Stephanie Rutter
La Trobe Institute for Molecular Science
Abstract
Introduction:
Apoptosis and the clearance of apoptotic cells are essential to maintain physiological homeostasis. Notably, uncleared apoptotic debris can release pro-inflammatory material and trigger unwanted inflammation. Previous work from our group identified a series of protein regulators that controls the efficient dismantling of apoptotic cells into large EVs known apoptotic bodies, and its role in facilitating cell clearance. Unexpectedly, recent microscopy analysis revealed a previously uncharacterized morphological step, whereby adherent apoptotic cells can retract and leave behind actin-rich membrane remnants, which round into adherent EVs, herein known as the “FOotprint Of Death” (FOOD). The goals of this project are to:
- To characterise the novel apoptotic cell morphology “The FOotprint of Death” (FOOD) that remains at the site of cell death
- To investigate the contents of FOOD-derived ApoEVs
- To investigate the function of FOOD-derived ApoEVs under homeostatic conditions
- To investigate the function of FOOD-derived ApoEVs under pathogenic conditions
Methods:
We established a variety of 3D imaging approaches to visualise FOOD formation including confocal microscopy, SEM, and lattice light-sheet microscopy (LLSM). Moreover, we developed a novel purification approach to isolate FOOD to perform proteomic analysis, and visualise intravesicle contents using TEM. In addition, we used a model of influenza A viral infection to induce apoptosis.
Results:
Confocal microscopy and SEM analysis revealed that a variety of cell types including fibroblasts, endothelial, and epithelial cells generate FOOD during intrinsic apoptosis. High-resolution LLSM revealed distinct morphological stages of FOOD formation including (i) the retraction of the cell where membranous material and F-actin fibres are deposited, (ii) the gradual exposure of phosphatidylserine on membranes, and (iii) the rounding of membranes into vesicle-like structures. Mechanistically, we identified ROCK1, a protein kinase and positive regulator of apoptotic membrane blebbing, as a potential regulator in FOOD formation. Proteomic analysis identified a series of proteins, including adhesion-associated proteins, enriched in FOOD. Finally, we identified the capacity for cells to generate FOOD during influenza A infection, and traffic viral components into FOOD vesicles.
Conclusion:
Collectively, this data has identified a new morphological step that occurs during apoptosis, coined FOOD, that also represents a novel mechanism for the generation of adherent extracellular vesicles. Overall, this work contributes to our understanding of the complex and highly coordinated changes to dying cell morphology.
I started my honors year in 2020, blissfully unaware that the year would turn into many days spent at home reading, rather than exciting days filled with experiments in the lab. Despite this, seeing how quickly the international scientific community collaborated to tackle a global crisis helped me realize that being in the lab and using the tools around me to answer biological questions was where I am meant to be.
My favourite Scientist is Rosalind Franklin, a brilliant X-ray crystallographer who is best known for discovering the structure of DNA. During her career, Rosalind was one of very few female scientists, and while her scientific contributions were overlooked during her lifetime, her pioneering work lead the way for many female scientists who followed in her footsteps. She is a great role model for all women and girls in STEM.
In my spare time I love to play beach volleyball, and to be out in nature hiking, exploring rock pools, or swimming in the ocean. This passion for the environment drew me to think about ways we can be more sustainable in research, so with a few colleagues we set up a Green Labs Group at La Trobe University, where we collaborate with other researchers to organise events and initiatives to promote sustainable laboratory practices!
Awards
1st Place
The Grand Prize recipient from each participating country wins a trip to Promega headquarters in Madison, WI USA to meet our R&D team, present their project and network with fellow award recipients.
Public votes are cast between 15 October - 15 November 2024 for each nominee. The candidate with the most votes wins.
Runner Up Awards
Four remaining Finalists will each receive a limited edition Promega Lab Set with LEGO® Elements and a Duffel Bag
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