In vivo localization of centrin in the green alga Chlamydomonas reinhardtii.
The green alga Chlamydomonas reinhardtii has been used as a model system to study flagellar assembly, centriole assembly, and cell cycle events. These processes are dynamic. Therefore, protein targeting and protein-protein interactions should be evaluated in vivo. To be able to study dynamic processes in C. reinhardtii in vivo, we have explored the use of the green fluorescent protein (GFP). A construct containing a fusion of centrin and GFP was incorporated into the genome as a single copy. The selected clone shows expression in 25-50% of the cells. Centrin-GFP was targeted in vivo to the nuclear basal body connectors and the distal connecting fibers. At the electron microscopic level, it was also localized to the flagellar transitional regions. EM data of transformants indicate that there are some abnormalities in the centrin-containing structures. The transitional region consists of only the transverse septum or has lesions in the H-piece. The distal connecting fibers are thinner and their characteristic crossbands seem to be incomplete. Deflagellation is not affected since more than 95% of the cells deflagellate. Also basal body segregation is not affected since cells with an abnormal flagellar number were not detected. Functional studies of the centrin-GFP fusion show the characteristic calcium-induced mobility shift in SDS-PAGE. Immunofluorescence revealed that during cell division, centrin-GFP remains associated with the basal bodies. In vivo localization of the fusion protein during cell division shows that in metaphase centrin-GFP appears as two opposing spots located close to the spindle poles. The distance between the spots increases as the cells progress through anaphase and then decreases during telophase. GFP is a useful tool to study dynamic processes in the cytoskeleton of C. reinhardtii.
Ruiz-Binder
Nayma E
NE
Universitaet zu Koeln, Botanisches Institut I, Koeln, Germany. nayma.ruiz@uni-koeln.de
Geimer
Stefan
S
Melkonian
Michael
M
D000818:Animals; D015139:Blotting, Southern; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002453:Cell Cycle; D016825:Chlamydomonas reinhardtii / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005407:Flagella / Q000378:metabolism / Q000502:physiology; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D007150:Immunohistochemistry; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D002135:Calcium-Binding Proteins; D002868:Chromosomal Proteins, Non-Histone; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C056608:caltractin; D049452:Green Fluorescent Proteins; D004247:DNA