Semi-automated Tracking of Chlamydomonas reinhardtii for Modeling Tropic Responses A. M. Folcik, S. Nassani, T. Haire, N. Patrawalla, N. Nezamoddini-Kachouie Faculty Advisor: Dr. Andrew Palmer, Dept. of Biological Sciences, Florida Institute of Technology BACKGROUND RESULTS Wild type Motility plays an important role in the colonization of eukaryotic hosts by unicellular pathogens such as Trypanosoma brucei, or by mutualists, such as the paraphyletic group of bacteria collectively referred to as Rhizobia. Considerable progress has been made in identifying how these organisms integrate various inputs and molecular pathways to coordinate movement. However, due to their relatively small size and rapid movement, tracking and modeling these systems have proven challenging, permitting only the most cursory of observations (i.e. motile vs non- motile, or ‘slow’). We have developed a standardized tracking procedure to evaluate motility with the model unicellular algae Chlamydomonas reinhardtii. Using this software, phototaxis, chemotaxis, and mutant motility strains were tested to determine both accuracy of the procedure as well as to collect data on the sensitivity of C. reinhardtii to various stimuli. This work provides new tools for evaluating and modeling motility in C. reinhardtii while establishing the methodology for conducting similar experiments on other unicellular microorganisms below the range of traditional cell motility tracking software. Using these methods, strategies aimed at regulating motility in these unicellular organisms may have considerable benefit to human health and agricultural yields, as well as other areas of research. A B C D https://web.mst.edu Figure 1. A. Image slice from cc124 72 hour culture. B. Rose-plot histogram of directionality of cc124 under no-light conditions showing uniform distribution of movement in all directions. C. Movement plot of tracks originating at set coordinate. D. Average velocity in μm/s of triplicate cc124 72 hour flasks. Error bars display standard error between the velocities from each track in analyzed video. Phototaxis Mutant strains A B A C B C D METHODS C. reinhardtii (cc124 or mutant strain) was suspended in 25 ml TAP media in triplicate. Figure 2. A. Average velocity in μm/s of triplicate cc124 72 hour cultures at varying light intensities (lux). B. Rose-plot histogram at 18840 lux. C. Rose-plot histogram at 6780 lux. D. Rose-plot histogram at 1119 lux. Figure 3. A. Analysis images showing tracks and velocities for cc1036, cc3663, and cc124 strains, respectively. B. Average velocity in μm/s of triplicate cc1036, cc3663, and cc124 strains. C. Rose-plot histogram of cc1036, cc3663, and cc124 strains. SUMMARY & CONCLUSIONS Cultures grown under 16:8 light cycle for 24, 48, or 72 hours respectively. We can now determine the velocity, directionality, and size of rapidly moving single celled organisms in response to a variety of stimuli or in response to mutations. Using this technique we were able to determine that short term light exposures did not induce phototaxis in C. reinhardtii suggesting longer exposures are required to integrate this information. Future experiments will allow us to determine the minimum exposure time required for the induction of phototaxis. We were also able to use this approach to map the velocities of known motility mutants for the first time. For example, the cc1036 and cc3663 strains were determined to have approximately zero or 50% motility, respectively, in comparison to cc124 average velocities. The ability to determine velocity and directionality changes, will ultimately help us model mutant lines or novel drug-like compounds for their ability to impact motility. Chemotaxis & Migration Tool ToupView used as video collecting software InfranView to convert AVI video file to JPEG image format slices VirtualDub used as converter for JPEG to TIF file formats Fiji with trackmate plugin for spot and track analysis R software used for assembly of Fiji data files for modelling Used for roseplot (histogram) graphing for directionality REFERENCES Saxton, M. J., & Jacobson, K. (1997). Single-particle tracking: applications to membrane dynamics. Annual review of biophysics and biomolecular structure, 26(1), 373-399. Silflow, C. D., & Lefebvre, P. A. (2001). Assembly and motility of eukaryotic cilia and flagella. Lessons from Chlamydomonas reinhardtii. Plant Physiology, 127(4), 1500-1507.