The investigation of acetate sensitivity within shf1 Chlamydomonas mutants

Background: Cilia and flagella are essential for human health. Defects in the assembly and function of these organelles are associated with a collection of disorders called ciliopathies. Studies have suggested that regulation of ciliary size is associated with external environmental factors. Although TOR signaling pathway has recently been implicated as playing a pivotal role in linking the cellular environment with determination of cell and organelle size, additional biological pathways involved in this process remain largely unknown.

Methods: Both wild-type and shf1 cells were grown in M media with continuous aeration and under light-dark cycle to an equal cell density. 20 mM acetate was added, and cells were removed in 30 minute intervals. Cells were then fixed with an equal volume of 1% glutaraldehyde. Cells were examined by phase contrast microscopy and electron microscopy. Flagellar lengths and cell body area of wild-type and shf1 mutants was determined.

Results: Short flagellar (shf) mutants of Chlamydomonas assemble flagella that are half the length of wild-type cells. Consistent with the observation that ciliary length and cell size are interconnected, shf1 cell volume is increased compared to wild-type cells. Interestingly, shf1 mutants are a flagellate when grown in the presence of acetate. To learn more about the acetate sensitivity, we examined the ultrastructure of shf1 mutants following the addition of acetate. Microscopic analysis revealed notable deformities in the flagellar ultrastructure. When viewing the ultrastructure of wild-type versus shf1 mutants in the presence of acetate, it was noted that flagella was shorter in shf1 mutants. When looking at the shf1 in acetate under cross section abnormal morphology was noted. The cross section no longer demonstrates round structures, as seen with wild-type, but instead, bleb-like structures were seen coming off the flagella, and the flagella was square shaped. “Rodlike” structures were also abnormal and were located between the microtubule doublets and the flagellar membrane. The basal body and the transition zone microtubules appear to be unaffected by the acetate.

Conclusion: Although shf1 assembles short flagella, their cell bodies are approximately twice the size of wild-type cells. This suggests that regulation of flagellar length and cell body size are coupled together. As originally reported, inclusion of acetate in growth media results in the disassembly of shf1 flagella. Ultrastructural analysis demonstrates a dramatic change to the morphology of shf1 flagella upon acetate treatment. It is intriguing to speculate that the “rodlike” structures present between the axonemes, and the flagellar membrane are axonemal fragments. Currently, we are examining the composition of these “rods” to determine their biochemical components.