04/10/2026
Doctoral Thesis Defense: Tyler Nelson
Date: Friday, April 10 at 2:00 pm
Location: SST 106 (+Zoom)
Host Department: Chemistry
Advisor: Anna-Karin Gustavsson
Committee: Anna-Karin Gustavsson, Kevin Kelly, Michael Diehl
Title: Development of single-molecule microscopy tools for study of nanoscale dynamics in live endothelial cells
Abstract:
The structural integrity of human tissues is built upon dynamic, nanoscale interactions on a cellular and sub-cellular level – cytoskeletal proteins like actin connect with focal adhesion complexes to anchor cells to a substrate, while cell-to-cell junctions, like in vascular endothelial cells, maintain structure in part through the concerted action of transmembrane adhesion proteins called cadherins. Proper functioning of these protein complexes is of vital consequence in many physiological systems, and dysfunction can lead to severe pathologies like cardiovascular disease, autoimmune disorder, even increased risk of cancer metastasis, but many of the details of these complexes are too small to be studied with conventional microscopy techniques. The overarching goal of this work was to create a high-performance, multimodal microscopy platform with advanced super-resolution imaging capabilities and apply it to investigate the biophysics behind these critical systems. Conventional single-molecule super-resolution microscopes are typically limited by uneven Gaussian illumination profiles, leading to suboptimal photophysics, and out-of-focus back- ground fluorescence, degrading performance. To overcome these limitations, I developed a 3D-capable super-resolution microscope featuring a light sheet for optical sectioning and a flat-field total internal reflection fluorescence (TIRF) illumination system. I validated this platform through high-precision, dual-channel 3D reconstructions of actin stress fibers and focal adhesion complexes. I then used the platform to investigate the nanoscale dynamics of VE-cadherin, an adherens junction protein crucial to the regulation of vascular permeability. By developing a custom analysis pipeline for highly parallelized single-particle tracking (SPT) in live human umbilical vein endothelial cells (HUVECs), I was able to quantify the diffusive behavior of VE-cadherin in response to targeted pharmacological perturbations of the actin network. Analysis revealed VE-cadherin’s sensitivity to different types of actin reorganization, including some unexpected responses suggesting a previously unknown relationship between SLK/LOK inhibition and junctional remodeling. Ultimately, this work establishes a robust technological foundation for advanced 3D super-resolution imaging and provides biophysical insights into the complex mechanoregulation of endothelial barriers.