Date of Award

Summer 8-16-2024

Level of Access Assigned by Author

Open-Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Physics

Advisor

Samuel T Hess

Second Committee Member

R Dean Astumian

Third Committee Member

David J Batuski

Additional Committee Members

Clarissa Henry

MacKenzie R. Stetzer

Abstract

Fluorescence photoactivation localization microscopy (FPALM) is a super-resolution imaging technique that surpasses the spatial resolution limitations of standard diffraction-limited microscopy. It has been demonstrated to be able to image biological structures with resolution down to a few tens of nanometers or better. Here we focus on utilizing FPALM to probe the intricate molecular mechanisms underlying muscle disease and influenza viral infection along with developing new methods to quantify the dynamics of biomolecules.

Muscular dystrophy is a progressive disease affecting muscle and neurological health in humans. Muscle cells adhere to their surrounding extracellular matrix (ECM) as well as the ECM at the myotendinous junction. Adhesion of muscle fibers to the ECM is critical for skeletal muscle function. The two major transmembrane receptors that anchor mature muscle cells to their ECM microenvironment are Dystroglycan (DG) and integrin alpha7 (Itga7). Mutations in each of these receptors lead to congenital muscular dystrophies. These processes/changes occur on smaller length scales, emphasizing the significance of imaging techniques that can capture information below the diffraction limit and offer valuable details on single molecule information. The super-resolution technique FPALM has showcased its ability to image detailed structures and dynamics of single molecules in living species. We demonstrate advancements of FPALM that have successfully obtained super-resolution images of dystroglycan and integrin proteins in muscle fibers of zebrafish. Our results demonstrate the successful imaging of single- and two-color imaging of muscle fibers using FPALM. This opens new avenues for answering biological and disease related questions in vivo at the nanoscale.

Influenza A virus (IAV) is a respiratory virus which continues to cause significant illness and mortality. The IAV protein Hemagglutinin (HA) forms clusters on the host cell membrane and viral membrane, and high-density HA clusters are necessary for viral entry through membrane fusion, but the mechanism behind HA clustering is not completely understood. It is important to identify the link between HA clusters and host cell components to understand the mechanism of HA clustering and how it can be disrupted. HA forms clusters in actin rich membrane regions, but again the interaction mechanism is unknown. We recently discovered HA interacts with the lipid phosphatidylinositol (4,5)-bisphosphate (PIP2), which is strongly connected to actin and the actin-binding protein cofilin, which have been implicated in viral assembly, budding, and release. Could PIP2 be the link between HA, actin, and cofilin? FPALM three color imaging reveals that PIP2 modulates the HA-cofilin associations. However, disruption of PIP2 with cetylpyridinium chloride (CPC) did not cause significant changes in HA-cofilin co-clustering. Several explanations for this surprising combination of results are discussed. New insight into the mechanism for HA to modulate host cell phosphoinositides and regulate cofilin activity may illuminate IAV assembly and budding, leading to discovery of new molecular targets for anti-IAV drug development.

FPALM is well-suited to determination of the spatial distribution of molecules, but to obtain molecular kinetics at similar length scales, we have developed a super-resolution localization version of fluorescence cross-correlation spectroscopy called Localization cross correlation spectroscopy (LoCCS). LoCCS can be applied to follow molecular interactions in cells, quantify colocalization and characterize co-diffusion, but with a 5-10x improvement in resolution. Results for diffusion of fluorescent beads in solution confirm and calibrate the method, while data for LoCCS of HA and PIP2 are quantified to determine whether HA and PIP2 diffuse together in living cells. Measurement of diffusion time and co-diffusion of HA- PIP2 will help identify the mechanism of HA-PIP2 interactions. We have observed that HA and PIP2 motions are affected by each other at the PM and that HA and PIP2 are associated in a time-dependent manner through cross-correlation measurements. The time dependent association implies that the interactions between HA and PIP2 are not static but rather dynamic.

Download

Available for download on Saturday, October 17, 2026

Files over 10MB may be slow to open. For best results, right-click and select "save as..."

Share