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- ItemOrchestrated control of filaggrin-actin scaffolds underpins cornification(London [u.a.] : Nature Publishing Group, 2018) Gutowska-Owsiak, Danuta; de La Serna, Jorge Bernardino; Fritzsche, Marco; Naeem, Aishath; Podobas, Ewa I.; Leeming, Michael; Colin-York, Huw; O’Shaughnessy, Ryan; Eggeling, Christian; Ogg, Graham S.Epidermal stratification critically depends on keratinocyte differentiation and programmed death by cornification, leading to formation of a protective skin barrier. Cornification is dynamically controlled by the protein filaggrin, rapidly released from keratohyalin granules (KHGs). However, the mechanisms of cornification largely remain elusive, partly due to limitations of the observation techniques employed to study filaggrin organization in keratinocytes. Moreover, while the abundance of keratins within KHGs has been well described, it is not clear whether actin also contributes to their formation or fate. We employed advanced (super-resolution) microscopy to examine filaggrin organization and dynamics in skin and human keratinocytes during differentiation. We found that filaggrin organization depends on the cytoplasmic actin cytoskeleton, including the role for α- and β-actin scaffolds. Filaggrin-containing KHGs displayed high mobility and migrated toward the nucleus during differentiation. Pharmacological disruption targeting actin networks resulted in granule disintegration and accelerated cornification. We identified the role of AKT serine/threonine kinase 1 (AKT1), which controls binding preference and function of heat shock protein B1 (HspB1), facilitating the switch from actin stabilization to filaggrin processing. Our results suggest an extended model of cornification in which filaggrin utilizes actins to effectively control keratinocyte differentiation and death, promoting epidermal stratification and formation of a fully functional skin barrier.
- ItemScanning electron microscopy preparation of the cellular actin cortex: A quantitative comparison between critical point drying and hexamethyldisilazane drying(San Francisco, California, US : PLOS, 2021) Schu, Moritz; Terriac, Emmanuel; Koch, Marcus; Paschke, Stephan; Lautenschläger, Franziska; Flormann, Daniel A.D.The cellular cortex is an approximately 200-nm-thick actin network that lies just beneath the cell membrane. It is responsible for the mechanical properties of cells, and as such, it is involved in many cellular processes, including cell migration and cellular interactions with the environment. To develop a clear view of this dense structure, high-resolution imaging is essential. As one such technique, electron microscopy, involves complex sample preparation procedures. The final drying of these samples has significant influence on potential artifacts, like cell shrinkage and the formation of artifactual holes in the actin cortex. In this study, we compared the three most used final sample drying procedures: critical-point drying (CPD), CPD with lens tissue (CPD-LT), and hexamethyldisilazane drying. We show that both hexamethyldisilazane and CPD-LT lead to fewer artifactual mesh holes within the actin cortex than CPD. Moreover, CPD-LT leads to significant reduction in cell height compared to hexamethyldisilazane and CPD. We conclude that the final drying procedure should be chosen according to the reduction in cell height, and so CPD-LT, or according to the spatial separation of the single layers of the actin cortex, and so hexamethyldisilazane.
- ItemVinculin binding angle in podosomes revealed by high resolution microscopy(San Francisco, CA : Public Library of Science, 2014) Walde, M.; Monypenny, J.; Heintzmann, R.; Jones, G.E.; Cox, S.Podosomes are highly dynamic actin-rich adhesive structures formed predominantly by cells of the monocytic lineage, which degrade the extracellular matrix. They consist of a core of F-actin and actin-regulating proteins, surrounded by a ring of adhesion-associated proteins such as vinculin. We have characterised the structure of podosomes in macrophages, particularly the structure of the ring, using three super-resolution fluorescence microscopy techniques: stimulated emission depletion microscopy, structured illumination microscopy and localisation microscopy. Rather than being round, as previously assumed, we found the vinculin ring to be created from relatively straight strands of vinculin, resulting in a distinctly polygonal shape. The strands bind preferentially at angles between 116° and 135°. Furthermore, adjacent vinculin strands are observed nucleating at the corners of the podosomes, suggesting a mechanism for podosome growth.
- ItemGuidance of mesenchymal stem cells on fibronectin structured hydrogel films(San Francisco, California, US : PLOS, 2014) Kasten, Annika; Naser, Tamara; Brüllhoff, Kristina; Fiedler, Jörg; Müller, Petra; Möller, Martin; Rychly, Joachim; Groll, Jürgen; Brenner, Rolf E.; Engler, Adam J.Designing of implant surfaces using a suitable ligand for cell adhesion to stimulate specific biological responses of stem cells will boost the application of regenerative implants. For example, materials that facilitate rapid and guided migration of stem cells would promote tissue regeneration. When seeded on fibronectin (FN) that was homogeneously immmobilized to NCO-sP(EO-stat-PO), which otherwise prevents protein binding and cell adhesion, human mesenchymal stem cells (MSC) revealed a faster migration, increased spreading and a more rapid organization of different cellular components for cell adhesion on fibronectin than on a glass surface. To further explore, how a structural organization of FN controls the behavior of MSC, adhesive lines of FN with varying width between 10 µm and 80 µm and spacings between 5 µm and 20 µm that did not allow cell adhesion were generated. In dependance on both line width and gaps, cells formed adjacent cell contacts, were individually organized in lines, or bridged the lines. With decreasing sizes of FN lines, speed and directionality of cell migration increased, which correlated with organization of the actin cytoskeleton, size and shape of the nuclei as well as of focal adhesions. Together, defined FN lines and gaps enabled a fine tuning of the structural organization of cellular components and migration. Microstructured adhesive substrates can mimic the extracellular matrix in vivo and stimulate cellular mechanisms which play a role in tissue regeneration.
- ItemCellular responses to beating hydrogels to investigate mechanotransduction([London] : Nature Publishing Group UK, 2019) Chandorkar, Yashoda; Castro Nava, Arturo; Schweizerhof, Sjören; Van Dongen, Marcel; Haraszti, Tamás; Köhler, Jens; Zhang, Hang; Windoffer, Reinhard; Mourran, Ahmed; Möller, Martin; De Laporte, LauraCells feel the forces exerted on them by the surrounding extracellular matrix (ECM) environment and respond to them. While many cell fate processes are dictated by these forces, which are highly synchronized in space and time, abnormal force transduction is implicated in the progression of many diseases (muscular dystrophy, cancer). However, material platforms that enable transient, cyclic forces in vitro to recreate an in vivo-like scenario remain a challenge. Here, we report a hydrogel system that rapidly beats (actuates) with spatio-temporal control using a near infra-red light trigger. Small, user-defined mechanical forces (~nN) are exerted on cells growing on the hydrogel surface at frequencies up to 10 Hz, revealing insights into the effect of actuation on cell migration and the kinetics of reversible nuclear translocation of the mechanosensor protein myocardin related transcription factor A, depending on the actuation amplitude, duration and frequency.