Session: Research Posters

Paper Number: 172897

Pemphigus Antibodies Induce Keratinocyte Cell-Cell Junction Stiffening and Early Rupture Under Large Strain 

Pemphigus vulgaris (PV), a rare autoimmune disease which causes skin blistering, alters the mechanics of desomosomal cell-cell adhesion complexes through antibody-induced disassembly. Changing junctional mechanics, specifically in the presence of mechanical load, may play a critical role in pathogenesis and progression of the disease, but are yet to be fully understood. In the presented study, we investigated the implications of varied strain levels on the junctional mechanics of adherent epithelial single cell pairs exposed to various PV antibodies. To perform mechanical characterization of the cell pairs, a previously developed single-cell adhesion micro tensile testing (SCAµTT) device was used to isolate and pair cells to reliably form junctions. The SCAµTT device additionally, through the use of two-photon polymerization (TPP) in fabrication, allowed for highly tunable structural stiffness, ensuring controlled force application to cell pairs. Briefly, cell pairs were positioned onto the SCAµTT device, allowed to adhere and form mature cell-cell junctions, then either exposed to several dosages and dwell times of a variety of PV antibodies or tested as controls. Using a modified atomic force microscopy (AFM) probe tip, the SCAµTT device was then actuated to strain the cell pair a defined distance at a specified strain rate. The applied force was determined by the deflection of a constituent beam of a known stiffness, allowing for the cell pair’s tensile stress to be calculated. Using a custom MATLAB script to perform digital image correlation of tensile test videos, beam deflection, stress, and cell pair strain were determined. Experiments were performed at low (~50%) and high (~200%) strain levels, as well as for cyclical strain cycles. To further explore the underlying mechanisms of the altered mechanical behaviors, intermediate filaments (keratin) and F-actin, were imaged. These proteins were selected due to their key roles in load transfer, cell stiffness, and structural integrity. Briefly, control and antibody treated samples were stained and subjected to similar tensile tests as described previously. Cell pairs during tests were fluorescently imaged using a confocal microscope. Analysis of images was then performed to quantify changes in fluorescent intensity and organization. Our experiments showed that mechanical behavior at low-strain conditions was not significantly altered in the presence of a wide array of PV antibodies. This finding suggests that desmosomal junctions do not take on an impactful role at these strain magnitudes. In the presence of high strain, however, it was found that exposure to the PV antibodies resulted in stiffer cell pairs, increased stress levels, and earlier rupture as compared to controls. Fluorescent image analysis revealed that cytoskeletal remodeling is hindered in the presence of the antibodies, thus suggesting a more vital role of the desmosome-keratin network at larger strain magnitudes. In sum, the presented study provides evidence for desmosomal disruption propagated by PV antibodies alters cell-cell junction mechanics towards a more fragile and stiffer behavior in the presence of tensile stress.

Presenting Author: Timothy Goldsmith Institute of Quantitative Health Science and Engineering, Michigan State University

Presenting Author Biography: Timothy graduated from the University of Nebraska-Lincoln with a Bachelor's of Science in Mechanical Engineering in 2024. He is pursuing a PhD degree in Biomedical Engineering at Michigan State University.

Authors:

Timothy Goldsmith Institute of Quantitative Health Science and Engineering, Michigan State University
Amir Ostadi Moghaddam Institute of Quantitative Health Science and Engineering, Michigan State University
Bahareh Tajvidi Safa Institute of Quantitative Health Science and Engineering, Michigan State University
Jordan Rosenbohm Department of Mechanical and Materials Engineering, University of Nebraska-Lincoln
Xiaowei Jin Department of Mechanical and Materials Engineering, University of Nebraska-Lincoln
Wei Ting Tan Institute of Quantitative Health Science and Engineering, Michigan State University
Kristina Seiffert-Sinha Department of Dermatology, University at Buffalo
Merced Leiker Department of Dermatology, University at Buffalo
Animesh A. Sinha Department of Dermatology, University at Buffalo
Ruiguo Yang Institute of Quantitative Health Science and Engineering, Michigan State University