Phenotypic and proteomic analysis of a Chlamydomonas mutant, shf1, defective in flagellar assembly

Marwa Elsayed, Amber Yu, Dilani Herath, Nedra Wilson

Research output: Contribution to conferencePosterpeer-review

Abstract

Introduction/Objectives: 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. To learn more about these pathways, we undertook a phenotypic and proteomic analysis of a mutant defective in assembling full-length flagella, shf1. These mutants assemble flagella that are half the length of wildtype. Interestingly, the flagella of these mutants are unstable in the presence of acetate.

Methods: Wildtype and shf1 cells were grown to equal density on a 12-hour light/dark cycle. Flagella and cell body sizes as well as acetate-induced changes were determined by microscopic analysis. Quantitative proteomic analysis was performed using label-free methods and analyzed using MaxQuant software. Statistical analysis was performed using two-tailed student’s t-test to identify proteins whose levels varied between wildtype and shf1 cell bodies and isolated flagella.

Results: As previously shown, shf1 cells assembled flagella that were half the length of wildtype cells. Surprisingly, the cell body volume of shf1 was increased up to twice that of wildtype. The inclusion of acetate in the media resulted in aflagellate shf1 cells and cells were seen to lose flagella within 30 minutes of addition of acetate. Proteomic analysis on isolated cell bodies and flagella identified 4,943 and 3,169 proteins, respectively. Preliminary analysis of the proteomic data demonstrated that 4% of the cell body proteins were present at levels that differed in a statistically significant manner. Similarly, a statistically significant difference in protein levels was seen for 9.8% of flagellar proteins.

Conclusions: 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 absence of shf1 flagella. These results suggest that acetate induces instability of the shf1 flagella leading to flagellar disassembly. Our preliminary data suggest that shf1 and wildtype cells have significant differences in protein composition and levels. Currently, we are examining the proteins that are statistically different between these two strains to learn more about the pathways regulating flagellar assembly and function.
Original languageAmerican English
Pages43
StatePublished - 16 Feb 2024
Event
Oklahoma State University Center for Health Sciences Research Week 2024
- Oklahoma State University Center for Health Sciences, Tulsa, United States
Duration: 13 Feb 202417 Feb 2024
https://medicine.okstate.edu/research/research_days.html

Conference

Conference
Oklahoma State University Center for Health Sciences Research Week 2024
Country/TerritoryUnited States
CityTulsa
Period13/02/2417/02/24
Internet address

Keywords

  • Chlamydomonas
  • cilia/flagella
  • proteome

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