Biology subjects

Kowalczyk, A. P.

Publications and source records attributed to Kowalczyk, A. P..

10 recordsLinked to original sources

bioRxiv2026

The Desmoplakin Hinge Domain Confers a Reversible Conformational Change in Response to Acute External Force

Desmosomes provide mechanical integrity to tissues that experience strain, yet how mechanical force is sensed and distributed within the desmosomal plaque remains poorly understood. Here we identify the hinge within the desmoplakin (DP) head domain is required for repositioning the DP tail in response to acute uniaxial mechanical stretch. This repositioning occurs within 30 seconds of applied stretch and reverses with release, indicating a dynamic reversible switch rather than a stable structural transition. We show this hinge-dependent repositioning correlates with the propagation of mechanical force to the desmosomal cadherins, consistent with the hinge's role in buffering tension at the cadherin interface during mechanical loading. Deletion or shortening of the hinge abolishes the stretch-induced response and compromises desmosome mechanical integrity. The repositioning of the DP tail in the calcium-dependent to hyper-adhesive transition was not impacted by alterations to the hinge, demonstrating that DP repositioning due to acute force or adhesive state is mechanistically distinct. Together, these findings define a reversible, hinge-dependent mechanotransduction pathway through which DP senses and relays mechanical force within the desmosome. This mechanism could act as a mechanical buffer by reducing the impulse of forces on the cadherins, and play a central role in mechanical resilience.

cell biology↗

bioRxiv2026

Adapting Upright Light Sheet Fluorescence Microscopy for Imaging at Air-Liquid Interface

Light sheet fluorescence microscopy (LSFM) is increasingly appreciated as the gold standard for gentle, volumetric imaging with fast acquisition speeds and/or long imaging durations. However, the often-constrained sample space of these microscopes has precluded a specific class of biological specimens from being studied with these tools: those requiring an air-liquid interface (ALI). Here, we present a device for robust imaging at ALI on an upright light sheet microscope with dipping objectives. We demonstrate the system using three relevant use-cases: ex vivo embryonic mouse salivary glands, human epidermal equivalent cultures, and in vivo adult Drosophila melanogaster brains. While the device presented is engineered for one specific light sheet microscope design, it provides a blueprint for easy adaptation to other systems. In doing so, it can potentially spur the use of LSFM for model systems that have so far been unable to take advantage of this powerful technology.

bioengineering↗

bioRxiv2026

Volume EM reveals three-dimensional architecture of the desmosome in epithelial cells and tissue models

Desmosomes are a type of cell-cell adhesive junction present in cardiac tissue and epithelial tissues such as the epidermis. These intercellular junctions anchor to the intermediate filament cytoskeleton, providing mechanical integrity to the tissues in which they reside. Our understanding of desmosome architecture has largely been influenced by observations of two-dimensional images obtained through conventional electron microscopy. Here, using focused ion beam scanning electron microscopy, we report the three-dimensional ultrastructure of desmosomes in A431 and S1 human mammary epithelial cells. We also reveal differences in desmosome ultrastructure at homo- and heterotypic junctions of human nasal airway epithelial cells. Quantitative analyses of these volume EM datasets reveal variations in desmosome size, shape, and organization. Importantly, we report the presence of discontinuities or "holes" within the desmosome outer dense plaque, a novel feature that is observed in either one or both halves of a desmosome. This study provides the first comprehensive description of the epithelial desmosome as a three-dimensional structure, and emphasizes the need to investigate the effects of dynamic morphogenetic processes and disease states on desmosome ultrastructure.

cell biology↗

bioRxiv2025

Cadherin Regulation of Endoplasmic Reticulum-Plasma Membrane Contact Sites

The spatial organization and dynamics of the endoplasmic reticulum (ER) govern when and where ER tubules engage with other organelles and the plasma membrane. We previously found that ER tubules are closely associated with desmosomes, but the mechanisms of ER recruitment to these adhesive intercellular junctions were unclear. Here, we demonstrate that adherens junctions recruit ER tubules to intercellular junctions in a manner dependent upon E-cadherin association with -catenin and vinculin. During cell-cell junction assembly, adherens junctions and ER appear nearly simultaneously at nascent cell-cell contacts, followed by desmosome formation. ER recruitment to cell-cell contacts allows the formation of ER-plasma membrane contact sites (ER-PMCS) and the assembly of a complex comprising adherens junctions, ER-PMCS, and desmosomes. Ablating adherens junctions disrupts this tripartite complex and perturbs global cellular lipid levels. Collectively, our findings identify cadherins as key organizers of ER-PMCS positioning and suggest that this complex integrates cellular mechanical elements with plasma membrane homeostasis.

cell biology↗

bioRxiv2024

Pemphigus vulgaris autoantibodies induce an ER stress response

Desmosomes are intercellular junctions that mediate cell-cell adhesion and are essential for maintaining tissue integrity. Pemphigus vulgaris (PV) is an autoimmune epidermal blistering disease caused by autoantibodies (IgG) targeting desmoglein 3 (Dsg3), a desmosomal cadherin. PV autoantibodies cause desmosome disassembly and loss of cell-cell adhesion, but the molecular signaling pathways that regulate these processes are not fully understood. Using high- resolution time-lapse imaging of live keratinocytes, we found that ER tubules make frequent and persistent contacts with internalizing Dsg3 puncta in keratinocytes treated with PV patient IgG. Biochemical experiments demonstrated that PV IgG activated ER stress signaling pathways, including both IRE1 and PERK pathways, in cultured keratinocytes. Further, ER stress transcripts were upregulated in PV patient skin. Pharmacological inhibition of ER stress protected against PV IgG-induced desmosome disruption and loss of keratinocyte cell-cell adhesion, suggesting that ER stress may be an important pathomechanism and therapeutically targetable pathway for PV treatment. These data support a model in which desmosome adhesion is integrated with ER function to serve as a cell adhesion stress sensor that is activated in blistering skin disease.

cell biology↗

bioRxiv2024

The transmembrane domain of the desmosomal cadherin desmoglein-1 governs lipid raft association to promote desmosome adhesive strength

Cholesterol- and sphingolipid-enriched domains called lipid rafts are hypothesized to selectively coordinate protein complex assembly within the plasma membrane to regulate cellular functions. Desmosomes are mechanically resilient adhesive junctions that associate with lipid raft membrane domains, yet the mechanisms directing raft association of the desmosomal proteins, particularly the transmembrane desmosomal cadherins, are poorly understood. We identified the desmoglein-1 (DSG1) transmembrane domain (TMD) as a key determinant of desmoglein lipid raft association and designed a panel of DSG1TMD variants to assess the contribution of TMD physicochemical properties (length, bulkiness, and palmitoylation) to DSG1 lipid raft association. Sucrose gradient fractionations revealed that TMD length and bulkiness, but not palmitoylation, govern DSG1 lipid raft association. Further, DSG1 raft association determines plakoglobin recruitment to raft domains. Super-resolution imaging and functional assays uncovered a strong relationship between the efficiency of DSG1TMD lipid raft association and the formation of morphologically and functionally robust desmosomes. Lipid raft association regulated both desmosome assembly dynamics and DSG1 cell surface stability, indicating that DSG1 lipid raft association is required for both desmosome formation and maintenance. These studies identify the biophysical properties of desmoglein transmembrane domains as key determinants of lipid raft association and desmosome adhesive function.

cell biology↗

bioRxiv2023

MARCH family E3 ubiquitin ligases selectively target and degrade cadherin family proteins

Cadherin family proteins play a central role in epithelial and endothelial cell-cell adhesion. The dynamic regulation of cell adhesion is achieved in part through endocytic membrane trafficking pathways that modulate cadherin cell surface levels. Here, we define the role for various MARCH family ubiquitin ligases in the regulation of cadherin degradation. We find that MARCH2 selectively downregulates VE-cadherin, resulting in loss of adherens junction proteins at cell borders and a loss of endothelial barrier function. Interestingly, N-cadherin is refractory to MARCH ligase expression, demonstrating that different classical cadherin family proteins are differentially regulated by MARCH family ligases. Using chimeric cadherins, we find that the specificity of different MARCH family ligases for different cadherins is conferred by the cadherin transmembrane domain. Further, juxta-membrane lysine residues are required for cadherin degradation by MARCH proteins. These findings expand our understanding of cadherin regulation and highlight a new role for mammalian MARCH family ubiquitin ligases in differentially regulating cadherin turnover.

cell biology↗

bioRxiv2022

Architecture and dynamics of a novel desmosome-endoplasmic reticulum organelle

The endoplasmic reticulum (ER) forms a dynamic network that contacts other cellular membranes to regulate stress responses, calcium signaling, and lipid transfer. Using high-resolution volume electron microscopy, we find that the ER forms a previously unknown association with keratin intermediate filaments and desmosomal cell-cell junctions. Peripheral ER assembles into mirror image-like arrangements at desmosomes and exhibits nanometer proximity to keratin filaments and the desmosome cytoplasmic plaque. ER tubules exhibit stable associations with desmosomes, and perturbation of desmosomes or keratin filaments alters ER organization and mobility. These findings indicate that desmosomes and the keratin cytoskeleton pattern the distribution of the ER network. Overall, this study reveals a previously unknown subcellular architecture defined by the structural integration of ER tubules with an epithelial intercellular junction. One-Sentence SummaryThe desmosome adhesive junction regulates the organization and dynamics of the endoplasmic reticulum network.

cell biology↗

bioRxiv2019

VE-cadherin endocytosis controls vascular integrity and patterning during development

Tissue morphogenesis requires dynamic intercellular contacts that are subsequently stabilized as tissues mature. The mechanisms governing these competing adhesive properties are not fully understood. Using gain- and loss-of-function approaches, we tested the role of p120-catenin (p120) and VE-cadherin (VE-cad) endocytosis in vascular development using mouse mutants that exhibit increased (VE-cadGGG/GGG) or decreased (VE-cadDEE/DEE) internalization. VE-cadGGG/GGG mutant mice exhibited reduced VE-cad-p120 binding, reduced VE-cad levels, microvascular hemorrhaging, and decreased survival. By contrast, VE-cadDEE/DEE mutants exhibited normal vascular permeability but displayed microvascular patterning defects. Interestingly, VE-cadDEE/DEE mutant mice did not require endothelial p120, demonstrating that p120 is dispensable in the context of a stabilized cadherin. In vitro, VE-cadDEE mutant cells displayed defects in polarization and cell migration that were rescued by uncoupling VE-cadDEE from actin. These results indicate that cadherin endocytosis coordinates cell polarity and migration cues through actin remodeling. Collectively, our results indicate that regulated cadherin endocytosis is essential for both dynamic cell movements and establishment of stable tissue architecture.\n\nSummary StatementThis study uses mouse genetic and in vitro approaches to demonstrate that cadherin endocytosis is critical for the formation of blood vessels during development by promoting actin-dependent collective cell migration, whereas the inhibition of this endocytosis by p120 binding is essential for vessel stabilization.

cell biology↗

bioRxiv2018

The Desmosome is a Mesoscale Lipid Raft-Like Membrane Domain

Desmogleins are cadherin family adhesion molecules essential for epidermal integrity. Previous studies have shown that desmogleins associate with lipid rafts, but the significance of this association was not clear. Here, we report that the desmoglein transmembrane domain (TMD) is the primary determinant of raft association. Further, we identify a novel mutation in the DSG1 TMD (G562R) that causes severe dermatitis, multiple allergies, and metabolic wasting (SAM) syndrome. Molecular modeling predicts that this G to R mutation shortens the DSG1 TMD, and experiments directly demonstrate that this mutation compromises both lipid raft association and desmosome incorporation. Finally, cryo-electron tomography (cryo-ET) indicates that the lipid bilayer within the desmosome is ~10% thicker than adjacent regions of the plasma membrane. These findings suggest that differences in bilayer thickness influence the organization of adhesion molecules within the epithelial plasma membrane, with cadherin TMDs recruited to the desmosome via establishment of a specialized mesoscale lipid raft-like membrane domain.

cell biology↗