Current Research
Overview: Disruption of mitochondrial dynamics can have significant detrimental effects to cells. Different organisms handle this disruption differently. For many multi-cellular organisms loss of fission causes significant developmental delays. In humans fission defects seem to result in severe developmental disabilities and death a few days after birth. Defects in fusion can result in childhood blindness or Charcot-Marie Tooth, a disease that manifests as muscle atrophy in the hands and feet. Defects in both processes alter the regulation of apoptosis, a process critical for maintaining healthy cells. It appears that alterations of mitochondrial dynamics seems to effect neurons the most, support for the latest research that suggests that fission and fusion may be involved in the progression of Alzheimer’s and Parkinson’s disease.
Project 1: Mitochondrial dysfunction plays a role in the progression of Parkinson’s Disease (PD), thus understanding mitochondrial dysfunction is one of the important keys to finding PD treatment. The study of the relationship between the cytoskeleton and fission and fusion in our model, Dictyostelium
discoideum suggests that insufficient fission can cause a tangle of interconnected mitochondria and insufficient fusion can cause mitochondrial aggregates that leads to a decrease in mitochondrial motility and potentially damaged organelles. To continue to understand the relationship between mitochondrial dynamics and PD, we are determining the rates of fission, fusion, and motility when overexpressing and under-expressing DJ-1 in D. discoideum. DJ-1 is a protein linked to PD and mitochondria, yet its function is poorly understood. Our results will help clarify its function and the relationship between DJ-1, dynamics, and mitochondrial dysfunction.
Project 2: Dictyostelium discoideum is a well-established mitochondrial model system for both disease and dynamics, yet we still do not understand the actual mechanisms of mitochondrial dynamics in this system. The FtsZ proteins, including FszA, found in D. discoideum are hypothesized to operate similarly to dynamin-related GTPases in humans, the driving force behind dynamics . The loss of these dynamics ultimately causes the mitochondria to malfunction, which can cause or contribute to neurodegenerative diseases such as Parkinson’s disease, and Alzheimer’s disease. Understanding the role FtsZ plays in D. discoideum mitochondrial dynamics will allow us to gain insight into the evolutionary shift from FtsZs to DRPs. To better understand the role of these proteins in mitochondrial dynamics we will determine the effect of overexpressing FszA-GFP and knocking-down FszA to determine whether the protein inhibits mitochondrial dynamics in D. discoideum by quantifying fission, fusion and motility rates. We will also analyze the effect on reactive oxygen species (ROS). Mitochondria are the largest producers of ROS, but they also have the most mechanisms to regulate ROS levels, we will determine if alteration of FszA and mitochondrial dynamics directly effects ROS.
Project 3: Mitochondrial dynamics, including motility, fission, and fusion are necessary for mitochondria to maintain proper morphology and function. It is known that mitochondrial dynamics are dependent on the cytoskeleton in mammals and yeast, but not much is known about the cytoskeleton’s role in dynamics in D. dicoideum. To determine this role we need to analyze the interactions between the cytoskeleton and the mitochondria during mitochondrial dynamics. We developed a live-cell cytoskeletal imaging assay that allowed us to visualize the mitochondria and the cytoskeleton simultaneously. The quantity and speed of moving mitochondria will be compared to its proximity to the cytoskeleton. In addition rates of fission and fusion will be compared to the distance the event occurred from the cytoskeleton.
Wavreille, 2006
Project 4: FszA and FszB are important to mitochondrial dynamics, but we do not understand their function. Our goal is to identify the protein partners of FszA and FszB by pulldown assays to gain a better understanding of the roles these proteins play in mitochondrial dynamics and in disease. Strains expressing FLAG-FszA and FLAG-FszB were created. The FLAG-FszA construct, mRNA, and protein were confirmed as present in our cells, though the FLAG-FszB construct, mRNA, nor protein could be clearly identified. Immunoprecipitation of FLAG tagged FtsZs using a FLAG antibody was used to pulldown FszA or FszB (depending on the strain) along with the binding proteins of each. The proteins pulled down by the FLAG antibodies were identified by UAMS Proteomics via mass spectrometry. The identified proteins were then sorted based on the biological processes they are involved in including translation, transcription, other cellular processes, proteins that may be involved in regulating the processes of fission and fusion, and possible mitochondrial dynamic machinery. Additional analysis of these proteins is now needed. We plan to upregulate and downregulate each identified protein and quantify the effect on mitochondrial dynamics. In addition we plan to isolate mitochondria and repeat the pull down to determine if we can identify any additional proteins of interest.