Cloning of Chlamydomonas p60 katanin and localization to the site of outer doublet severing during deflagellation.
Katanin, a heterodimeric microtubule-severing protein that localizes to sites of microtubule organization, can mediate in vitro the ATP-dependent disassembly of both taxol-stabilized microtubules and axonemal doublet microtubules. In the unicellular biflagellate alga Chlamydomonas, katanin has been implicated in deflagellation, a highly specific process that involves a Ca(2+)-signal transduction pathway starting at the plasma membrane and culminating in the severing of axonemal outer doublet microtubules and excision of both flagella from the cell body. Previously, we showed that the microtubule severing activity of deflagellation and katanin's 60 kD catalytic subunit (termed p60) purified with the flagellar basal body complex (FBBC). Additional evidence supporting the involvement of katanin in deflagellation came from the observation that an antibody against human p60 katanin significantly inhibited FBBC-associated microtubule-severing activity. Here we report the cloning of p60 katanin from Chlamydomonas reinhardtii. Immunogold electron microscopy places Chlamydomonas p60 at several locations within the basal body apparatus and associated structures. Importantly, we find a dense accumulation of colloidal gold labeling the distal end of the flagellar transition zone, the site of outer doublet severing during deflagellation. These results suggest that, in addition to a potential involvement in the deflagellation pathway, katanin-mediated microtubule-severing may be associated with multiple processes in Chlamydomonas.
Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia 30322-3030, USA.
T A
TA
Lohret
L
L
Zhao
L M
LM
Quarmby
D000251:Adenosine Triphosphatases / Q000032:analysis / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D016825:Chlamydomonas reinhardtii / Q000235:genetics* / Q000502:physiology / Q000648:ultrastructure; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D005407:Flagella / Q000502:physiology* / Q000648:ultrastructure; D019084:Fluorescent Antibody Technique, Indirect; D000074201:Katanin; D016253:Microscopy, Immunoelectron; D008870:Microtubules / Q000737:chemistry*; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D000251:Adenosine Triphosphatases; D000074201:Katanin