1617-Pos Contribution of desmoplakin isoforms to desmosome architecture Krishna A. Patel, Collin M. Ainslie, Alexa L. Mattheyses. Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL, USA. Desmosomes are cell-cell junctions that maintain the integrity of tissues that experience significant mechanical stress, such as the skin and heart. Desmosomal plaque proteins are connected to the intermediate filaments by desmoplakin (DP), and genetic variants in DP are associated with several skin and heart pathologies, including erythrokeratodermia and arrhythmogenic cardiomyopathy. DP contains three domains: an N-terminal plakin head, a central rod, and an intermediate filament-binding C-terminal tail. Furthermore, DP has three splice isoforms (DPI, DPIa, and DPII) that differ only in the length of the rod domain and have distinct tissue-specific expression patterns. DPI is the primary cardiac isoform and is present in all desmosomes, whereas DPIa and DPII are expressed in stratified epithelia. Although DP’s binding partners and spliced isoforms are known, their spatial arrangement and functional implications within the desmosome remain unclear. The largest isoform, DPI, has a head to tail length of 180 nm. Our previous work suggests that the long axis of DP is arranged not perpendicular to the plasma membrane but at an acute angle. However, it is unclear if the length of the rod domain influences the architecture of DP. In this project, we established individual EGFP-tagged isoforms (DPI-mEGFP, DPIa-mEGFP, and DPII-mEGFP) in wild-type and DP-knockout human epidermal keratinocytes (HaCaTs) containing varying DP rod domain lengths. In these stable cell lines, DP architecture will be quantified by super-resolution STORM microscopy, and desmosome adhesive strength will be evaluated by dispase assay. Together, this work sets the stage to understand how DP structure guides desmosome function through the regulation of junction strength by differentially expressed desmoplakin isoforms.

1618-Pos A correlative quantitative phase contrast and fluorescence superresolution microscope for imaging molecules in their cellular context Yujin Bao1, Zach Marin1, Chuyue Zhang1, Karla M. Neugebauer1, David Baddeley2, Michael Shribak3, Joerg Bewersdorf1. 1Yale School of Medicine, New Haven, CT, USA, 2Auckland Bioengineering Institute, The University of Auckland, Auckland, New Zealand, 3Marine Biological Laboratory, Woods Hole, MA, USA. Fluorescence super-resolution microscopy (SRM), such as single-molecule localization microscopy (SMLM), is widely used in cell biology. Its power lies in the capability to localize specific molecules of interest at nanoscale precision. However, in exchange for high specificity, SRM is blind to the cellular context the molecules are embedded in. To address this disadvantage, we integrated label-free orientation-independent differential interference contrast (OI-DIC) microscopy, a powerful quantitative phase contrast method, into a SMLM instrument. This added imaging modality allows users to precisely quantify global cellular composition from the refractive index differences at diffraction-limited resolution. In our presentation, we describe the technical realization of our correlative OI-DIC/SMLM instrument and demonstrate that it enables both three-dimensional super-resolution imaging and the visualization of quantitative cellular context with high sensitivity and contrast. We characterize the two imaging modalities using beads and DNA origami and show correlative images of microtubules in COS-7 cells and other applications. Our correlative …