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Title: Organization, dynamics and adaptation of the photosynthetic machinery in cyanobacteria
Author: Casella, Selene
ISNI:       0000 0004 7428 7163
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2018
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Cyanobacteria are the oldest oxygenic phototrophs on Earth. In the cyanobacterial cell, oxygenic photosynthesis typically takes place in the specialised intracellular membranes, namely thylakoid membranes (TMs), analogous to higher plants. The cyanobacterial photosynthetic machinery embedded in thylakoid lipid bilayers typically consists of a series of membrane integral multi-subunit complexes, including photosystem I (PSI), photosystem II (PSII), cytochrome (Cyt) b6f and ATP synthase (ATPase) complexes. Cyanobacterial thylakoid membranes also act as the site that harbours the components of respiratory electron transport chains, comprising type-I NAD(P)H dehydrogenase-like complex (NDH-1), succinate dehydrogenase (SDH), Cyt oxidase and alternative oxidase. Remarkable macromolecular crowding and close protein-protein contacts within the cyanobacterial thylakoid membrane result in the dense packing of photosynthetic components. Nowadays, information about the structures and functions of individual supercomplexes, in both photosynthetic and respiratory chains, are available. However, their spatial organization and dynamics have not been well understood experimentally. Here, we study the native organisation and mobility of PSI, PSII, ATPase and Cyt b6f complexes in the TMs from the cyanobacterium Synechococcus elongatus PCC 7942. We also describe the interferences on these dynamics from external (red light, temperature) or internal parameters (supramolecular organization, protein size) in vivo. We first, determined the native arrangement and dense packing of PSI, PSII and Cyt b6f within the thylakoid membrane using atomic force microscopy. We calculated that the 75% of the TMs is composed of proteins, in which PSI and PSII complexes appear interspersed. Then, we fused fluorescent proteins to the complexes of interest, maintaining the expression of genes under the control of their endogenous promoters. Genetic, biochemical, structural and functional characterisation of the mutants were carried out, confirming the functional tagging. Live-cell fluorescence imaging using total internal reflection fluorescence and confocal microscopy were performed to study the global distribution of the supercomplexes. We revealed that, in normal growth conditions, PSI, ATPase and Cyt b6f have a higher heterogeneous distribution compared with PSII along the TMs. From the fluorescence pictures, we extracted the relative quantification of the photosynthetic complexes. We visualised for the first time the reorganization in defined patches of ATPase and Cyt b6f complexes upon red light illumination. We also characterised in detail the distinct rearrangement of PSI and PSII complexes. Using fluorescence recovery after photobleaching (FRAP), we monitored the movement of the complexes within the crowded thylakoid membrane environment. This work revealed that in normal growth conditions, the photosynthetic complexes are mobile within a time frame of 90 s. It also showed that the velocity of diffusion of PSI, PSII and Cyt b6f can be reduced by red light. On the other hand, we observed that in red light adapted cells, the PSII complex arranges in discrete patches which are highly mobile within a time frame of 13 min. Lastly, we provide a quantitative description of the interference of temperature, supramolecular organisation and protein size on the in vivo diffusion dynamics of photosynthetic complexes. Our findings provide evidence about the clustering distribution and mobility of the photosynthetic complexes, and indicate the differential responses of photosynthetic complexes to the same environmental stimulus. Knowledge of the cyanobacterial thylakoid membranes could be extended to other cell membranes, such as chloroplasts, given the close evolutionary links between chloroplasts and cyanobacteria, as well as mitochondrial membranes. Understanding the organisation and dynamics of photosynthetic membranes is essential for rational design and construction of artificial photosynthetic systems to underpin bioenergy development.
Supervisor: Liu, Luning Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral