Structural and functional dissection of a cell cycle chromatin switch in green algae
EMSL Project ID
60332
Abstract
The long-term goal of this project is to understand the dynamic genetic programming which underlies cell growth and proliferation in green algae. This project addresses a long-standing enigma of how the retinoblastoma tumor suppressor complex (RBC), a conserved chromatin-associated cell cycle regulator, modulates cell cycle progression in a non-canonical manner that is independent of transcription in the model green alga Chlamydomonas reinhardtii (Chlamydomonas). Mounting evidence points to the RBC as a direct modulator of chromatin states which can govern the irreversible decision for a cell to undergo DNA replication and mitosis. The RBC core in Chlamydomonas has three protein subunits--a retinoblastoma-related protein called MAT3 that acts as a cell cycle repressor--and its target, a DNA binding heterodimer and cell cycle activator composed of related proteins, E2F1 and DP1. Mutations in the genes encoding any of these three subunits lead to cell size phenotypes caused by either too much or too little cell division activity (depending on whether the repressor activator subunits are missing). The activity of the RBC is controlled by cell cycle mediated phosphorylation, but the structural and functional consequences of this phosphorylation are unknown. Under this proposal we will take advantage of special expertise and resources at EMSL to interrogate the structure of the Chlamydomonas RBC. Our objectives are i) Cryo-EM structural characterization of unmodified recombinant RBC in its DNA bound and unbound states; ii) Cryo-EM structural characterization of recombinant RBC in its phosphorylated activator state that will be achieved through one or more in vitro methods; iii) proteomic and phospho-proteomic characterization of native RBC immunopurified from key cell cycle time points in synchronized Chlamydomonas cultures. If successful, we will be able to produce and validate a cutting-edge functional and structural map of a dynamic and enigmatic protein complex that will have impact across the fields of structural biology, chromatin biology and synthetic biology. Moreover, Chlamydomonas is a DOE flagship species and model for algal systems biology and bioenergy, and the insights we gain in Chlamydomonas can be translated to other green algal systems that are being developed as bioenergy feedstocks where traits such as cell size and proliferation status can have significant impacts on biomass or bioproduct productivity.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2022-10-01
End Date
N/A
Status
Active
Released Data Link
Team
Principal Investigator
Co-Investigator(s)
Team Members