1. Student: Nathan King Professor: Dr. Dae-Sik Lim Project Supervisor: Minchul Kim Activity of Merlin (NF-2) in Novel Tumor Pathway

2. Hippo Pathway: Drosophila controls organ size in animals through the regulation of cell proliferation and apoptosis Stimulation at connection sites with other cells signal transduced by successive protein phosphorylations

3. Mutant Effects Knockout Drosophila of Hippo Pathway proteins results in cancerous epithelial tissues This results in deformities in development of organs

4. Hippo Pathway: Homo sapien Homologous pathway exist in human cells Similar cancerous phenotypes result from knockouts

5. Mechanical Environments Hippo stimulation at connection sites with other cells Mechanical environment : - Contact with ECM - Adhesion between adjecent cells - Flow of fluid surrounding cells

6. ad Environmental Signaling

7. Mechanotransduction Actin stiffening accumulates nuclear Yorkie / YAP Loss of actin integrity / stress fibres inactivates Yorkie / YAP Actin stiffening induces organ overgrowth, which requires Yorkie ECM stiffness can control MSC differentiation lineage choice, which require YAP regulation This regulation does not seem to require canonical Hippo pathway, however Lats1/2 is required. The responsible mechanisms were not identified

8. Novel Cancer Pathway Novel pathway that stimulates YAP/TAZ to act as sensors and mediators of mechanical cues instructed by the cellular microenvironment which regulate cell differentiation and organ growth

9. My Project Is NF-2 (Merlin) involved in the novel actin density-mediated growth control pathway? Reasons: Research has shown NF-2 activation in growth regulating pathways that exclude MST1/2 Merlin is associated closely with actin

10. Methods Procedure: 1.Ligation of NF-2 ΔBB into pMSCV pure vector 2.Create retrovirus with new vector 3.Infect NIH3T3 cells for integration of engineered vector into genome 4.Test for expression of NF-2 ΔBB protein using Western Blot 5. Run subsequent experiments using novel pathway Main Idea: Use of Bluebox Deletion of NF-2 gene as dominant negative to mimic knockout of NF-2 and observe resulting effects on the actin stimulation pathway

11. Results Selected colonies 1 and 2 for genome sequencing and viral infection

12. Results 95% match, account for deletion of BlueBox Sequencing data showed successful ligation in bacterial colony number 1

13. Results NIH3T3 infection: Cells continue to grow after addition of puromycin antibiotic, proving success of infection No initial difference is seen in growth of control vs. mutant cell lines

14. Results Western Blot: NF-2 with BlueBox Deletion not expressed Endogenous NF-2

15. Results Error Analysis: Sequencing data Viral infection NF-2 endogenous expression There is most likely a mutation in the sequence of the gene. Trial and Error: Next step is to check the sequence of the original gene used for any errors that could cause it to not be expressed.