Mechanotransduction through hemidesmosomes during aging and longevity.

Hemidesmosomes are structural protein complexes localized at the interface of tissues with high mechanical demand and shear forces. Beyond tissue anchoring, hemidesmosomes have emerged as force-modulating structures important for translating mechanical cues into biochemical and transcriptional adaptation (i.e. mechanotransduction) across tissues. Here, we discuss the recent insights into the roles of hemidesmosomes in age-related tissue regeneration and aging in C. elegans, mice and humans. We highlight the emerging concept of preserved dynamic mechanoregulation of hemidesmosomes in tissue maintenance and healthy aging.

Diagnostic and prognostic role of protein and ultrastructural alterations at cell-extracellular matrix junctions in neoplastic progression of human oral malignancy.

Despite advancements in technology and increase in favorable outcomes associated with oral cancer, early detection remains the most significant factor in limiting mortality. The current study aimed to develop early diagnostic and prognostic markers for oral tumorigenesis. Protein and ultrastructural alterations at cell-extracellular matrix (ECM) adhesion junctions were examined concurrently using immunohistochemistry (IHC) and transmission electron microscopy (TEM) on progressive grade of oral carcinomas (n = 285). The expression of hemidesmosome (HD) proteins-integrin ß4, BP180, and laminin-5 increased in hyperplasia as compared to normal, and significantly increased further, as the disease progressed. TEM analysis in parallel tissues revealed a significant decrease in HD number and increase in the length of basal lamina (BL) in hyperplasia. With cancer progression, the severity of ultrastructural alterations increased gradually and significantly. Overexpression of HD proteins, decrease in HD number and increase in BL length significantly correlated with nodal metastasis, local recurrence, and recurrence-free survival of patients. Concurrent use of IHC and TEM can add value to early recognition of neoplastic changes in primary carcinomas of oral cavity. In this regard, altered expression of integrin ß4 and laminin-5, loss of HDs, and increased BL length could offer criteria for early diagnosis and prognosis of oral malignancy.

Post-translational modifications to hemidesmosomes in human airway epithelial cells following diacetyl exposure.

Diacetyl (DA; 2,3-butanedione) is a highly reactive alpha (α)-diketone. Inhalation exposure to DA can cause significant airway epithelial cell injury, however, the mechanisms of toxicity remain poorly understood. The purpose of these experiments was to assess for changes in abundance and distribution of hemidesmosome-associated proteins following DA exposure that contribute to DA-induced epithelial toxicity. Human bronchial epithelial cells were grown in submerged cultures and exposed to three occupationally-relevant concentrations of DA (5.7, 8.6, or 11.4 mM) for 1 h. Following DA exposure, epithelial cells were cultured for 4 days to monitor for cell viability by MTT and WST-1 assays as well as for changes in cellular distribution and relative abundance of multiple hemidesmosome-associated proteins, including keratin 5 (KRT5), plectin (PLEC), integrin alpha 6 (ITGα6) and integrin beta 4 (ITGß4). Significant toxicity developed in airway epithelial cells exposed to DA at concentrations ≥ 8.6 mM. DA exposure resulted in post-translational modifications to hemidesmosome-associated proteins with KRT5 crosslinking and ITGß4 cleavage. Following DA exposure at 5.7 mM, these post-translational modifications to KRT5 resolved with time. Conversely, at DA concentrations ≥ 8.6 mM, modifications to KRT5 persisted in culture with decreased total abundance and perinuclear aggregation of hemidesmosome-associated proteins. Significant post-translational modifications to hemidesmosome-associated proteins develop in airway epithelial cells exposed to DA. At DA concentrations ≥ 8.6 mM, these hemidesmosome modifications persist in culture. Future work targeting hemidesmosome-associated protein modifications may prevent the development of lung disease following DA exposure.

Pancreatic ductal adenocarcinoma cells employ integrin α6ß4 to form hemidesmosomes and regulate cell proliferation.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis due to its aggressive progression, late detection and lack of druggable driver mutations, which often combine to result in unsuitability for surgical intervention. Together with activating mutations of the small GTPase KRas, which are found in over 90% of PDAC tumours, a contributory factor for PDAC tumour progression is formation of a rigid extracellular matrix (ECM) and associated desmoplasia. This response leads to aberrant integrin signalling, and accelerated proliferation and invasion. To identify the integrin adhesion systems that operate in PDAC, we analysed a range of pancreatic ductal epithelial cell models using 2D, 3D and organoid culture systems. Proteomic analysis of isolated integrin receptor complexes from human pancreatic ductal epithelial (HPDE) cells predominantly identified integrin α6ß4 and hemidesmosome components, rather than classical focal adhesion components. Electron microscopy, together with immunofluorescence, confirmed the formation of hemidesmosomes by HPDE cells, both in 2D and 3D culture systems. Similar results were obtained for the human PDAC cell line, SUIT-2. Analysis of HPDE cell secreted proteins and cell-derived matrices (CDM) demonstrated that HPDE cells secrete a range of laminin subunits and form a hemidesmosome-specific, laminin 332-enriched ECM. Expression of mutant KRas (G12V) did not affect hemidesmosome composition or formation by HPDE cells. Cell-ECM contacts formed by mouse and human PDAC organoids were also assessed by electron microscopy. Organoids generated from both the PDAC KPC mouse model and human patient-derived PDAC tissue displayed features of acinar-ductal cell polarity, and hemidesmosomes were visible proximal to prominent basement membranes. Furthermore, electron microscopy identified hemidesmosomes in normal human pancreas. Depletion of integrin ß4 reduced cell proliferation in both SUIT-2 and HPDE cells, reduced the number of SUIT-2 cells in S-phase, and induced G1 cell cycle arrest, suggesting a requirement for α6ß4-mediated adhesion for cell cycle progression and growth. Taken together, these data suggest that laminin-binding adhesion mechanisms in general, and hemidesmosome-mediated adhesion in particular, may be under-appreciated in the context of PDAC. Proteomic data are available via ProteomeXchange with the identifiers PXD027803, PXD027823 and PXD027827.

Wnt/ß-Catenin Signaling Stabilizes Hemidesmosomes in Keratinocytes.

Hemidesmosomes (HDs) are adhesion complexes that promote epithelial-stromal attachment in stratified and complex epithelia, including the epidermis. In various biological processes, such as differentiation and migration of epidermal keratinocytes during wound healing or carcinoma invasion, quick assembly and disassembly of HDs are prerequisites. In this study, we show that inhibition of Wnt/ß-catenin signaling disturbs HD organization in keratinocytes. Screening with inhibitors identified the depletion of HD components and HD-like structures through Wnt inhibition, but keratinocyte differentiation was not affected. Wnt inhibition significantly diminished plectin and type XVII collagen expression in the basal side of Wnt-inhibited cells and the dermo-epidermal junction of the Wnt-inactive murine basal epidermis. Similar to Wnt inhibition, PLEC-knockout cells or cells with plectin-type XVII collagen binding defects showed type XVII collagen reduction in the basal side of the cells, implying the possible involvement of Wnt/ß-catenin signaling in HD assembly. Atypical protein kinase C inhibition ameliorated the phenotypes of Wnt-inhibited cells. These findings show that Wnt/ß-catenin signaling regulates the localization of HD components in keratinocytes and that the atypical protein kinase C pathway is involved in Wnt inhibition‒induced HD disarrangement. Our study suggests that the Wnt signaling pathway could be a potential therapeutic target for treating HD-defective diseases, such as epidermolysis bullosa.

BP180/Collagen XVII: A Molecular View.

BP180 is a type II collagenous transmembrane protein and is best known as the major autoantigen in the blistering skin disease bullous pemphigoid (BP). The BP180 trimer is a central component in type I hemidesmosomes (HD), which cause the adhesion between epidermal keratinocytes and the basal lamina, but BP180 is also expressed in several non-HD locations, where its functions are poorly characterized. The immunological roles of intact and proteolytically processed BP180, relevant in BP, have been subject to intensive research, but novel functions in cell proliferation, differentiation, and aging have also recently been described. To better understand the multiple physiological functions of BP180, the focus should return to the protein itself. Here, we comprehensively review the properties of the BP180 molecule, present new data on the biochemical features of its intracellular domain, and discuss their significance with regard to BP180 folding and protein-protein interactions.

Hemidesmosome-Related Keratin Filament Bundling and Nucleation.

The epithelial cytoskeleton encompasses actin filaments, microtubules, and keratin intermediate filaments. They are interconnected and attached to the extracellular matrix via focal adhesions and hemidesmosomes. To study their interplay, we inhibited actin and tubulin polymerization in the human keratinocyte cell line HaCaT by latrunculin B and nocodazole, respectively. Using immunocytochemistry and time-lapse imaging of living cells, we found that inhibition of actin and tubulin polymerization alone or in combination induced keratin network re-organization albeit differently in each situation. Keratin filament network retraction towards the nucleus and formation of bundled and radial keratin filaments was most pronounced in latrunculin-B treated cells but less in doubly-treated cells and not detectable in the presence of nocodazole alone. Hemidesmosomal keratin filament anchorage was maintained in each instance, whereas focal adhesions were disassembled in the absence of actin filaments. Simultaneous inhibition of actin and tubulin polymerization, therefore, allowed us to dissect hemidesmosome-specific functions for keratin network properties. These included not only anchorage of keratin filament bundles but also nucleation of keratin filaments, which was also observed in migrating cells. The findings highlight the fundamental role of hemidesmosomal adhesion for keratin network formation and organization independent of other cytoskeletal filaments pointing to a unique mechanobiological function.

Efficacy of Long-term Use of 0.3% Sodium Hyaluronate Eye Drops for Traumatic Corneal Abrasion: A Randomized Controlled, Pilot Trial.

PURPOSE: Traumatic corneal abrasion (TCA) causes damage to both corneal epithelium and the underlying hemidesmosomal junctions. Delayed recovery of hemidesmosomal junctions causes symptomatic episodes. However, there is no recommended treatment for recovery of hemidesmosomal junctions, indicating that a blank period exists in TCA treatment. In this study, the efficacy of long-term use of sodium hyaluronate on recovery of hemidesmosomal junctions during the blank period in TCA healing was investigated. METHODS: In this prospective, randomized control pilot study, 60 patients with TCA were enrolled. The patients were randomized 1:1 to receive 0.3% sodium hyaluronate eye drops for 3 months (HA group) or observation alone (control group) after complete corneal epithelium recovery. The primary and secondary outcomes were the cumulative incidence of major and minor symptomatic episodes during a 12-month follow-up, respectively. RESULTS: Fifty-six subjects (29 in the HA group and 27 in the control group) completed the 12-month follow-up. The 12-month cumulative incidence rate of major symptomatic episodes was 20.7% in the HA group and 18.5% in the control group. No significant difference was found between the 2 groups (P = 0.838). The 12-month cumulative incidence rate of minor symptomatic episodes was 48.3% and 37.0% in the HA and control groups, respectively, with no significant difference (P = 0.397). CONCLUSIONS: Approximately one-fifth of patients with TCA experience major symptomatic episodes again within their 1-year follow-up. Long-term use of sodium hyaluronate in the period of recovery of hemidesmosomal junctions has no benefit to it.

A Hemidesmosome-to-Cytoplasm Translocation of Small Heat Shock Proteins Provides Immediate Protection against Heat Stress.

Small heat shock proteins (sHSPs) are important regulators for maintaining protein homeostasis in response to stresses. However, the strategies used by constitutively expressed sHSPs to control their activities in normal versus stressed conditions are still not fully understood. Here we show that the constitutively expressed HSP-43 in the C. elegans epidermis is stored within the basal C. elegans hemidesmosomes (CeHDs) under normal conditions and is rapidly released into the cytoplasm to exert protective functions upon heat stress. The association with CeHDs protects HSP-43 from degradation or toxic cytoplasmic aggregation in unstressed situations. Our study reveals a rapid and specific translocation-based heat shock response of the sHSPs working through hemidesmosomes. It refreshes our knowledge about the stress-resistant functions of stable cellular adhesions and provides insight into the activity-control strategies of sHSPs. It also underlines the importance of structural integrity of the cells on stress resistance and damage control.

Comparative interactomics analysis reveals potential regulators of α6ß4 distribution in keratinocytes.

The integrin α6ß4 and cytoskeletal adaptor plectin are essential components of type I and type II hemidesmosomes (HDs). We recently identified an alternative type II HD adhesion complex that also contains CD151 and the integrin α3ß1. Here, we have taken a BioID proximity labeling approach to define the proximity protein environment for α6ß4 in keratinocytes. We identified 37 proteins that interacted with both α6 and ß4, while 20 and 78 proteins specifically interacted with the α6 and ß4 subunits, respectively. Many of the proximity interactors of α6ß4 are components of focal adhesions (FAs) and the cortical microtubule stabilizing complex (CMSC). Though the close association of CMSCs with α6ß4 in HDs was confirmed by immunofluorescence analysis, CMSCs have no role in the assembly of HDs. Analysis of the ß4 interactome in the presence or absence of CD151 revealed that they are strikingly similar; only 11 different interactors were identified. One of these was the integrin α3ß1, which interacted with α6ß4 more strongly in the presence of CD151 than in its absence. These findings indicate that CD151 does not significantly contribute to the interactome of α6ß4, but suggest a role of CD151 in linking α3ß1 and α6ß4 together in tetraspanin adhesion structures.

The intracellular domain of BP180/collagen XVII is intrinsically disordered and partially folds in an anionic membrane lipid-mimicking environment.

The trimeric transmembrane collagen BP180, also known as collagen XVII, is an essential component of hemidesmosomes at the dermal-epidermal junction and connects the cytoplasmic keratin network to the extracellular basement membrane. Dysfunction of BP180 caused by mutations in patients with junctional epidermolysis bullosa or autoantibodies in those with bullous pemphigoid leads to severe skin blistering. The extracellular collagenous domain of BP180 participates in the protein's triple-helical folding, but the structure and functional importance of the intracellular domain (ICD) of BP180 are largely unknown. In the present study, we purified and characterized human BP180 ICD. When expressed in Escherichia coli as glutathione-S-transferase or 6 × histidine tagged fusion protein, the BP180 ICD was found to exist as a monomer. Analysis of the secondary structure content by circular dichroism spectroscopy revealed that the domain is intrinsically disordered. This finding aligned with that of a bioinformatic analysis, which predicted a disordered structure. Interestingly, both anionic detergent micelles and lipid vesicles induced partial folding of the BP180 ICD, suggesting that in its natural environment, the domain's folding and unfolding may be regulated by interaction with the cell membrane or accompanying proteins. We hypothesize that the intrinsically disordered structure of the ICD of BP180 contributes to the mechanism that allows the remodeling of hemidesmosome assembly.

Role of BP230 autoantibodies in bullous pemphigoid.

Bullous pemphigoid (BP) is an autoimmune disease associated with subepidermal blistering due to autoantibodies directed against BP180 and BP230. BP180 is currently considered as the major pathogenic autoantigen. However, previous clinical findings suggested that anti-BP230 autoantibodies alone can cause skin lesions in animal models and many BP patients. The characteristics of BP230 and the pathogenic roles of anti-BP230 antibodies have been proposed. First, at the molecular level, BP230 mediates the attachment of keratin intermediate filaments to the hemidesmosomal plaque and interacts with other constituents of hemidesmosomes. Second, the presence of BP230 autoantibodies may correlate with specific clinical features of BP. The immunoglobulin (Ig)G autoantibodies from BP patients react mainly against the C-terminus of BP230, while the IgE autoantibodies are still inconclusive. Third, in vivo, autoantibodies against BP230 involved in the disease may not only induce the inflammatory response but also impair the structural stability of hemidesmosomes. This article reviews recently published work about the role of BP230 and its antibodies, including IgG and IgE, aiming to find clues of its clinical association and lay the foundation for the research on the pathogenicity of antibodies against BP230.

Epidermal control of axonal attachment via ß-spectrin and the GTPase-activating protein TBC-10 prevents axonal degeneration.

Neurons are subjected to strain due to body movement and their location within organs and tissues. However, how they withstand these forces over the lifetime of an organism is still poorly understood. Here, focusing on touch receptor neuron-epidermis interactions using Caenorhabditis elegans as a model system, we show that UNC-70/ß-spectrin and TBC-10, a conserved GTPase-activating protein, function non-cell-autonomously within the epidermis to dynamically maintain attachment of the axon. We reveal that, in response to strain, UNC-70/ß-spectrin and TBC-10 stabilize trans-epidermal hemidesmosome attachment structures which otherwise become lost, causing axonal breakage and degeneration. Furthermore, we show that TBC-10 regulates axonal attachment and maintenance by inactivating RAB-35, and reveal functional conservation of these molecules with their vertebrate orthologs. Finally, we demonstrate that ß-spectrin functions in this context non-cell-autonomously. We propose a model in which mechanically resistant epidermal attachment structures are maintained by UNC-70/ß-spectrin and TBC-10 during movement, preventing axonal detachment and degeneration.

Keratin intermediate filaments: intermediaries of epithelial cell migration.

Migration of epithelial cells is fundamental to multiple developmental processes, epithelial tissue morphogenesis and maintenance, wound healing and metastasis. While migrating epithelial cells utilize the basic acto-myosin based machinery as do other non-epithelial cells, they are distinguished by their copious keratin intermediate filament (KF) cytoskeleton, which comprises differentially expressed members of two large multigene families and presents highly complex patterns of post-translational modification. We will discuss how the unique mechanophysical and biochemical properties conferred by the different keratin isotypes and their modifications serve as finely tunable modulators of epithelial cell migration. We will furthermore argue that KFs together with their associated desmosomal cell-cell junctions and hemidesmosomal cell-extracellular matrix (ECM) adhesions serve as important counterbalances to the contractile acto-myosin apparatus either allowing and optimizing directed cell migration or preventing it. The differential keratin expression in leaders and followers of collectively migrating epithelial cell sheets provides a compelling example of isotype-specific keratin functions. Taken together, we conclude that the expression levels and specific combination of keratins impinge on cell migration by conferring biomechanical properties on any given epithelial cell affecting cytoplasmic viscoelasticity and adhesion to neighboring cells and the ECM.

Tetraspanin CD151 and integrin α3ß1 contribute to the stabilization of integrin α6ß4-containing cell-matrix adhesions.

Tetraspanin CD151 has been suggested to regulate cell adhesion through its association with laminin-binding integrins α3ß1 and α6ß4; however, its precise function in keratinocyte adhesion remains elusive. In this study, we investigated the role of CD151 in the formation and maintenance of laminin-associated adhesions. We show that CD151, through binding to integrin α3ß1, plays a critical role in the stabilization of an adhesion structure with a distinct molecular composition of hemidesmosomes with tetraspanin features. These hybrid cell-matrix adhesions, which are formed early during cell adhesion and spreading and at later stages of cell spreading, are present in the central region of the cells. They contain the CD151-α3ß1/α6ß4 integrin complexes and the cytoskeletal linker protein plectin, but are not anchored to the keratin filaments. In contrast, hemidesmosomes, keratin filament-associated adhesions that contain integrin α6ß4, plectin, BP180 (encoded by COL17A1) and BP230 (encoded by DST), do not require CD151 for their formation or maintenance. These findings provide new insights into the dynamic and complex regulation of adhesion structures in keratinocytes and the pathogenic mechanisms underlying skin blistering diseases caused by mutations in the gene for CD151.

Hemidesmosomes and Focal Adhesions Treadmill as Separate but Linked Entities during Keratinocyte Migration.

Hemidesmosomes anchor the epidermal keratin filament cytoskeleton to the extracellular matrix. They are crucial for the mechanical integrity of skin. Their role in keratinocyte migration, however, remains unclear. Examining migrating primary human keratinocytes, we find that hemidesmosomes cluster as ordered arrays consisting of multiple chevrons that are flanked by actin-associated focal adhesions. These hemidesmosomal arrays with intercalated focal adhesions extend from the cell rear to the cell front. New hemidesmosomal chevrons form subsequent to focal adhesion assembly at the cell's leading front, whereas chevrons and associated focal adhesions disassemble at the cell rear in reverse order. The bulk of the hemidesmosome-focal adhesion composite, however, remains attached to the substratum during cell translocation. Similar hemidesmosome-focal adhesion patterns emerge on X-shaped fibronectin-coated micropatterns, during cell spreading and in leader cells during collective cell migration. We further find that hemidesmosomes and focal adhesions affect each other's distribution. We propose that both junctions are separate but linked entities, which treadmill coordinately to support efficient directed cell migration and cooperate to coordinate the dynamic interplay between the keratin and actin cytoskeleton.

Integrin α6ß4 Recognition of a Linear Motif of Bullous Pemphigoid Antigen BP230 Controls Its Recruitment to Hemidesmosomes.

Mechanical stability of epithelia requires firm attachment to the basement membrane via hemidesmosomes. Dysfunction of hemidesmosomal proteins causes severe skin-blistering diseases. Two plakins, plectin and BP230 (BPAG1e), link the integrin α6ß4 to intermediate filaments in epidermal hemidesmosomes. Here, we show that a linear sequence within the isoform-specific N-terminal region of BP230 binds to the third and fourth FnIII domains of ß4. The crystal structure of the complex and mutagenesis analysis revealed that BP230 binds between the two domains of ß4. BP230 induces closing of the two FnIII domains that are locked in place by an interdomain ionic clasp required for binding. Disruption of BP230-ß4 binding prevents recruitment of BP230 to hemidesmosomes in human keratinocytes, revealing a key role of this interaction for hemidesmosome assembly. Phosphomimetic substitutions in ß4 and BP230 destabilize the complex. Thus, our study provides insights into the architecture of hemidesmosomes and potential mechanisms of regulation.

Integrin α6ß4E variant is associated with actin and CD9 structures and modifies the biophysical properties of cell-cell and cell-extracellular matrix interactions.

Integrin α6ß4 is an essential, dynamic adhesion receptor for laminin 332 found on epithelial cells, required for formation of strong cell-extracellular matrix (ECM) adhesion and induced migration, and coordinated by regions of the ß4C cytoplasmic domain. ß4E, a unique splice variant of ß4 expressed in normal tissue, contains a cytoplasmic domain of 231 amino acids with a unique sequence of 114 amino acids instead of ß4C's canonical 1089 amino acids. We determined the distribution of α6ß4E within normal human glandular epithelium and its regulation and effect on cellular biophysical properties. Canonical α6ß4C expressed in all basal cells, as expected, while α6ß4E expressed within a subset of luminal cells. α6ß4E expression was induced by three-dimensional culture conditions, activated Src, was reversible, and was stabilized by bortezomib, a proteasome inhibitor. α6ß4C expressed in all cells during induced migration, whereas α6ß4E was restricted to a subset of cells with increased kinetics of cell-cell and cell-ECM resistance properties. Interestingly, α6ß4E presented in "ringlike" patterns measuring ∼1.75 × 0.72 microns and containing actin and CD9 at cell-ECM locations. In contrast, α6ß4C expressed only within hemidesmosome-like structures containing BP180. Integrin α6ß4E is an inducible adhesion isoform in normal epithelial cells that can alter biophysical properties of cell-cell and cell-ECM interactions.

Life before and beyond blistering: The role of collagen XVII in epidermal physiology.

Type XVII collagen (COL17) is a transmembranous protein that is mainly expressed in the epidermal basal keratinocytes. Epidermal-dermal attachment requires COL17 expression at the hemidesmosomes of the epidermal basement membrane zone because congenital COL17 deficiency leads to junctional epidermolysis bullosa and acquired autoimmunity to COL17 induces bullous pemphigoid. Recently, in addition to facilitating epidermal-dermal attachment, COL17 has been reported to serve as a niche for hair follicle stem cells, to regulate proliferation in the interfollicular epidermis and to be present along the non-hemidesmosomal plasma membrane of epidermal basal keratinocytes. This review focuses on the physiological properties of COL17 in the epidermis, its role in maintaining stem cells and its association with signalling pathways. We propose possible solutions to unanswered questions in this field.

CCAR-1 affects hemidesmosome biogenesis by regulating unc-52/perlecan alternative splicing in the C. elegans epidermis.

Hemidesmosomes are epithelial-specific attachment structures that maintain tissue integrity and resist tension. Despite their importance, how hemidesmosomes are regulated at the post-transcriptional level is poorly understood. Caenorhabditiselegans hemidesmosomes (CeHDs) have a similar structure and composition to their mammalian counterparts, making C. elegans an ideal model for studying hemidesmosomes. Here, we focus on the transcription regulator CCAR-1, identified in a previous genetic screen searching for enhancers of mutations in the conserved hemidesmosome component VAB-10A (known as plectin in mammals). Loss of CCAR-1 function in a vab-10(e698) background results in CeHD disruption and muscle detachment from the epidermis. CCAR-1 regulates CeHD biogenesis, not by controlling the transcription of CeHD-related genes, but by affecting the alternative splicing of unc-52 (known as perlecan or HSPG2 in mammals), the predicted basement extracellular matrix (ECM) ligand of CeHDs. CCAR-1 physically interacts with HRP-2 (hnRNPR in mammals), a splicing factor known to mediate unc-52 alternative splicing to control the proportions of different UNC-52 isoforms and stabilize CeHDs. Our discovery underlines the importance of post-transcriptional regulation in hemidesmosome reorganization. It also uncovers previously unappreciated roles of CCAR-1 in alternative splicing and hemidesmosome biogenesis, shedding new light on the mechanisms through which mammalian CCAR1 functions in tumorigenesis.