Biology subjects

Ainslie, C. M.

Publications and source records attributed to Ainslie, C. M..

3 recordsLinked to original sources

bioRxiv2026

N-cadherin orientational order decreases with mechanical load at cardiomyocyte adherens junctions

Adherens junctions physically connect neighboring cells and are built around classical cadherins, homophilic transmembrane proteins that link to the actin cytoskeleton. Classical cadherins can organize into ordered arrays in vitro, but whether they do so in cells remains to be established. Here, we use fluorescence polarization microscopy to show that the classical cadherin N-cadherin is orientationally ordered at cardiomyocyte cell-cell junctions. Whereas the desmosomal cadherin desmoglein 2 was similarly ordered across junction types, N-cadherin order was spatially heterogeneous. Order was lowest where organized myofibrils terminate at high-load, vinculin-enriched axial junctions and highest at low-load, vinculin-poor lateral junctions. This inverse relationship between order and mechanical load suggests that robust cadherin-mediated adhesion does not require ectodomain order. Our findings provide evidence that a classical cadherin is orientationally ordered in cells and show that mechanically active adhesions adopt distinct organizational strategies according to local mechanical demands. Summary StatementAt cardiomyocyte junctions, N-cadherin is ordered where mechanical load is low but disordered where load is high, suggesting that cadherin organization adapts to local force conditions.

cell biology↗

bioRxiv2025

Super-Resolution Imaging Reveals Stretch-Induced Architectural Rearrangement of Desmoplakin in Desmosomes

Desmosomes (DSMs) are intercellular junctions essential for providing mechanical resilience to tissues, particularly the epidermis. Desmoplakin (DP) is a key DSM protein which anchors plaque proteins to keratins, thereby ensuring tissue integrity under mechanical stress. Clinically, DP mutations impair keratinocyte adhesion and structural integrity, leading to skin fragility disorders. However, how mechanical forces influence DSM architecture is poorly understood. We hypothesized that physiological stretch could alter DP architecture in DSMs. To test this, we subjected normal human epidermal keratinocytes (NHEKs) and DP-knockout human keratinocytes expressing either DPI-mEGFP, DP1a-mEGFP, or DP2-mEGFP to mechanical stretch using the Flexcell system (13% uniaxial strain for 30 minutes). Direct stochastic optical reconstruction microscopy (dSTORM) was used to visualize DP architecture with 20 nm resolution. We found mechanical stretch significantly increased the distance between DP cytoplasmic tails compared to static controls across all cell lines. In contrast, there was no significant change in the N-terminal head domain under stretch, highlighting the tail domain as the primary site of mechanical adaptation. This work enhances our understanding of how DSMs and DP isoforms respond to biomechanical forces, revealing that the C-term of DP undergoes a strain-induced conformational shift, reorganizing the DSM architecture in response to physiological stress. Ultimately, elucidating the spatial and biomechanical behavior of DP will deepen our understanding of its contribution to dermatological health and disease.

cell biology↗

bioRxiv2025

Desmoplakin tail domain position in the desmosomal plaque is isoform dependent

Desmoplakin (DP) is the anchoring subunit of desmosomes, macromolecular junctions that provide mechanical integrity to the skin and heart. DP has three isoforms, DPI, DPIa, and DPII that arise from alternative splicing. The isoforms are structurally identical excluding the length of their central rod domain. As desmosomes are macromolecular complexes, the precise arrangement of their component proteins, or architecture, is essential to maintain physiological function. Alterations of the tissue-specific expression of DP isoforms underlies rare human diseases impacting the skin and heart. Overall DP is oriented with its head domain closest to the plasma membrane and tail domain extending into the cytosol. However, the differences in the architecture of the DP isoforms within the desmosomal plaque remains unknown. Here, we sought to define the architectural arrangement of each DP isoform. To address this, we utilized direct stochastic optical reconstruction microscopy (dSTORM) and analysis of DP KO HaCaT cells stably expressing DPI, DPIa, or DPII with a C-terminal mEGFP tag. Our results show the DP head domain position in the desmosomal plaque is isoform independent and the DP tail domain position correlates with rod length. The tail domain of DPI, the isoform with the longest rod, is furthest from the plasma membrane and that of DPII, the isoform with the shortest rod, is closest. We propose a variable tail location model to describe the architectural arrangement of each isoform. In this model, the DP isoforms are arranged with their rod domains parallel at an angle between 21{degrees} to 25{degrees} from the plasma membrane. These results provide valuable insight into the role of DP isoforms in desmosomal architecture and function.

cell biology↗