First person – Danielle Buglak and Kathleen Holmes Free

Danielle Buglak

How would you explain the main findings of your paper in lay terms?

K.H.: The tailed sperm cell we're all familiar with generally has three parts: the head, the tail and the neck. The head holds the genetic information, the tail propels the genetic information to the egg and the neck stably links the head and the tail. When the sperm's neck fails to properly connect the head to the tail, the tail can separate from the head, which leads to infertility. Using the sperm of the fruit fly Drosophila as a model system, Danielle and I describe how the architecture of the neck is remodeled during sperm development. Our work reveals that the sperm's neck develops multiple points of attachment to the head. We then show that these multiple points of attachment are critical for the neck's stability and, by extension, fertility.

D.B.: Most sperm cells comprise a head containing the genetic material and a tail that propels the sperm cell during swimming. Connecting the head and tail is the sperm cell neck; failures at the sperm neck cause the head and tail to separate and lead to male infertility. In this study, we carefully map several key components of the sperm neck over time in the fruit fly Drosophila melanogaster. We show that the sperm neck is heavily remodeled during development and that the head and tail are connected via multiple attachment sites in the sperm neck. We also highlight a novel role for a structure called the proximal centriole-like (PCL) in facilitating this attachment.

Were there any specific challenges associated with this project? If so, how did you overcome them?

K.H.: I think the biggest challenge I ran into with this project was my own inexperience. This started as an exploratory project and I was a postbaccalaureate fellow, straight out of college with very little lab experience. I made countless mistakes over the course of this project and learned so much. The biggest challenge I remember having was not really understanding the significance of my work. It took me a long time to start seeing my data as something that could end up in a paper.

D.B.: Although I came into the Rusan lab with a lot of experience in nucleus–centriole linkage, I had worked only in tissue culture, mice and zebrafish. This project was my first time working with Drosophila, so I had a lot to catch up on! My co-authors, Katie and Brian, were instrumental in teaching me everything I needed to know about flies and sperm development.

When doing the research, did you have a particular result or ‘eureka’ moment that has stuck with you?

K.H.: I think the moment that stuck with me the most was doing SIM of the PCL at different stages of spermatid development. I remember sitting at the microscope collecting images and realizing that the PCL was getting further away from the nucleus throughout development. I couldn't believe what I was seeing. In hindsight, that experiment took my project from exploratory towards hypothesis-driven and it was the first time I could see my work providing any significant mechanistic insight into the stability of the head–tail connection apparatus (HTCA).

D.B.: A lot of gene mutations that lead to male infertility have very obvious defects during sperm development. The poc1 mutants we used in this study were different. We knew that these flies were infertile and that something was going wrong during development, but morphologically the spermatids looked very normal for most of development. It wasn't until we looked closely at a specific marker for the sperm neck, Spag4, that it became clear that the sperm neck was unstable in later stages of sperm development. From that point on, everything clicked into place and we knew exactly what experiments we needed to finish the story.

Why did you choose Journal of Cell Science for your paper?

K.H.: We were looking to publish our work in a journal that would reach a broad audience of cell biologists and Journal of Cell Science seemed like a good fit!

D.B.: JCS has a fantastic history of publishing high quality papers in cell biology.

Kathleen Holmes

Have you had any significant mentors who have helped you beyond supervision in the lab? How was their guidance special?

K.H.: Danielle, my co-author, was an amazing mentor over the course of this project. She joined the lab about halfway through the second year of my fellowship and she was instrumental in helping me understand this project and getting it to where it is now. I could not have done this without her. I miss talking to her about our unanswered HTCA questions and figuring out how we would test them. I ended up following in Danielle's footsteps and now I am a graduate student in the same program where she completed her PhD in at UNC Chapel Hill.

D.B.: My supervisors Dr Anne Carlson, Dr Vicki Bautch and Dr Nasser Rusan have all been instrumental in teaching me how to think and communicate like a scientist. My undergraduate academic advisor Dr Candy DeBerry was the person who really got me excited about careers in science and encouraged me to apply to graduate school. I have also had the privilege to work with numerous undergraduate and postbaccalaureate students throughout my career including my co-author Katie, and they have all brought to the lab an infectious energy and excitement that continuously reinvigorates my passion for science. I'm certain I have learned more from them than they have from me!

What motivated you to pursue a career in science, and what have been the most interesting moments on the path that led you to where you are now?

K.H.: This project was my most significant motivation towards pursuing a career in science. When I joined the Rusan lab, I thought I wanted to go to medical school. I was quickly hooked by the constant challenge of doing research and decided to do a PhD instead. I'm not sure that any one moment sticks out as particularly interesting, but as a young scientist I have never found research boring. Day-to-day lab work becomes full of routine tasks, but when you're truly passionate about your project there is never a shortage of interesting questions to ponder while you flip flies or mix buffers.

D.B.: Science was always my favorite subject in school, but I didn't know what kinds of career paths were available to me until college. During my freshman year, my advisor Dr Candy DeBerry showed me for the first time living HeLa cells under a microscope, which sparked my interest in cell biology. My lab internship with Dr Anne Carlson at the University of Pittsburgh solidified my desire to pursue a PhD.

Who are your role models in science? Why?

K.H.: I don't like emulating extraordinary people like Nobel Prize winners or famous scientists, because becoming one of them isn't my goal as a young woman in science. My science role models are women like my current PI, Katherine Baldwin, and the many women who came before her, who have managed to be both brilliant scientists and remarkable mothers. At this stage of my career, it's a lot of pressure to try to be extraordinary, I am more focused on trying to be great at my job without sacrificing too many things that bring my life joy and meaning.

D.B.: All of the mentors I have had throughout my career have been incredible role models. I also have a great admiration for the countless disabled scientists, like Dr Bonnielin Swenor, who have worked tirelessly to increase accessibility in science.

What's next for you?

K.H.: I just finished the first year of my PhD so my journey has just begun. My next step is to work hard on a new project which hopefully will lead me to defend my thesis in about 4 years.

D.B.: I'm excited to start working on my next project in the Rusan lab studying how nuclear envelope proteins function to link the sperm head and tail.

Tell us something interesting about yourself that wouldn't be on your CV

K.H.: I have a couple of hobbies apart from being a graduate student. I love to cook and bake, so I'm always bringing baked goods to share with the lab. I also love listening to podcasts about everything from random corners of the science world to economics and history. As a result, I accumulate a lot of random knowledge which helps me approach the puzzles I'm presented with in the lab from unique angles. It also makes me pretty good at trivia.

D.B.: I'm a retired adrenaline junkie that now prefers quieter activities like crochet and knitting. Most of my free time is spent making blankets and sweaters. I'm also a huge Nancy Drew nerd and love RPG games.