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Search for Palladin, an Actin-Associated Protein, in Pig Retinal Pigmented Epithelium and Its Role in Epithelial-Mesenchymal Transition

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Search for Palladin, an Actin-Associated Protein, in Pig Retinal Pigmented Epithelium and Its Role in Epithelial-Mesenchymal Transition Bellarmine University ScholarWorks@Bellarmine Undergraduate Theses Undergraduate Works 5-10-2021 Search for Palladin, an Actin-Associated Protein, in Pig Retinal Pigmented Epithelium and its Role in Epithelial-Mesenchymal Transition Katrina Powell [email protected] Follow this and additional works at: https://scholarworks.bellarmine.edu/ugrad_theses Part of the Biological Phenomena, Cell Phenomena, and Immunity Commons, Biotechnology Commons, Cell Anatomy Commons, Cell Biology Commons, Eye Diseases Commons, Laboratory and Basic Science Research Commons, Medical Cell Biology Commons, Molecular Biology Commons, Optometry Commons, and the Structural Biology Commons Recommended Citation Powell, Katrina, "Search for Palladin, an Actin-Associated Protein, in Pig Retinal Pigmented Epithelium and its Role in Epithelial-Mesenchymal Transition" (2021). Undergraduate Theses. 74. https://scholarworks.bellarmine.edu/ugrad_theses/74 This Honors Thesis is brought to you for free and open access by the Undergraduate Works at ScholarWorks@Bellarmine. It has been accepted for inclusion in Undergraduate Theses by an authorized administrator of ScholarWorks@Bellarmine. For more information, please contact [email protected], [email protected]. Powell 1 Search for Palladin, an Actin-Associated Protein, in Pig Retinal Pigmented Epithelium and its Role in Epithelial-Mesenchymal Transition Katrina Powell Spring 2021 A Senior Honors Thesis Presented in Partial Fulfillment of the Requirements of the Bellarmine University Honors Program Advisor: Dr. Steven D. Wilt Reader: Dr. Paul Kiser Reader: Dr. Carrie M. Doyle Reader: Dr. Kathryn West Powell 2 TABLE OF CONTENTS LIST OF FIGURES…………………………………………………………………………….3 ABSTRACT……………………………………………………………………………………4 INTRODUCTION……………………………………………………………………………...5 Anatomy of the Eye…………………………………………………………………….5 Retinal Pigmented Epithelium………………………………………………....7 Palladin…………………………………………………………………………………8 Cytoskeleton Elements…………………………………………………………………10 Actin……………………………………………………………………………11 Microtubules and Intermediate Filaments …………………………………….12 Epithelial Mesenchymal Transition…………………………………………………....12 Proliferative Vitreoretinopathy………………………………………………………....14 METHODS…………………………………………………………………………………......16 Primary Cell Tissue Culture of Retinal Pigmented Epithelium………………………...16 Vitreous Exposure………………………………………………………………17 Semi-Quantitative Reverse Transcription Polymerase Chain Reaction……………...…17 Immunoblotting………………………………………………………………………....18 RESULTS……………………………………………………………………………………….21 DISCUSSION…………………………………………………………………………………...27 ACKNOWLEDGEMENT………………………………………………………………………32 REFERENCES………………………………………………………………………………….33 Powell 3 LIST OF FIGURES Figure 1. “Right Eye (Viewed from Above).” A Guide To the Many Parts of the Human Eye and How They Function, Papin and Pretorius Optometrists, www.ppoptometrists.co.za/eye- care/anatomy-of-the-eye/ ……………………………………………………………….7 Figure 2. Sonoda, S. (2016, July). Diagram of Outer Retina [Shows the retinal pigmented epithelium between the Bruch’s membrane and neural retina]. Retrieved March 20, 2021, from https://www.researchgate.net/publication/305276317_Methods_for_culturing_retinal_pig ment_epithelial_cells_a_review_of_current_protocols_and_future_recommendations …8 Figure 3. George, R. D. (2007, January). Palladin an Alpha-Actin Binding Protein [Digital image]. Retrieved February 25, 2021, from https://www.researchgate.net/publication/6606858_Palladin_Mutation_Causes_Famili al_Pancreatic_Cancer_and_Suggests_a_New_Cancer_Mechanism……………………………….9 Figure 4. W, E. M. (2009, December 15). Structure of PALLD protein based on PYMOL rendering [Digital image]. Retrieved March 20, 2021, from https://en.wikipedia.org/wiki/Palladin#/media/File:Protein_PALLD_PDB_2dm2.png….10 Figure 5. Cytoskeleton elements in RPE Tarau, I., Berlin, A., Curcio, C. A., & Arch, T. (2019, July 22). The Cytoskeleton of the RPE [Cross-sectional view of the RPE and color coated Filaments]. Retrieved February 25, 2021, from https://www.mdpi.com/1422- 0067/20/14/3578/htm……………………………………………………………………11 Figure 6. Kalluri, R., & Weinberg, R. A. (2009, June 1). The Basics of Epithelial-Mesenchymal Transition [Digital image]. Retrieved February 25, 2021, from https://www.jci.org/articles/view/39104/figure/1……………………………………….13 Figure 7. Brady, C. J., & Kaiser, R. S. (2015, October 5). Proliferative Vitreoretinopathy: Re- detachment [Digital image]. Retrieved March 20, 2021, from https://www.reviewofophthalmology.com/article/pvr-an-update-on-prevention-- management……………………………………………………………………………….14 Figure 8. Control vs Vitreous Exposed RPE Cells…………………………………………….22 Figure 9: Control RPE with PALLD Monoclonal Primary Antibody………………………….23 Figure 10. Control and Vitreous Exposed RPE with Monoclonal Primary Antibody………….24 Figure 11. Control and Vitreous Exposed RPE with Polyclonal Primary Antibody………………25 Figure 12. Control and Vitreous Exposed RPE with Actin Monoclonal Primary Antibody……26 Figure 13. RT-PCR of Control and Vitreous Exposed Cells……………………………………27 Powell 4 ABSTRACT This study investigates the expression of Palladin, a phosphoprotein product of the PALLD gene, in the retinal pigmented epithelium (RPE). The RPE is a necessary layer below the retina of the eye. It plays a role it transporting and absorbing nutrients between the retina and the blood vessels below in the choroid. Palladin is an actin cross-linking protein and plays a role in cell adhesion and motility. It is found predominantly in muscle tissue helping the actin arrays form to keep the structure and shape of the cell. Published reports have demonstrated that a down regulation of Palladin in colon cancer cells results in a reorganization of the actin cytoskeleton, causing the cells to lose their typical shape, become proliferative and migratory. This process is otherwise known as epithelial-mesenchymal transition (EMT). In this process, the epithelial cells lose the cuboidal shape and gain a mesenchymal oval-like shape. The adhesions to nearby cells are lost and the cells can now migrate and proliferate away from other retinal pigmented epithelial cells. A similar EMT phenomenon is observed when the RPE is exposed to the vitreous humor in patients with proliferative vitreoretinopathy (PVR). PVR occurs when the RPE proliferates and forms epiretinal membranes above the retina. This causes retinal detachment and blindness. In this study, the expression of Palladin is investigated in primary cultures of pig retinal pigmented epithelium in both normal and vitreous-exposed cells. The cells are observed for any phenotypic transformation. It is expected that the vitreous exposed cells will not have the normal epithelial phenotype. The cells will be expected to be oval-shaped and disconnected from each other. Reverse transcription polymerase chain reaction (RT-PCR) and western blotting were performed to demonstrate mRNA and protein expression, respectively. Protein expression was tested using two different Palladin primary antibodies. The first antibody used was the PALLD monoclonal antibody which is more specific to just Palladin. The second antibody used was the Powell 5 PALLD polyclonal primary antibody. This antibody is less specific to Palladin. Palladin was seen expressed in the RPE cells; however, there was no notable difference in the expression of Palladin mRNA in vitreous-exposed cells versus the control cells. This indicates that while the protein is there, epithelial mesenchymal transition is not occurring and therefore no up or down regulation of Palladin is seen. Palladin is at normal expression rates. The immunoblotting analysis was inconclusive. More research will be necessary to see if Palladin plays a role in the RPE. Use of a different primary antibody may help achieve cohesive results in protein expression. Repetition of this experiment would need to be completed to obtain corroborating results. INTRODUCTION Anatomy of the Eye The eye may seem to have a simple anatomy but it is actually a complex organ that is tasked with many essential functions. The eye itself is asymmetrical when removed from the socket. The sclera is the white layer that surrounds the entire exterior of the eyeball giving the eye wall support. The sclera turns into the cornea more anteriorly. The cornea is a transparent layer that protects the pupil and iris. This layer gives the clearest image to the retinal photoreceptor layer since it is the first lens of the eye. Below the cornea lies the first chamber of fluid in the eye called the anterior chamber. This chamber is filled with the aqueous humor. The second barrier to this chamber is the iris. This circular pigmented muscle is what gives the eye its color. The muscle contracts and relaxes the pupil to allow additional or reduced light in. The pupil therefore is where light enters the interior of the eye and is the aperture of the iris. Powell 6 Behind the iris is the posterior chamber that is also filled with aqueous humor. The lens lies behind the iris and is held in place by ligaments called zonula fibers and then connected to the anterior portion of the eye by the ciliary body. The ciliary body consists of muscles that contract in order to change the shape of the lens in a process called accommodation. This allows an image to be focused on the retina. Behind the lens is the largest chamber called the vitreous body filled with the vitreous humor. This humor is more gelatinous than the aqueous humor.4 The main difference between the humors is that the vitreous humor is 99% water with collagen and hyaluronic
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