Defective temporal and spatial control of flagellar assembly in a mutant of Chlamydomonas reinhardtii with variable flagellar number.

Abstract

Wild-type Chlamydomonas reinhardtii carry two flagella per cell that are used for both motility and mating. We describe a mutant, vfl-1, in which the biflagellate state is disrupted such that the number of flagella per cell ranges from 0 to as many as 10. vfl-1 cells possess the novel ability to assemble new flagella throughout the G1 portion of the cell cycle, resulting in an average increase of about 0.05 flagella per cell per hour. Such uncoupling of the flagellar assembly cycle from the cell cycle is not observed in other mutants with abnormal flagellar number. Rather than being located in an exclusively apical position characteristic of the wild type, vfl-I flagella can be at virtually any location on the cell surface, vfl-1 cells display abnormally wide variations in cell size, probably owing to extremely unequal cell divisions. Various ultrastructural abnormalities in the flagellar apparatus are also present, including missing or defective striated fibers and reduced numbers of rootlet microtubules. The pleiotropic defects observed in vfl-1 result from a recessive Mendelian mutation mapped to Chromosome VIII.The basal body developmental cycle of the unicellular biflagellate green alga, Chlamydomonas reinhardtii, represents a clear case in which organelle replication is coupled to cell replication. In the basal body cycle, new basal bodies form at determined times and places during interphase, and they segregate equally at cell division to yield biflagellate daughter cells (2,8). Through the analysis of mutants with defects in the basal body cycle, we hope to gain insight into the mechanisms of basal body biogenesis and segregation, and also into the mechanisms by which cell and organelle cycles are coordinated. In this communication we describe a mutant, vfl-1, in which the temporal and spatial regulation of the basal body cycle is defective. As a result, vfl-1 cells have the novel property of assembling new flagella continuously throughout much of the cell cycle.

The ultrastructure of theChlamydomonas reinhardtiibasal apparatus: identification of an early marker of radial asymmetry inherent in the basal body

“…Mispositioned or absent s-and d-fibers would probably result in a mispositioning of the microtubular roots. This is the case in vfl1 mutants, in which the normal cruciate array of microtubular flagellar roots has not been observed (Adams et al, 1985). In vfl1 mutants, cell motility is also severely impaired, with even biflagellate cells showing aberrant spinning or tumbling movements, indicating that the flagella do not beat in opposite directions (Adams et al, 1985).…”

“…In Chlamydomonas, a kinesin-II protein, FLA10p (also called KHP1), is present at the pbbs (Vashishtha et al, 1996) and VFL1p, which is located within the lumen of bbs, was also shown to be present at the pbbs (Silflow et al, 2001). In the Chlamydomonas vfl1 mutant (Adams et al, 1985), the correct rotational orientation of the bbs is impaired (Silflow et al, 2001). By careful immunogold electron microscopy, Silflow et al showed that the position of VFL1p in the basal body is rotationally asymmetric.…”

“…7). In vfl1, the dcf is either absent or incomplete (Adams et al, 1985). Mispositioned or absent s-and d-fibers would probably result in a mispositioning of the microtubular roots.…”

Three-dimensional Organization of Basal Bodies from Wild-Type and δ-Tubulin Deletion Strains ofChlamydomonas reinhardtii

“…In the variable flagellar mutants, vfl1, vfl2, and vfl3 the distal striated fiber is either absent or only partially assembled (Wright et al, 1983Adams et al, 1985). The dense plate that normally is present beneath the distal striated fiber is also absent.…”

An evolutionarily conserved leucine-rich repeat protein CLERC is a centrosomal protein required for spindle pole integrity

“…In particular, the variable flagella number mutant, vfl1, was observed to have pleiotropic effects on flagellar number and localization, the timing of flagellar outgrowth during the cell cycle, and the positioning of the cleavage furrow at cytokinesis. 26 Defects in the number and localization of basal bodies and in their associated striated fibers were also observed at the ultrastructural level. Silflow et al, 27 cloned the vfl1 gene that encodes a protein of 128 kDa with five leucine-rich repeat sequences near the NH 2 terminus and a large α-helical coiled-coil domain at the COOH terminus.…”