Purification And Characterization Of Outer Arm Dynein From Trypanosoma Brucei

The flagellar motility of kinetoplastids, which is essential for viability, virulence, cell morphology, and cytokinesis, exhibits a bending wave that uniquely propagates from the flagellum's tip to its base, rather than base-to-tip as in all other eukaryotes (including humans), driven by thousands of dynein arms decorated along the axoneme. Much experimental evidence and many biophysical models suggest that the coordinated activity of axonemal dynein motor proteins underlies the properties of flagellar beating, including bending wave propagation direction. We hypothesize that the tip-to-base flagellar bending wave propagation direction in kinetoplastids, including Trypanosoma brucei, is due to unique biophysical behaviors of T. brucei's dynein motors, as an individual or as a team, along the axoneme, particularly in how the motors generate force and how the information about the force transmits via microtubules to the associated dyneins. We purified the T. brucei outer arm dynein (OAD) motors from a flagellar attachment zone protein knocked down strain (FLAM3 RNA interference) to facilitate isolation of flagellum from the cell body using a combination of high salt concentration, ATP buffers, and a His6 affinity tag we added to the C-terminus of OAD light chain 2 (LC2). We characterized the ATPase activity and microtubule gliding motility of the purified motors and are using the epitope tagging of the LC2 with eGFP (enhanced green fluorescence protein) and biotin to conduct further single-molecule biophysical assays. Ultimately, we expect that the characterization of T. brucei axonemal dyneins will provide both mechanistic insights into the unique kinetoplastid dynein-driven tip-to-base flagellar motility and help to establish flagellar proteins as potential candidates for pan-kinetoplastid drug development.