MicroRNA-205 promotes hair regeneration by modulating mechanical properties of hair follicle stem cells.

Stiffness and actomyosin contractility are intrinsic mechanical properties of animal cells required for the shaping of tissues. However, whether tissue stem cells (SCs) and progenitors located within SC niche have different mechanical properties that modulate their size and function remains unclear. Here, we show that hair follicle SCs in the bulge are stiff with high actomyosin contractility and resistant to size change, whereas hair germ (HG) progenitors are soft and periodically enlarge and contract during quiescence. During activation of hair follicle growth, HGs reduce contraction and more frequently enlarge, a process that is associated with weakening of the actomyosin network, nuclear YAP accumulation, and cell cycle reentry. Induction of miR-205, a novel regulator of the actomyosin cytoskeleton, reduces actomyosin contractility and activates hair regeneration in young and old mice. This study reveals the control of tissue SC size and activities by spatiotemporally compartmentalized mechanical properties and demonstrates the possibility to stimulate tissue regeneration by fine-tuning cell mechanics.

Epidermal stratification requires retromer-mediated desmoglein-1 recycling.

Sorting transmembrane cargo is essential for tissue development and homeostasis. However, mechanisms of intracellular trafficking in stratified epidermis are poorly understood. Here, we identify an interaction between the retromer endosomal trafficking component, VPS35, and the desmosomal cadherin, desmoglein-1 (Dsg1). Dsg1 is specifically expressed in stratified epidermis and, when properly localized on the plasma membrane of basal keratinocytes, promotes stratification. We show that the retromer drives Dsg1 recycling from the endo-lysosomal system to the plasma membrane to support human keratinocyte stratification. The retromer-enhancing chaperone, R55, promotes the membrane localization of Dsg1 and a trafficking-deficient mutant associated with a severe inflammatory skin disorder, enhancing its ability to promote stratification. In the absence of Dsg1, retromer association with and expression of the glucose transporter GLUT1 increases, exposing a potential link between Dsg1 deficiency and epidermal metabolism. Our work provides evidence for retromer function in epidermal regeneration, identifying it as a potential therapeutic target.

Organotypic Human Skin Cultures Incorporating Primary Melanocytes.

Three-dimensional (3D) human organotypic skin cultures provide a physiologically relevant model that recapitulates in vivo skin features. Most commonly, organotypic skin cultures are created by seeding isolated epidermal keratinocytes onto a collagen/fibroblast plug and lifting to an air liquid interface. These conditions are sufficient to drive stratification and differentiation of the keratinocytes to form an epidermal-like sheet with remarkable similarities to human epidermis. Coupled with genetic or pharmacological treatments, these cultures provide a powerful tool for elucidating keratinocyte biology. Recent focus has been placed on increasing the utility of organotypic skin cultures by incorporating other cell types that are present in the skin, such as melanocytes, immune cells, and other cells. Here we describe a step-by-step protocol for the isolation of neonatal human epidermal keratinocytes and melanocytes from foreskins, and the creation of organotypic skin cultures that include both cell types. We also describe methods that can be used to assess melanocyte behavior in these organotypic cultures, including methods for whole mount staining, measurement of melanocyte dendricity, staining for pigment, and 5-bromo-2'-deoxyuridine (BrdU) labeling for identification of proliferating cells. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolation of primary cells Alternate Protocol: Isolation of primary cells using differential trypsinization Basic Protocol 2: Organotypic culture protocol Support Protocol 1: Culture and maintenance of NHEKs and melanocytes Support Protocol 2: Lentiviral transduction of melanocytes Support Protocol 3: Retroviral transduction of NHEKs Support Protocol 4: Whole mount immunostaining protocol Support Protocol 5: Measuring melanocyte dendricity Support Protocol 6: Fontana-Masson staining protocol Support Protocol 7: BrdU labeling and staining.

The Desmosome-Keratin Scaffold Integrates ErbB Family and Mechanical Signaling to Polarize Epidermal Structure and Function.

While classic cadherin-actin connections in adherens junctions (AJs) have ancient origins, intermediate filament (IF) linkages with desmosomal cadherins arose in vertebrate organisms. In this mini-review, we discuss how overlaying the IF-desmosome network onto the existing cadherin-actin network provided new opportunities to coordinate tissue mechanics with the positioning and function of chemical signaling mediators in the ErbB family of receptor tyrosine kinases. We focus in particular on the complex multi-layered outer covering of the skin, the epidermis, which serves essential barrier and stress sensing/responding functions in terrestrial vertebrates. We will review emerging data showing that desmosome-IF connections, AJ-actin interactions, ErbB family members, and membrane tension are all polarized across the multiple layers of the regenerating epidermis. Importantly, their integration generates differentiation-specific roles in each layer of the epidermis that dictate the form and function of the tissue. In the basal layer, the onset of the differentiation-specific desmosomal cadherin desmoglein 1 (Dsg1) dials down EGFR signaling while working with classic cadherins to remodel cortical actin cytoskeleton and decrease membrane tension to promote cell delamination. In the upper layers, Dsg1 and E-cadherin cooperate to maintain high tension and tune EGFR and ErbB2 activity to create the essential tight junction barrier. Our final outlook discusses the emerging appreciation that the desmosome-IF scaffold not only creates the architecture required for skin's physical barrier but also creates an immune barrier that keeps inflammation in check.

Plectin pulls it together, coupling the cortical actin and intermediate filament cytoskeletons.

The integration of cytoskeletal/adhesive networks is critical to epithelial mechanobiology. In this issue, Prechova et al. (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202105146) demonstrate that the cytolinker protein plectin is essential for the construction of a cortical cytoskeletal architecture required for epithelial tensional homeostasis.

Translational implications of Th17-skewed inflammation due to genetic deficiency of a cadherin stress sensor.

Desmoglein 1 (Dsg1) is a cadherin restricted to stratified tissues of terrestrial vertebrates, which serve as essential physical and immune barriers. Dsg1 loss-of-function mutations in humans result in skin lesions and multiple allergies, and isolated patient keratinocytes exhibit increased proallergic cytokine expression. However, the mechanism by which genetic deficiency of Dsg1 causes chronic inflammation is unknown. To determine the systemic response to Dsg1 loss, we deleted the 3 tandem Dsg1 genes in mice. Whole transcriptome analysis of embryonic Dsg1-/- skin showed a delay in expression of adhesion/differentiation/keratinization genes at E17.5, a subset of which recovered or increased by E18.5. Comparing epidermal transcriptomes from Dsg1-deficient mice and humans revealed a shared IL-17-skewed inflammatory signature. Although the impaired intercellular adhesion observed in Dsg1-/- mice resembles that resulting from anti-Dsg1 pemphigus foliaceus antibodies, pemphigus skin lesions exhibit a weaker IL-17 signature. Consistent with the clinical importance of these findings, treatment of 2 Dsg1-deficient patients with an IL-12/IL-23 antagonist originally developed for psoriasis resulted in improvement of skin lesions. Thus, beyond impairing the physical barrier, loss of Dsg1 function through gene mutation results in a psoriatic-like inflammatory signature before birth, and treatment with a targeted therapy significantly improved skin lesions in patients.

Desmosomes polarize and integrate chemical and mechanical signaling to govern epidermal tissue form and function.

The epidermis is a stratified epithelium in which structural and functional features are polarized across multiple cell layers. This type of polarity is essential for establishing the epidermal barrier, but how it is created and sustained is poorly understood. Previous work identified a role for the classic cadherin/filamentous-actin network in establishment of epidermal polarity. However, little is known about potential roles of the most prominent epidermal intercellular junction, the desmosome, in establishing epidermal polarity, in spite of the fact that desmosome constituents are patterned across the apical to basal cell layers. Here, we show that desmosomes and their associated intermediate filaments (IFs) are key regulators of mechanical polarization in epidermis, whereby basal and suprabasal cells experience different forces that drive layer-specific functions. Uncoupling desmosomes and IF or specific targeting of apical desmosomes through depletion of the superficial desmosomal cadherin, desmoglein 1, impedes basal stratification in an in vitro competition assay and suprabasal tight junction barrier functions in 3D reconstructed epidermis. Surprisingly, disengaging desmosomes from IF also accelerated the expression of differentiation markers, through precocious activation of the mechanosensitive transcriptional regulator serum response factor (SRF) and downstream activation of epidermal growth factor receptor family member ErbB2 by Src family kinase (SFK)-mediated phosphorylation. This Dsg1-SFK-ErbB2 axis also helps maintain tight junctions and barrier function later in differentiation. Together, these data demonstrate that the desmosome-IF network is a critical contributor to the cytoskeletal-adhesive machinery that supports the polarized function of the epidermis.

Scaling up single-cell mechanics to multicellular tissues - the role of the intermediate filament-desmosome network.

Cells and tissues sense, respond to and translate mechanical forces into biochemical signals through mechanotransduction, which governs individual cell responses that drive gene expression, metabolic pathways and cell motility, and determines how cells work together in tissues. Mechanotransduction often depends on cytoskeletal networks and their attachment sites that physically couple cells to each other and to the extracellular matrix. One way that cells associate with each other is through Ca2+-dependent adhesion molecules called cadherins, which mediate cell-cell interactions through adherens junctions, thereby anchoring and organizing the cortical actin cytoskeleton. This actin-based network confers dynamic properties to cell sheets and developing organisms. However, these contractile networks do not work alone but in concert with other cytoarchitectural elements, including a diverse network of intermediate filaments. This Review takes a close look at the intermediate filament network and its associated intercellular junctions, desmosomes. We provide evidence that this system not only ensures tissue integrity, but also cooperates with other networks to create more complex tissues with emerging properties in sensing and responding to increasingly stressful environments. We will also draw attention to how defects in intermediate filament and desmosome networks result in both chronic and acquired diseases.

Keratinocyte cadherin desmoglein 1 controls melanocyte behavior through paracrine signaling.

The epidermis is the first line of defense against ultraviolet (UV) light from the sun. Keratinocytes and melanocytes respond to UV exposure by eliciting a tanning response dependent in part on paracrine signaling, but how keratinocyte:melanocyte communication is regulated during this response remains understudied. Here, we uncover a surprising new function for the keratinocyte-specific cell-cell adhesion molecule desmoglein 1 (Dsg1) in regulating keratinocyte:melanocyte paracrine signaling to promote the tanning response in the absence of UV exposure. Melanocytes within Dsg1-silenced human skin equivalents exhibited increased pigmentation and altered dendrite morphology, phenotypes which were confirmed in 2D culture using conditioned media from Dsg1-silenced keratinocytes. Dsg1-silenced keratinocytes increased melanocyte-stimulating hormone precursor (Pomc) and cytokine mRNA. Melanocytes cultured in media conditioned by Dsg1-silenced keratinocytes increased Mitf and Tyrp1 mRNA, TYRP1 protein, and melanin production and secretion. Melanocytes in Dsg1-silenced skin equivalents mislocalized suprabasally, reminiscent of early melanoma pagetoid behavior. Together with our previous report that UV reduces Dsg1 expression, these data support a role for Dsg1 in controlling keratinocyte:melanocyte paracrine communication and raise the possibility that a Dsg1-deficient niche contributes to pagetoid behavior, such as occurs in early melanoma development.

Desmosomes: Essential contributors to an integrated intercellular junction network.

The development of adhesive connections between cells was critical for the evolution of multicellularity and for organizing cells into complex organs with discrete compartments. Four types of intercellular junction are present in vertebrates: desmosomes, adherens junctions, tight junctions, and gap junctions. All are essential for the development of the embryonic layers and organs as well as adult tissue homeostasis. While each junction type is defined as a distinct entity, it is now clear that they cooperate physically and functionally to create a robust and functionally diverse system. During evolution, desmosomes first appeared in vertebrates as highly specialized regions at the plasma membrane that couple the intermediate filament cytoskeleton at points of strong cell-cell adhesion. Here, we review how desmosomes conferred new mechanical and signaling properties to vertebrate cells and tissues through their interactions with the existing junctional and cytoskeletal network.

Techniques to stimulate and interrogate cell-cell adhesion mechanics.

Cell-cell adhesions maintain the mechanical integrity of multicellular tissues and have recently been found to act as mechanotransducers, translating mechanical cues into biochemical signals. Mechanotransduction studies have primarily focused on focal adhesions, sites of cell-substrate attachment. These studies leverage technical advances in devices and systems interfacing with living cells through cell-extracellular matrix adhesions. As reports of aberrant signal transduction originating from mutations in cell-cell adhesion molecules are being increasingly associated with disease states, growing attention is being paid to this intercellular signaling hub. Along with this renewed focus, new requirements arise for the interrogation and stimulation of cell-cell adhesive junctions. This review covers established experimental techniques for stimulation and interrogation of cell-cell adhesion from cell pairs to monolayers.

Desmosomal cadherin association with Tctex-1 and cortactin-Arp2/3 drives perijunctional actin polymerization to promote keratinocyte delamination.

The epidermis is a multi-layered epithelium that serves as a barrier against water loss and environmental insults. Its morphogenesis occurs through a tightly regulated program of biochemical and architectural changes during which basal cells commit to differentiate and move towards the skin's surface. Here, we reveal an unexpected role for the vertebrate cadherin desmoglein 1 (Dsg1) in remodeling the actin cytoskeleton to promote the transit of basal cells into the suprabasal layer through a process of delamination, one mechanism of epidermal stratification. Actin remodeling requires the interaction of Dsg1 with the dynein light chain, Tctex-1 and the actin scaffolding protein, cortactin. We demonstrate that Tctex-1 ensures the correct membrane compartmentalization of Dsg1-containing desmosomes, allowing cortactin/Arp2/3-dependent perijunctional actin polymerization and decreasing tension at E-cadherin junctions to promote keratinocyte delamination. Moreover, Dsg1 is sufficient to enable simple epithelial cells to exit a monolayer to form a second layer, highlighting its morphogenetic potential.

Adherens Junctions and Desmosomes Coordinate Mechanics and Signaling to Orchestrate Tissue Morphogenesis and Function: An Evolutionary Perspective.

Cadherin-based adherens junctions (AJs) and desmosomes are crucial to couple intercellular adhesion to the actin or intermediate filament cytoskeletons, respectively. As such, these intercellular junctions are essential to provide not only integrity to epithelia and other tissues but also the mechanical machinery necessary to execute complex morphogenetic and homeostatic intercellular rearrangements. Moreover, these spatially defined junctions serve as signaling hubs that integrate mechanical and chemical pathways to coordinate tissue architecture with behavior. This review takes an evolutionary perspective on how the emergence of these two essential intercellular junctions at key points during the evolution of multicellular animals afforded metazoans with new opportunities to integrate adhesion, cytoskeletal dynamics, and signaling. We discuss known literature on cross-talk between the two junctions and, using the skin epidermis as an example, provide a model for how these two junctions function in concert to orchestrate tissue organization and function.

A rim-and-spoke hypothesis to explain the biomechanical roles for cytoplasmic intermediate filament networks.

Textbook images of keratin intermediate filament (IF) networks in epithelial cells and the functional compromization of the epidermis by keratin mutations promulgate a mechanical role for this important cytoskeletal component. In stratified epithelia, keratin filaments form prominent radial spokes that are focused onto cell-cell contact sites, i.e. the desmosomes. In this Hypothesis, we draw attention to a subset of keratin filaments that are apposed to the plasma membrane. They form a rim of filaments interconnecting the desmosomes in a circumferential network. We hypothesize that they are part of a rim-and-spoke arrangement of IFs in epithelia. From our review of the literature, we extend this functional role for the subplasmalemmal rim of IFs to any cell, in which plasma membrane support is required, provided these filaments connect directly or indirectly to the plasma membrane. Furthermore, cytoplasmic IF networks physically link the outer nuclear and plasma membranes, but their participation in mechanotransduction processes remain largely unconsidered. Therefore, we also discuss the potential biomechanical and mechanosensory role(s) of the cytoplasmic IF network in terms of such a rim (i.e. subplasmalemmal)-and-spoke arrangement for cytoplasmic IF networks.

Research Techniques Made Simple: Methodology and Applications of Förster Resonance Energy Transfer (FRET) Microscopy.

Classical biochemical techniques have contributed a great deal to our understanding of the mechanisms regulating fundamental biological processes. However, these approaches are typically end-point, population-based assays and are often insufficient in examining transient molecular events. Förster resonance energy transfer (FRET) microscopy is a powerful technique capable of investigating dynamic interactions between proteins and a plethora of biochemical signaling events based on the development of specific biosensors. This technique exploits the principle that when FRET occurs, energy from a donor fluorophore is transferred to an acceptor fluorophore only when certain conditions are met. These include dependence on both distance and fluorophore orientation. In this article, applications of FRET microscopy to protein interactions and modifications are discussed, and examples are given of the types of biosensors that can be developed. There are a number of methods to measure FRET. The most common modalities and specific advantages and shortcomings for each are reviewed. Finally, general considerations and guidelines for choosing a method are discussed.

The desmoplakin-intermediate filament linkage regulates cell mechanics.

The translation of mechanical forces into biochemical signals plays a central role in guiding normal physiological processes during tissue development and homeostasis. Interfering with this process contributes to cardiovascular disease, cancer progression, and inherited disorders. The actin-based cytoskeleton and its associated adherens junctions are well-established contributors to mechanosensing and transduction machinery; however, the role of the desmosome-intermediate filament (DSM-IF) network is poorly understood in this context. Because a force balance among different cytoskeletal systems is important to maintain normal tissue function, knowing the relative contributions of these structurally integrated systems to cell mechanics is critical. Here we modulated the interaction between DSMs and IFs using mutant forms of desmoplakin, the protein bridging these structures. Using micropillar arrays and atomic force microscopy, we demonstrate that strengthening the DSM-IF interaction increases cell-substrate and cell-cell forces and cell stiffness both in cell pairs and sheets of cells. In contrast, disrupting the interaction leads to a decrease in these forces. These alterations in cell mechanics are abrogated when the actin cytoskeleton is dismantled. These data suggest that the tissue-specific variability in DSM-IF network composition provides an opportunity to differentially regulate tissue mechanics by balancing and tuning forces among cytoskeletal systems.

Desmosome regulation and signaling in disease.

Desmosomes are cell-cell adhesive organelles with a well-known role in forming strong intercellular adhesion during embryogenesis and in adult tissues subject to mechanical stress, such as the heart and skin. More recently, desmosome components have also emerged as cell signaling regulators. Loss of expression or interference with the function of desmosome molecules results in diseases of the heart and skin and contributes to cancer progression. However, the underlying molecular mechanisms that result in inherited and acquired disorders remain poorly understood. To address this question, researchers are directing their studies towards determining the functions that occur inside and outside of the junctions and the extent to which functions are adhesion-dependent or independent. This review focuses on recent discoveries that provide insights into the role of desmosomes and desmosome components in cell signaling and disease; wherever possible, we address molecular functions within and outside of the adhesive structure.

Automated analysis of cell-matrix adhesions in 2D and 3D environments.

Cell-matrix adhesions are of great interest because of their contribution to numerous biological processes, including cell migration, differentiation, proliferation, survival, tissue morphogenesis, wound healing, and tumorigenesis. Adhesions are dynamic structures that are classically defined on two-dimensional (2D) substrates, though the need to analyze adhesions in more physiologic three-dimensional (3D) environments is being increasingly recognized. However, progress has been greatly hampered by the lack of available tools to analyze adhesions in 3D environments. To address this need, we have developed a platform for the automated analysis, segmentation, and tracking of adhesions (PAASTA) based on an open source MATLAB framework, CellAnimation. PAASTA enables the rapid analysis of adhesion dynamics and many other adhesion characteristics, such as lifetime, size, and location, in 3D environments and on traditional 2D substrates. We manually validate PAASTA and utilize it to quantify rate constants for adhesion assembly and disassembly as well as adhesion lifetime and size in 3D matrices. PAASTA will be a valuable tool for characterizing adhesions and for deciphering the molecular mechanisms that regulate adhesion dynamics in 3D environments.

Activation of Rac by Asef2 promotes myosin II-dependent contractility to inhibit cell migration on type I collagen.

Non-muscle myosin II (MyoII) contractility is central to the regulation of numerous cellular processes, including migration. Rho is a well-characterized modulator of actomyosin contractility, but the function of other GTPases, such as Rac, in regulating contractility is currently not well understood. Here, we show that activation of Rac by the guanine nucleotide exchange factor Asef2 (also known as SPATA13) impairs migration on type I collagen through a MyoII-dependent mechanism that enhances contractility. Knockdown of endogenous Rac or treatment of cells with a Rac-specific inhibitor decreases the amount of active MyoII, as determined by serine 19 (S19) phosphorylation, and negates the Asef2-promoted increase in contractility. Moreover, treatment of cells with blebbistatin, which inhibits MyoII activity, abolishes the Asef2-mediated effect on migration. In addition, Asef2 slows the turnover of adhesions in protrusive regions of cells by promoting large mature adhesions, which has been linked to actomyosin contractility, with increased amounts of active ß1 integrin. Hence, our data reveal a new role for Rac activation, promoted by Asef2, in modulating actomyosin contractility, which is important for regulating cell migration and adhesion dynamics.

Fluorescence resonance energy transfer microscopy as demonstrated by measuring the activation of the serine/threonine kinase Akt.

This protocol describes procedures for performing fluorescence resonance energy transfer (FRET) microscopy analysis by three different methods: acceptor photobleaching, sensitized emission and spectral imaging. We also discuss anisotropy and fluorescence lifetime imaging microscopy-based FRET techniques. By using the specific example of the FRET probe Akind (Akt indicator), which is a version of Akt modified such that FRET occurs when the probe is activated by phosphorylation, indicating Akt activation. The protocol provides a detailed step-by-step description of sample preparation, image acquisition and analysis, including control samples, image corrections and the generation of quantitative FRET/CFP ratio images for both sensitized emission and spectral imaging. The sample preparation takes 2 d, equipment setup takes 2-3 h and image acquisition and analysis take 6-8 h.