Isolation and Phenotypic Characterization of Chlamydomonas Mutants Defective in Cytokinesis.

Abstract

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...

ChlamydomonasDIP13 and human NA14: a new class of proteins associated with microtubule structures is involved in cell division

“…Vfl2 and vfl3 mutants, defective in centrin or the distal connecting fiber, respectively, also show random segregation of basal bodies (Wright et al, 1983) and thus have multiple flagella but, in contrast to DIP13 RNAi transformants, are mononucleate and of normal size. At present, DIP13 RNAi transformants appear to be similar to oca1 and oca2 cytokinesis mutants described previously (Hirono and Yoda, 1997). Cultures of these mutants contain large abnormallyshaped cells with multiple flagella; each pair of flagella is connected to one nucleus.…”

The Chlamydomonas cell cycle

“…The specific relationship between DIP13/NA14 and centrosomal function remains to be determined. Three mutants isolated in screens to identify cytokinesis defects have been described in Chlamydomonas, cyt1, oca1 and oca2 (Warr, 1968;Hirono and Yoda, 1997). cyt1 cells are multinucleate and multiflagellate, much like vfl2 and vfl3 (Warr, 1968).…”

“…oca1 and oca2 were characterized based on the morphology of division and nuclear number. At moderate frequency they form large, multinucleate and multiflagellate cells, and often show slow or arrested cleavage‐furrow formation (Hirono and Yoda, ). The oca1 and oca2 mutations have not been further characterized or cloned.…”

The Vfl1 Protein in Chlamydomonas Localizes in a Rotationally Asymmetric Pattern at the Distal Ends of the Basal Bodies

“…Cells were examined by phase-contrast microscopy to determine flagellar number. To determine nuclear number, cells were fixed and stained with DAPI as described by Hirono and Yoda (1997). Cells were examined by epifluorescence microscopy using a UV filter.…”