From New Method to Intraflagellar Transport and Photosynthesis, a Single Molecule Investigation

Intraflagellar transport mediates ciliary and flagellar function by taking protein complexes from the cell body to the end of the cilia or flagellum. Many of the mechanisms and processes that control the reorganization and return of the various protein complexes are not well understood. In particular the process of kinesin recycling to the basal body, the location of IFT cargo reorganization, and the role of dynein in regulating turnaround time are all unresolved phenomena. Through the use of single molecule localization techniques and computer simulations, these issues were addressed. We present experimental evidence of (i) kinesin leaving the flagellar tip via free diffusion, (ii) the BBSome, a known IFT cargo, remaining tethered to the flagellar membrane during reorganization, and (iii) dynein also remaining with the IFT complex and cargo, tethered to the membrane. Furthermore, the same principles and techniques used to study IFT are applied to study energy capture and transfer of the phycobilisome, a photosynthetic antenna complex found in cyanobacteria. We determine that many intact PBS have two quantum bleaching units. By determining the separation between the centers of fluorescence for these two bleaching units, we localize the relative site of terminal emitters in the core of the PBS. The results indicate that the sites for terminal emission get further apart with increased excitation intensity, which may be a possible mechanism for energy dissipation.