New mutations in flagellar motors identified by whole genome sequencing in Chlamydomonas

Abstract

BackgroundThe building of a cilium or flagellum requires molecular motors and associated proteins that allow the relocation of proteins from the cell body to the distal end and the return of proteins to the cell body in a process termed intraflagellar transport (IFT). IFT trains are carried out by kinesin and back to the cell body by dynein.MethodsWe used whole genome sequencing to identify the causative mutations for two temperature-sensitive flagellar assembly mutants in Chlamydomonas and validated the changes using reversion analysis. We examined the effect of these mutations on the localization of IFT81, an IFT complex B protein, the cytoplasmic dynein heavy chain (DHC1b), and the dynein light intermediate chain (D1bLIC).ResultsThe strains, fla18 and fla24, have mutations in kinesin-2 and cytoplasmic dynein, respectively. The fla18 mutation alters the same glutamic acid (E24G) mutated in the fla10-14 allele (E24K). The fla18 strain loses flagella at 32?C more rapidly than the E24K allele but less rapidly than the fla10-1 allele. The fla18 mutant loses its flagella by detachment rather than by shortening. The fla24 mutation falls in cytoplasmic dynein and changes a completely conserved amino acid (L3243P) in an alpha helix in the AAA5 domain. The fla24 mutant loses its flagella by shortening within 6 hours at 32?C. DHC1b protein is reduced by 18-fold and D1bLIC is reduced by 16-fold at 21?C compared to wild-type cells. We identified two pseudorevertants (L3243S and L3243R), which remain flagellated at 32?C. Although fla24 cells assemble full-length flagella at 21?C, IFT81 protein localization is dramatically altered. Instead of localizing at the basal body and along the flagella, IFT81 is concentrated at the proximal end of the flagella. The pseudorevertants show wild-type IFT81 localization at 21?C, but proximal end localization of IFT81 at 32?C.ConclusionsThe change in the AAA5 domain of the cytoplasmic dynein in fla24 may block the recycling of IFT trains after retrograde transport. It is clear that different alleles in the flagellar motors reveal different functions and roles. Multiple alleles will be important for understanding structure-function relationships.

Role for intraflagellar transport in building a functional transition zone

“…This indicates that despite no significant reduction in ciliary length, IFT assembly/function within the cilium is impaired. From these experiments, we conclude that dynein-2 is required for the maintenance of metazoan non-motile (primary) cilia, consistent with observations obtained from motile cilia in the unicellular green alga, C. reinhardtii [70][71][72]. Such a function may depend on the cell type, however, as IFT is required for ciliogenesis but not for axonemal maintenance in Trypanosoma brucei motile cilia and mouse spermatozoa [73,74].…”

“…Interestingly, our ts-che-3 mutant is partially defective at the permissive temperature, indicating that while dynein-2 is required for ciliary maintenance, ciliary structure is maintained despite the reduced dynein-2 activity. A similar requirement for retrograde IFT trafficking was observed by analysing temperature-sensitive dynein-2 mutants in Chlamydomonas [70,71], although IFT is not required for the maintenance of Trypanosoma or mouse sperm flagella [73,74]. It will be interesting to determine whether mammalian primary cilia show a dependence on IFT, similar to what we observe.…”

Evidence That an Unconventional Actin Can Provide Essential F-Actin Function and That a Surveillance System Monitors F-Actin Integrity inChlamydomonas

“…First, the novel overlapping-pool sequencing strategy that we used greatly facilitated the identification of the causative mutations in our collection of LatB-sensitive mutants. Other recent studies have used high-throughput, wholegenome sequencing either of individual mutants after prior meiotic mapping (Dutcher et al 2012;Lin et al 2013) or of bulked segregants after backcrossing (Alford et al 2013;Tulin and Cross 2014) to localize the mutations of interest to genomic regions of 0.3-2 Mb, and in many cases to identify the actual causative mutation, a major improvement over methods that had been available previously. The overlapping-pool approach offers a substantial further improvement.…”

Molecular techniques to interrogate and edit the Chlamydomonas nuclear genome

“…With the advent of next‐generation sequencing, whole mutant genomes can be sequenced to identify causal mutations (Dutcher et al ., ; Lin et al ., ,b; Tulin and Cross, ). Individual mutants or bulked segregants can be sequenced to identify alterations in the genome that led to the observed phenotype (see Alford et al ., supplemental figure for visualizations of bulked segregant mapping).…”

“…Mutant mapping data can be useful for narrowing down the genomic regions that should be searched for mutations, especially if there are many SNPs between the reference genome and the mutant strain. With this technique, a causative mutation can be identified quickly and accurately (Dutcher et al, 2012;Lin et al, 2013b).…”