Estimating Z-ring radius and contraction in dividing Escherichia coli.
We present a fluorescence recovery after photobleaching-based method for monitoring the progression of septal Z-ring contraction in dividing Escherichia coli cells. In a large number of cells undergoing division, we irreversibly bleached cytosolically expressed Enhanced Green Fluorescent Protein on one side of the septal invagination and followed the fluorescence relaxation on both sides of the septum. Since the relaxation time depends on the cross-sectional area of the septum, it can be used to determine the septal radius r. Assuming that the fraction of the observed cells with r-values in a given interval reflects the duration of that interval in the division process we could derive an approximate time-course for the contraction event, as a population average. By applying the method repeatedly on individual cells, the contraction process was also followed in real time. On a population average level, our data are best described by a linear contraction process in time. However, on the single cell level the contraction processes display a complex behaviour, with varying levels of activity. The proposed approach provides a simple yet versatile method for studying Z-ring contraction in vivo, and will help to elucidate its underlying mechanisms.
Department of Applied Physics, Royal Institute of Technology, Stockholm, Sweden.
Johan
J
Strömqvist
Karl
K
Skoog
Daniel O
DO
Daley
Jerker
J
Widengren
Gunnar
G
von Heijne
D001426:Bacterial Proteins / Q000378:metabolism*; D001431:Bacteriological Techniques / Q000379:methods; D002455:Cell Division; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism / Q000502:physiology*; D005453:Fluorescence; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D046911:Macromolecular Substances / Q000378:metabolism*; D038761:Photobleaching; D055503:Protein Multimerization; D013997:Time Factors
D001426:Bacterial Proteins; D003598:Cytoskeletal Proteins; C071525:FtsZ protein, Bacteria; D046911:Macromolecular Substances; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins