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Author: Fritzsche, Marco × WGL Institute: IPHT ×

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Now showing 1 - 3 of 3
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    Statistical Analysis of Scanning Fluorescence Correlation Spectroscopy Data Differentiates Free from Hindered Diffusion
    (Washington, DC : Soc., 2018-7-20) Schneider, Falk; Waithe, Dominic; Lagerholm, B. Christoffer; Shrestha, Dilip; Sezgin, Erdinc; Eggeling, Christian; Fritzsche, Marco
    Cells rely on versatile diffusion dynamics in their plasma membrane. Quantification of this often heterogeneous diffusion is essential to the understanding of cell regulation and function. Yet such measurements remain a major challenge in cell biology, usually due to low sampling throughput, a necessity for dedicated equipment, sophisticated fluorescent label strategies, and limited sensitivity. Here, we introduce a robust, broadly applicable statistical analysis pipeline for large scanning fluorescence correlation spectroscopy data sets, which uncovers the nanoscale heterogeneity of the plasma membrane in living cells by differentiating free from hindered diffusion modes of fluorescent lipid and protein analogues.
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    Orchestrated 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.
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    The cortical actin network regulates avidity-dependent binding of hyaluronan by the lymphatic vessel endothelial receptor LYVE-1
    (Bethesda, Md. : ASBMB Publications, 2020) Stanly, Tess A.; Fritzsche, Marco; Banerji, Suneale; Shrestha, Dilip; Schneider, Falk; Eggeling, Christian; Jackson, David G.
    Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) mediates the docking and entry of dendritic cells to lymphatic vessels through selective adhesion to its ligand hyaluronan in the leukocyte surface glycocalyx. To bind hyaluronan efficiently, LYVE-1 must undergo surface clustering, a process that is induced efficiently by the large cross-linked assemblages of glycosaminoglycan present within leukocyte pericellular matrices but is induced poorly by the shorter polymer alone. These properties suggested that LYVE-1 may have limited mobility in the endothelial plasma membrane, but no biophysical investigation of these parameters has been carried out to date. Here, using super-resolution fluorescence microscopy and spectroscopy combined with biochemical analyses of the receptor in primary lymphatic endothelial cells, we provide the first evidence that LYVE-1 dynamics are indeed restricted by the submembranous actin network. We show that actin disruption not only increases LYVE-1 lateral diffusion but also enhances hyaluronan- binding activity. However, unlike the related leukocyte HA receptor CD44, which uses ERM and ankyrin motifs within its cytoplasmic tail to bind actin, LYVE-1 displays little if any direct interaction with actin, as determined by co-immunoprecipitation. Instead, as shown by super-resolution stimulated emission depletion microscopy in combination with fluorescence correlation spectroscopy, LYVE-1 diffusion is restricted by transient entrapment within submembranous actin corrals. These results point to an actin-mediated constraint on LYVE-1 clustering in lymphatic endothelium that tunes the receptor for selective engagement with hyaluronan assemblages in the glycocalyx that are large enough to cross-bridge the corral-bound LYVE-1 molecules and thereby facilitate leukocyte adhesion and transmigration. © 2020 Stanly et al.