Cell Communication in Filamentous Cyanobacteria
Conrad Mullineaux
Professor of Microbiology
School of Biological and Chemical Sciences
Queen Mary, University of London
Mile End Road,
London E1 4NS
UK
This project involves collaborations with the Instituto de Bioquimica Vegetal y Fotosintesis, Sevilla, Spain (Enrique Flores, Antonia Herrero, Vicente Mariscal) and David G. Adams (University of Leeds). We are interested in multicellularity in cyanobacteria. Filamentous cyanobacteria are true multicellular organisms, in which cells in a filament communicate and co-operate. Anabaena and related species form specialised nitrogen-fixing cells called heterocysts. These cells must exchange metabolites (sugars and amino acids) with the neighbouring vegetative cells. But how do molecules move from cell to cell in the filament? We can look at this by loading the cytoplasm with fluorescent molecules, then using confocal fluorescence microscopy and Fluorescence Recovery after Photobleaching to observe the movement of the molecule from cell to cell.
Confocal fluorescence micrograph showing a filament of Anabaena cylindrica (PCC7122) loaded with calcein
Green fluorescence from calcein in the cytoplasm, red fluorescence from chlorophyll in the thylakoid membranes. The enlarged cell with low chlorophyll fluorescence is a heterocyst.
Calcein-AM can be obtained from Molecular Probes. It is hydrophobic enough to traverse the plasma membrane and enter the cytoplasm, where it is hydrolysed by esterases to produce a hydrophilic green fluorescent molecule of 623 Da. FRAP measurements can then be used to observe and quantify the movement of calcein from cell to cell.
The video clips below show FRAP measurements of calcein movement in filaments of Anabaena cylindrica. All movies are shown speeded up 6 X. Frames are 70 microns across. The first frames show two-colour images of the filaments (showing chlorophyll fluorescence as well as calcein fluorescence). Then we show calcein fluorescence before and after bleaching fluorescence in a single cell in the centre of the frame. The subsequent images show fluorescence redistribution as calcein flows from cell to cell. Where molecular exchange is rapid, the bleach already spreads through several cells before the first post-bleach image is recorded.
A. Calcein exchange among vegetative cells (cells grown in medium containing nitrate).
B. Calcein exchange among vegetative cells (cells grown without nitrate).
C. Calcein exchange between a heterocyst and vegetative cells
Molecular exchange can be quantified in terms of an "Exchange Coefficient" E, which relates the concentrations of the molecule in two neighbouring cells (C1 and C2) to the net flux from cell to cell.
Net flux from Cell 1 to Cell 2 = E (C1 - C2). E is a measure of the permeability of the cell junction to the molecule. Our measurements show that when filaments differentiate to form heterocysts, E increases among vegetative cells. However, exchange between vegetative cells and heterocysts is slower. This is probably a price paid to reduce the influx of oxygen into the heterocyst cytoplasm (oxygen poisons the nitrogenase and thus prevents nitrogen fixation).
Our studies indicate that there are channels that allow small molecules to diffuse from cytoplasm to cytoplasm. We are using specific Anabaena mutants to investigate how these channels are formed and regulated. We have identified a protein known as SepJ or FraG (the product of the open-reading frame alr2338 in Anabaena 7120) as a strong candidate for the protein that forms the intercellular channels.
A key publication on this topic:
Mullineaux, C.W., Mariscal, V., Nenninger, A., Khanum, H., Herrero, A., Flores, E. and Adams, D.G. (2008) Mechanism of intercellular molecular exchange in heterocyst-forming cyanobacteria. EMBO Journal 27, 1299-1308.
