The Effect of Caldesmon Overexpression on Epithelial-Mesenchymal Transition in Normal Murine Mammary Gland Epithelial Cells
Open Access
- Author:
- Virgi, Gage Anthony
- Area of Honors:
- Chemical Engineering
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Esther Winter Gomez, Thesis Supervisor
Darrell Velegol, Thesis Honors Advisor - Keywords:
- TGFB
Caldesmon
EMT
Epithelial-Mesenchymal Transition
Cytoskeleton
NMuMG
Focal Adhesions
Smooth Muscle Actin
Stress Fibers
Traction Force - Abstract:
- The purpose of this thesis was to examine how the overexpression of caldesmon affected epithelial-mesenchymal transition (EMT) in normal murine mammary gland epithelial cells. EMT has been implicated in a number of biological processes including wound healing, tissue fibrosis, and cancer metastasis. EMT is characterized by a loss of cell polarity and cell-cell adhesion affinity and leads to the formation of cells possessing of mesenchymal phenotype that have increased motility and contractility. During this transition, actin filaments begin to bundle to form stress fibers and become linked to the extracellular matrix through focal adhesion assemblies. Caldesmon is an actin binding protein that has been found to stabilize stress fiber formation in epithelial cells. Specifically, the effect of caldesmon overexpression on different aspects of EMT was analyzed including stress fiber formation, α-smooth muscle actin expression, focal adhesion formation, and traction force generation. Cells were transfected with a caldesmon wildtype plasmid to overexpress caldesmon and a green fluorescent protein plasmid as a control. The cells were then grown on polyacrylamide gels with a stiffness of 1800 Pa with either transforming growth factor (TGF)-β1 solution or control solution. The cells were then either analyzed using immunofluorescence microscopy or traction force microscopy. The formation of stress fibers was found to be qualitatively higher in cells treated with TGFβ1 than the control treated cells. The expression of α-smooth muscle actin was significantly higher in cells treated with TGFβ1 where caldesmon was overexpressed than the other treatments. Focal adhesion numbers and sizes were slightly higher for cells where caldesmon was overexpressed for both TGFβ1 and control treatments. Finally, treatment with TGFβ1 increased the magnitude of traction forces for both caldesmon overexpression on normal caldesmon expression.