Standing

Undergraduate

Type of Proposal

Oral Research Presentation

Faculty

Faculty of Science

Faculty Sponsor

Lisa Porter

Abstract/Description of Original Work

How cells regulate their growth and division involves a tightly controlled integration of many mechanisms. In cells, Tuberin (gene – TSC2) is a protein in the Tuberous Sclerosis Complex (TSC) that modulates cellular growth, size, and proliferation. Mutations in the proteins forming the TSC can cause Tuberous Sclerosis Complex, an autosomal dominant disorder characterized by multisystem pathologies that is often associated with benign hamartomas in the brain, kidney, lungs and skin. The focus of my research is to clarify the role of Tuberin in the regulation of cell size and proliferation at the G2/M cell cycle checkpoint. During late G2, Tuberin retains Cyclin B1 (gene – CCNB1), a mitotic cyclin, in the cytoplasm thereby prolonging mitotic onset. We constructed six TSC2 mutants that harbour clinically relevant mutations which are known to destabilize the TSC. Interestingly, these mutations fall within the Tuberin Cyclin B1 binding domain. Whether or not these mutations disrupt the regulation of the G2/M checkpoint is a key question of this project. This is studied by over-expressing the mutants with GFP tagged Cyclin B1 in Tuberin null cells. The resultant phenotypes are analyzed by flow cytometry, immunoprecipitation, and immunofluorescence. To aid in the temporal study of the cell cycle, I aim to validate successful CRISPR/Cas9-mediated knock-in of an iRFP tag within the TSC2 gene of HEK293 cells. This new cell line will be a powerful tool to dissect the roles of Tuberin in regulating cellular growth and division and can provide deep understanding of proliferative diseases like TSC and cancers.

Availability

March 29 (12-3); March 31 (12-3); April 1 (12-3)

Special Considerations

Only presenter: Ali Nadi

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Applying CRISPR/Cas9 and fluorescent tools to dissect the role of Tuberin in cell cycle regulation

How cells regulate their growth and division involves a tightly controlled integration of many mechanisms. In cells, Tuberin (gene – TSC2) is a protein in the Tuberous Sclerosis Complex (TSC) that modulates cellular growth, size, and proliferation. Mutations in the proteins forming the TSC can cause Tuberous Sclerosis Complex, an autosomal dominant disorder characterized by multisystem pathologies that is often associated with benign hamartomas in the brain, kidney, lungs and skin. The focus of my research is to clarify the role of Tuberin in the regulation of cell size and proliferation at the G2/M cell cycle checkpoint. During late G2, Tuberin retains Cyclin B1 (gene – CCNB1), a mitotic cyclin, in the cytoplasm thereby prolonging mitotic onset. We constructed six TSC2 mutants that harbour clinically relevant mutations which are known to destabilize the TSC. Interestingly, these mutations fall within the Tuberin Cyclin B1 binding domain. Whether or not these mutations disrupt the regulation of the G2/M checkpoint is a key question of this project. This is studied by over-expressing the mutants with GFP tagged Cyclin B1 in Tuberin null cells. The resultant phenotypes are analyzed by flow cytometry, immunoprecipitation, and immunofluorescence. To aid in the temporal study of the cell cycle, I aim to validate successful CRISPR/Cas9-mediated knock-in of an iRFP tag within the TSC2 gene of HEK293 cells. This new cell line will be a powerful tool to dissect the roles of Tuberin in regulating cellular growth and division and can provide deep understanding of proliferative diseases like TSC and cancers.