Of the uncloned ODA genes required for outer dynein arm assembly in Chlamydomonas, ODA5 and ODA10 are of particular interest because they do not encode known subunits of the outer arm or the outer dynein arm-docking complex (ODA-DC), and because genetic studies suggest their products interact. Beginning with a tagged oda5 allele, we isolated genomic and cDNA clones of the wild-type gene. ODA5 predicts a novel, 66-kDa coiled-coil protein. Immunoblotting indicates Oda5p is an axonemal component that assembles onto the axoneme independently of the outer arm and ODA-DC and is uniquely missing in oda5 and oda10 axonemes. Oda5p is released from the axoneme by extraction with 0.6 M KCl, but the soluble Oda5p does not cosediment with the outer dynein arm/ODA-DC in sucrose gradients. Quantitative mass spectrometry by using isotope coded affinity tagging revealed that a previously unidentified adenylate kinase is reduced 35-50% in oda5 flagella. Direct enzymatic assays demonstrated a comparable reduction in adenylate kinase activity in oda5 flagella, and also in oda10 flagella, but not in flagella of other oda mutants. We propose that Oda5p is part of a novel axonemal complex that is required for outer arm assembly and anchors adenylate kinase in proximity to the arm.
ABSTRACT. Two genetically independent Chlamydomonas mutants, ocal and oca2, that display abnormal cell division were isolated by DNAinsertional mutagenesis. The culture of these mutants contained large abnormally-shaped cells with multiple pairs of flagella. DAPIstaining showed that those aberrant cells carried the same number of nuclei as that of flagella pairs. Time-lapse video microscopy revealed the following characteristics of the cell division process in the mutants: i) although the mutants, like wild-type cells, had a potential to divide into eight daughter cells by successive three rounds of division cycle, they frequently failed to cleave and formed a fused cell in the course of division; ii) a cell often grew into an extremely large cell with many pairs of flagella; iii) the large cell was suddenly divided into a number of daughter cells by simultaneously forming multiple cleavage furrows; iv) alternatively, an extremely large cell stopped dividing although many cleavage furrows were formed on its surface. These observations suggest that these mutants are partially deficient in the progression of furrowing, and that Chlamydomonas is capable of undergoing cytokinesis between many pairs of nuclei simultaneously, as in the cellularization process of insect eggs.Cytokinesis in animal cells takes place through the constriction of a contractile ring situated at the cleavage furrow. It is usually tightly coupled with mitosis in that the progression of mitosis is prerequisite to the contractile ring formation. However, how the position and timing of the cleavage furrow formation are controlled is largely unknown, although many protein factors involved in cytokinesis have been identified, including actin and myosin as the main componentsof the contractilering (1,2, 5, 7, ll, 13, 14, 15).Studies using mutants would be important for the elucidation of this problem, which must involve interactions between a number of different proteins. Wehave thus started isolating cytokinesis mutants from the biflagellate alga Chlamydomonas reinhardtii, of which the cell division machinery has been considered to be similar to that of animal cells. Its nucleus divides by the process of mitosis in which the basal bodies function as centrosomes (4), and its cell body divides by means of a cleavage furrow where actin is localized (3, 8).Here we report the isolation and initial characterization of two Chlamydomonasmutants displaying abnormal cell division, produced by insertion of plasmid DNA into the nuclear genome.These mutants appear to be partially deficient in progression of cytokinesis and, unexpectedly, have an ability to undergo a novel mode of cell division in which a single multi-nuclear cell is simultaneously divided into multiple daughter cells. MATERIALS AND METHODS Strains.The Chlamydomonas reinhardtii 137c (wild type), nitl/cwl5, and the newly isolated mutants, ocal and oca2, were used. These mutants were isolated from cells transformed by the method of Kindle (10) using glass beads. Cells were treated with autolysin...
No abstract
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.