Live Cell Fluorescence Imaging Of Cyanobacteria Photosynthetic Membrane Proteins Dynamic Changes And Physiological Function

This work consists of three parts. Firstly, two types of live cell fluorescentimaging techniques -fluorescence recovery after photobleaching (FRAP) andfluorescence resonance energy transfer (FRET) were developed in livingcyanobacteria cells using laser scanning confocal microscope. Secondly, FRAP wasused to detect the diffusion of phycobilisomes on thylakoid membranes and thedriving force (or the regulatory factors) of this diffusion. Thirdly, FRET was used todetect the interaction of two subunits of NAD(P)H dehydrogenase complex incyanobacteria.The arriving of the post-genome era requires the studying of the structures,characteristics and interactions of biological macromolecules especially proteinsturning from in vitro to in vivo. This study used two model cyanobacteria-Synechococcus sp. PCC7942 and Synechocystis. PCC6803 to develop FRAP andFRET techniques in living cyanobacteria cells, which can be used to study thedynamics of photosynthetic membrane proteins.Studies using FRAP in living cyanobacteria cells have shown thatphycobilisomes diffuse rapidly on thylakoid membranes, and this diffusion is requiredfor state transitions in cyanobacteria. However, the driving force and the regulatoryfactors of phycobilisome diffusion are yet unknown. FRAP is a technique widely usedin cell biology to observe the dynamics of biological systems, including the diffusionof membrane components. This technique involves the use of a highly-focusedconfocal laser spot to selectively bleach the fluorophores in a small region of the cell.The diffusion of the fluorophores can then be monitored by observing the spread andrecovery of the bleach. In this study, we use FRAP and 77K fluorescence spectra toinvestigate the effects of 1, 4-benzoquinone (BQ), one of the benzoquinone analogues,on state transitions and phycobilisomes mobility in Synechococcus sp. PCC7942. Theresults show that BQ can induce a transition from state 2 to state 1 in the absence ofactinic light and cells treated with BQ were locked in state 1. At the same time, BQaffects the diffusion of phycobilisomes drastically. Most phycobilisomes areimmobilized on thyalkoid membranes. It is suggested that the redox state of PQ poolcan regulate the phycobilisome diffusion in cyanobacteria.NAD(P)H dehydrogenase complexes(NDH) is another photosynthetic membranecomplex found in thylakoid membranes in cyanobacteria and green plants in recentyears. It has specific functions in respiration and cyclic electron flow as well as inactive CO2 uptake. Attempts to isolate intact NDH complexes from cyanobacteria orplant chloroplasts have met severe difficulties due to the fragility of the enzyme andthe low quantity present in the thylakoid membrane. Also, the intact subunitscomposition is yet unknown. In this work, two variants of green fluorescentprotein(GFP)-yellow fluorescent protein(YFP) and cyan fluorescent protein(CFP)were used to label D4 and CupB subunits of NDH complexes respectively. ThenFRET was used to detect the interactions of these two subunits and theconformational changes of NDH complexes in different physiological conditions.Three different methods including sensitized emission, acceptor photobleaching anddonor photobleaching dynamics were used to detect FRET signals. However, theresults showed that many factors can interfere FRET studies in cyanobacteria. Theautofluorescence of the cell is high and the expression level of the fluorescent proteinsis low relative to the cell autofluorecence, which makes the ratio of signal to noise toolow to be detected. In this study, we also found a photo-activation-like phenomenon inthe range of 465-565 nm using 405 nm laser for XYT scanning. This may be relatedto the activity of carotenoids, which can protect the photosystems in the high lightmenace.