Book Description

"Many physiological processes, including angiogenesis, neurodevelopment and wound healing, rely on the directed movement of cells through the extracellular matrix (ECM). Cell migration is also a fundamental process involved in cancer metastasis. Indeed, proteins that enhance focal adhesion and actin cytoskeletal dynamics are often upregulated in invasive and metastatic cancer cells. In this thesis, we show that the adapter proteins ShcA (p46/52 isoforms) and lipoma-preferred partner (LPP) are required for the migration and invasion of ErbB2-overexpressing breast cancer cells in response to transforming growth factor [beta] (TGF[beta]). Live-cell microscopy techniques reveal that ShcA and LPP are both required for TGF[beta]-enhanced assembly and disassembly of adhesions. Moreover, p46/52ShcA must be phosphorylated on three key tyrosine residues (Y239/Y240/Y313) and LPP must interact with the actin cytoskeleton through its [alpha]-actinin binding domain (ABD) to mediate these effects. Using a BioID proximity labeling approach, we show that p46/52ShcA exists in a complex with various adhesion and actin cytoskeletal proteins, including paxillin and LPP. Total internal reflection fluorescence (TIRF) and 3D super-resolution iPALM microscopy confirm that p46/52ShcA is a novel component of adhesions and its localization to these structures precedes LPP.In addition to acting as a scaffold, the ECM provides biophysical cues that direct cell migration. We demonstrate that LPP is required for ErbB2+ breast cancer cells to sense substrate stiffness. Cells expressing wildtype LPP exhibit enhanced migration rates on intermediate stiffnesses (30-50 kPa), and slower migration rates on soft (10 kPa) and stiff (90kPa) substrates; in contrast, cells lacking LPP expression migrate at a constant speed. ErbB2+ cells also modulate invasive activity based on substrate stiffness. In particular, cells invade maximally on soft (5 kPa) and hard (100 kPa) substrates where migration is significantly reduced. This is the first study to demonstrate that LPP mediates mechanosensitivity in breast cancer cells.Breast cancer is a highly heterogenous disease with considerable cellular, molecular and pathological differences between patients. We find that LPP also plays an important role during TGF[beta]-enhanced migration and invasion of triple-negative breast cancer (TNBC) cells. Human MDA-MB-231 cells with lower levels of LPP expression fail to exhibit TGF[beta]-enhanced migration and invasion. Mouse 4T1 cells, and 4T1 derivatives that preferentially metastasize to the lungs (4T1-526) and live (4T1-2776), also fail to exhibit TGF[beta]-enhanced migration and invasion when LPP expression is reduced. Consequently, 4T1-2776 cells lacking LPP develop fewer liver metastases following splenic injection.Many of experimental results described in this thesis were obtained with live-cell fluorescence microscopy. Fluorescence microscopy provides a convenient, selective and sensitive way to observe live-cell dynamics; however, phototoxicity is a significant limitation of this technique. In this thesis, we show that much of the phototoxicity and photobleaching experienced with live-cell fluorescence imaging occurs as a result of “illumination overhead” (IO). This occurs when a sample is illuminated but fluorescence emission is not being captured by the microscope camera. As a result, we developed a workflow to optimize imaging conditions on any standard microscope. The workflow includes a guide on how to (1) determine the maximum image exposure time for a dynamic process, (2) optimize excitation light intensity, and (3) assess cell health with mitochondrial markers"--