Quantitative proteomic and phenotypic analysis of Chlamydomonas wild-type and lf4 mutant

Background: Normal health and development require cilia and flagella, which are essential organelles that are highly conserved across different species. The molecular mechanisms that regulate their assembly state, however, remain largely unknown. In Chlamydomonas, mutations in any of five long flagella (lf) genes result in cells that assemble flagella more than twice the length of wild-type cells. To learn more about the role of one of these genes, LF4 (which encodes a MAP kinase) we used a phenotypic analysis along with a global proteomic approach to identify differentially expressed proteins in the flagella and cell bodies of lf4 and wild-type cells.

Methods: To determine the cell body volumes, cells were fixed with an equal volume of 1% glutaraldehyde and examined by phase contrast microscopy. Cell volume was determined with the following equation (4/3π[length/2][width/2]2). Quantitative LC-MS/MS was performed on equal amounts of protein from purified cell bodies and flagella. Resulting data was analyzed using MaxQuant (Version 1.5.2.8) and Perseus (version 1.5.5.3). In order to examine the effect of rapamycin on cell growth, cells were cultured on agar plates containing rapamycin and exposed to constant light.

Results: As determined by microscopic analysis, there was a significant increase in the size of lf4 cell bodies compared to wild-type cells. As shown by analysis of the quantitative LC-MS/MS spectra, 66% of the total proteins were common between the two strains with 11% and 12% of flagella proteins and cell body proteins respectively being differentially expressed. Enrichment analysis revealed a decrease in proteins involved in protein synthesis but an increased expression of proteins involved in energy production in lf4 cell bodies compared to wild type. Differential expression of flagellar proteins was also seen in these two strains. For example, lf4 flagella had an increased expression of microtubule motor proteins while wild type had increased expression of proteins associated with organelles. Cells grown in the presence of rapamycin exhibited a dose-dependent decrease in viability and cell growth on both strains, although the effect was less pronounced in lf4.

Conclusions: The observation that lf4 cell bodies are larger than wild-type cells suggest that regulation of both flagellar length and cell body size are impaired. The LF4 gene product could regulate both flagella and cell body size. The increase in flagella and cell body size are surprising given the decreased expression of proteins involved in transcription and translation. The increase in protein levels for proteins involved in energy production could reflect an increased energy requirement necessary for flagellar motility with the lf4 flagella.