Susan K. Dutcher

Flagellar assembly in two hundred and fifty easy-to-follow steps

The eukaryotic flagellum is a complex biochemical machine that moves cells or moves materials over the surface of cells, such as in the mammalian esophagus, oviduct or in protozoa. It is composed of over 250 polypeptides that must be assembled into a number of different structures and each structure must be attached with an exact periodicity along the microtubules. Once the flagellum is assembled, each of the components must act in concert and in three dimensions to produce a complex waveform. This review provides an outline of the composition and function of the different structures found in the flagella of Chlamydomonas.

The tubulin fraternity: alpha to eta.

Microtubules perform essential and diverse functions in eukaryotic cells. They are required for spindles during mitosis and meiosis, for axonal transport, for organelle positioning, and for cilia and flagella. gamma-tubulin is necessary to initiate the assembly of alpha-beta tubulin heterodimers into microtubule polymers. Recent genetic analyses and database searches have added four new members of the tubulin superfamily, which now includes alpha-, beta-, gamma-, delta-, epsilon-, zeta-, and eta-tubulin.

Pharmacological and genetic evidence for a role of rootlet and phycoplast microtubules in the positioning and assembly of cleavage furrows in Chlamydomonas reinhardtii

In Chlamydomonas reinhardtii, specialized cytoskeletal structures known as rootlet microtubules are present throughout interphase and mitosis. During cytokinesis, an array of microtubules termed the phycoplast is nucleated from rootlet microtubules and forms coincidentally with the cleavage furrow [Johnson and Porter, 1968: J. Cell Biol. 38:403-425; Holmes and Dutcher, 1989: J. Cell Sci. 94:273-285; Gaffel and el-Gammel, 1990: Protoplasma 156:139-148; Schibler and Huang, 1991: J. Cell Biol. 113:605-614]. We have obtained two independent lines of evidence that support the hypothesis that the rootlet and phycoplast microtubules play a direct role in cleavage furrow placement and assembly. First, the destabilization of spindle and phycoplast microtubules by pharmacological agents was accompanied by the aberrant distribution of actin and a failure of cytokinesis. Second, we characterized mutant strains that failed to complete cytokinesis properly. Actin and myosin were mislocalized to additional rootlet microtubules in the cyt2-1 strain, and this mislocalization was correlated with the presence of additional cleavage furrows. This evidence suggests that microtubules are necessary for the correct positioning and assembly of functional cleavage furrows in C. reinhardtii.

A Unified Taxonomy for Ciliary Dyneins

The formation and function of eukaryotic cilia/flagella require the action of a large array of dynein microtubule motor complexes. Due to genetic, biochemical, and microscopic tractability, Chlamydomonas reinhardtii has become the premier model system in which to dissect the role of dyneins in flagellar assembly, motility, and signaling. Currently, 54 proteins have been described as components of various Chlamydomonas flagellar dyneins or as factors required for their assembly in the cytoplasm and/or transport into the flagellum; orthologs of nearly all these components are present in other ciliated organisms including humans. For historical reasons, the nomenclature of these diverse dynein components and their corresponding genes, mutant alleles, and orthologs has become extraordinarily confusing. Here, we unify Chlamydomonas dynein gene nomenclature and establish a systematic classification scheme based on structural properties of the encoded proteins. Furthermore, we provide detailed tabulations of the various mutant alleles and protein aliases that have been used and explicitly define the correspondence with orthologous components in other model organisms and humans.

Mating and tetrad analysis in Chlamydomonas reinhardtii.

Publisher Summary The ease of detecting mutations in haploid organisms is straightforward; any newly arising recessive mutation is apparent because there is no wild-type allele to mask the mutant phenotype. Thus, large-scale screens and/or selections can be performed for the isolation of mutations affecting the flagella of Chlamydomonas . Two different mating types exist in C. reinhardtii : mating type plus (mt + ) and mating type minus (mt - ). These mating types behave as alleles at a genetic locus on linkage group VI. Chlamydomonas reinhardtii is a haploid organism. Haploid organisms have several advantages over diploid organisms when dissecting a process by a mutational approach. Mating is initiated when the cells are starved for nutrients and specifically when they are starved for nitrogen. Recognition occurs between cells of opposite mating types at two levels. The initial recognition occurs by interactions between hydroxyprolinerich glycoproteins, agglutinins, in the membrane of the flagella of the two cell types. This process is known as tipping. It leads to an increase in the intracellular levels of cyclic AMP, which stimulates the formation of a fertilization tube on the surface of the mt + cell. Mutations that affect flagellar function, length, or number do not block the ability of C. reinhardtii strains to mate. Short flagella or paralyzed flagella may reduce the efficiency of mating, but do not block mating. Flagella are required for conventional mating procedures. Mutations that produce cells that lack flagella require additional steps to ensure mating. Because the first recognition events involve the tipping of flagella of the two mating types, this step must be bypassed. When meiosis is completed and the meiotic progeny have been collected, they can be analyzed as the progeny from individual meiotic events (a tetrad) or individually (random progeny). One advantage of tetrad analysis is the ability to map a gene with respect to its centromere as well as with respect to other genes.

Flagella in prokaryotes and lower eukaryotes.

During the past year, significant advances have been made in the understanding of both prokaryotic and eukaryotic flagella. About 50 genes are dedicated to the assembly and operation of bacterial flagella. Recent discoveries have advanced our understanding of how these genes are regulated and how their products assemble into a functional, rotating organelle. The dynein arms of eukaryotic flagella are now also better understood. Several genes that are found in the mechanochemical macroassemblies have been cloned, and other loci have been identified, suggesting that there is even greater complexity than first expected.

Purification of basal bodies and basal body complexes from Chlamydomonas reinhardtii.

Publisher Summary The microtubule organizing center (MTOC) in Chlamydomonas is defined as the pair of basal bodies, the rootlet microtubules, the distal and proximal striated fibers, and the nucleobasal body connectors (NBBCs). In Chlamydomonas , three different protocols can be used to obtain isolated basal body complexes. All of the isolated complexes include basal bodies and proximal fibers, but they differ in whether they contain other structures. Removal of the cell wall is a required step in preparing basal bodies. Autolysin is an enzyme produced by vegetative and gametic cells for removing the cell wall. Vegetative autolysin is used in the shedding of the mother cell wall following cell division. Gametic autolysin is used in the shedding of the cell wall prior to cytoplasmic fusion of mating cells. The steps required for preparation of autolysins have been mentioned in this chapter, along with the solutions and isolation of basal body complexes and of flagellum-basal body complexes. The isolated complexes can be treated with pressure and then centrifuged to separate basal bodies and proximal fibers from the flagella and other fiber systems. The presence of ethylene-diaminetetraacetic acid (EDTA) in this protocol is important to retaining the flagella and basal bodies together in the early steps. Isolation of nucleobasal body-flagellum complexes is a method for isolating a more complex structure that contains nuclei. This protocol relies on the use of autolysin or on cell wall-defective strains. The chapter also discusses components of basal body complexes. The isolation of mutations that affect the MTOC may be accomplished by classic means. Potential mutant phenotypes may include flagellar assembly defects and conditional lethality. The advent of insertional mutagenesis makes it possible to screen for mutations that are tagged with transforming DNA, which should facilitate the cloning of the locus of interest.

Genetic Properties of Linkage Group XIX in Chlamydomonas Reinhardtii

A unique linkage group has been identified in Chlamydomonas. To date, all mutations that have been mapped to linkage group XIX affect flagellar and basal body functions. Linkage group XIX shows several other striking genetic properties. First, the genetic map of this linkage group is circular. Genetic circularity can be achieved because the chromosome is a physically circular molecule or because of constraints on the types of recombination events that occur. A linear molecule that shows complete chromatid interference cannot be distinguished from a circular molecule. Complete chromatid interference is defined as the property that every chromatid is always involved in an even number of recombination events. If interference is not complete, three factor crosses will distinguish between a circular chromosome and a linear chromosome. Experiments of this type are underway (S.K. Dutcher, work in progress). Second, recombination levels on linkage group XIX are affected by temperature; recombination on 12 other linkage groups in Chlamydomonas is not affected by changes in temperature during any part of the meiotic life cycle (S.K. Dutcher, ms. in prep.). Patterns of interference and recombination on linkage group XIX are also different from other linkage groups. Basal bodies/centrioles are cellular organelles that are precisely replicated and partitioned in cell division. This fidelity distinguishes basal bodies/centrioles from all other cellular organelles, with the exception of the nucleus and the chromosomes. Because of the odd genetics of linkage group XIX and the strict replication and segregation of basal bodies, it is intriguing to speculate on the location of linkage group XIX. There are numerous reports in the literature of nucleic acid being associated with basal bodies. Both RNA and DNA have been reported to be localized to these structures. To date no unique species has been identified. Lwoff has suggested that basal bodies are genetically autonomous, and Sagan has suggested that they could have a symbiotic origin. Could linkage group XIX be located in the basal body and not in the nucleus? No definitive answer is available to this question. The number of chromosomes in the nucleus of Chlamydomonas has not been determined reliably. Linkage group XIX segregates as expected for a nuclear chromosome and appears to contain a region that behaves genetically as a centromere. However, any genetic information that is partitioned at meiosis in a regular manner and is present in a limited number of copies could resemble a nuclear chromosome in its segregational properties.(ABSTRACT TRUNCATED AT 400 WORDS)

Isolation and characterization of dominant tunicamycin resistance mutations in Chlamydomonas reinhardtii (Chlorophyceae)

Seven independent mutations that confer resistance to the nucleoside antibiotic tunicamycin in the unicellular green alga Chlamydomonas reinhardtii were isolated in wild‐type diploid cells. Upon resolution to haploidy, all the mutations segregated as single mutations and were semi‐dominant when retested in heterozygous diploids. In addition, the TUN1‐3 allele affects the ability of cells to agglutinate during conjugation in the absence of tunicamycin. The seven mutations map to a single locus on linkage group VIII. These mutations may be useful as a selectable marker in transformation studies of Chlamydomonas and in studies of processes that require asparagine‐linked glycosylation.