. Following Anaphase II, the spindle fibers detach from the chromosomes and (by a process too complicated to discuss here) move in between the chromosomes so that they can be discarded from the cell within a central residual body

          In mutant C. elegans, there are many errors in this process. Before divisions, spindle fibers are formed and migrate to the poles too early. In Metaphase I, chromosomes sometimes appear to be misaligned, while Anaphase I appears relatively normal if not slightly uneven. In Metaphase II, the cytoplasmic bridge between sets of chromosomes is rarely maintained, and Anaphase II shows an uneven division, if division occurs at all. The most striking error in mutant spermatogenesis is that spermatids are not formed; instead, the meiosis II is not completed and cells do not divide. Instead, multinucleate, disorganized spermatocytes are formed. Instead of producing neat spermatids, which retain no tubulin or actin, mutant worms produce non-functional, massive cells that have from 2-4 nuclei and lots of cellular waste. Clearly, the SPE-26 protein is essential in both tubulin and actin function during spermatogenesis.

          In conclusion, my research suggests that SPE-26 is necessary for establishing bipolarity of dividing spermatocytes, producing a residual body for cellular waste, and producing spermatids. Actin analysis offered insights into how the dividing sperm “pinched” off of the residual body in order to divide, and tubulin analysis showed how the spindle fibers attached to chromosomes in order to ultimately separate them into spermatids. All preps confirmed that spe-26 mutant C. elegans fail to make spermatids, meaning that these worms are sterile. While I studied many images of spermatogenesis throughout my project, I still need a lot more data in order to make concrete claims. I did not have enough images of control worms to confidently quantify my results, and some abnormalities that I observed in my preps could have been due to errors in the preparation, not due to the worm’s genetics. I learned a lot while completing this project, including how to conduct a variety of immunopreps, how to use the epifluorescent microscope, and how to use photoshop to analyze my images. I am grateful for the opportunity to work in Dr. Shakes’ lab this summer, and I look forward to continuing this project in the fall!