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Dissection of keratin network formation, turnover and reorganization in living murine embryos.

Schwarz N, Windoffer R, Magin TM, Leube RE - Sci Rep (2015)

Bottom Line: Epithelial functions are fundamentally determined by cytoskeletal keratin network organization.However, our understanding of keratin network plasticity is only based on analyses of cultured cells overexpressing fluorescently tagged keratins.This mouse model will help to further dissect keratin network dynamics in its native tissue context during physiological and also pathological events.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cellular Anatomy, RWTH Aachen University, Aachen, Germany.

ABSTRACT
Epithelial functions are fundamentally determined by cytoskeletal keratin network organization. However, our understanding of keratin network plasticity is only based on analyses of cultured cells overexpressing fluorescently tagged keratins. In order to learn how keratin network organization is affected by various signals in functional epithelial tissues in vivo, we generated a knock-in mouse that produces fluorescence-tagged keratin 8. Homozygous keratin 8-YFP knock-in mice develop normally and show the expected expression of the fluorescent keratin network both in fixed and in vital tissues. In developing embryos, we observe for the first time de novo keratin network biogenesis in close proximity to desmosomal adhesion sites, keratin turnover in interphase cells and keratin rearrangements in dividing cells at subcellular resolution during formation of the first epithelial tissue. This mouse model will help to further dissect keratin network dynamics in its native tissue context during physiological and also pathological events.

No MeSH data available.


Related in: MedlinePlus

Detection of Krt8-YFP by fluorescence microscopy in living intestinal mucosa.(a–a′′) Two different z planes (a, a′) from a stack of 19 focal planes (maximum intensity projection of all planes in a′′) are shown to depict the subapical dense network in the enterocytes and the laterally restricted filaments below (see also corresponding animation in Supplementary Movie 2). (b–b′) Images taken from a high-resolution stack of 13 focal planes showing an oblique surface views of the dense subapical network (b) and the network below that is restricted to the cortical juxtamembraneous domain of adjacent intestinal epithelial cells (b′; complete image series in Supplementary Movie 3). lu, lumen. Scale bars, 10 μm.
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f3: Detection of Krt8-YFP by fluorescence microscopy in living intestinal mucosa.(a–a′′) Two different z planes (a, a′) from a stack of 19 focal planes (maximum intensity projection of all planes in a′′) are shown to depict the subapical dense network in the enterocytes and the laterally restricted filaments below (see also corresponding animation in Supplementary Movie 2). (b–b′) Images taken from a high-resolution stack of 13 focal planes showing an oblique surface views of the dense subapical network (b) and the network below that is restricted to the cortical juxtamembraneous domain of adjacent intestinal epithelial cells (b′; complete image series in Supplementary Movie 3). lu, lumen. Scale bars, 10 μm.

Mentions: To test the suitability of the Krt8-YFP reporter for in vivo imaging, we examined freshly prepared, non-fixed intestinal mucosa. The resulting fluorescence images (Fig. 3a–b′; Supplementary Movies 2, 3) revealed details that were clearly superior to those obtainable by standard immunofluorescence techniques. Examples include the dense subapical network and the clearly distinguishable filament bundles below the lateral membranes of adjacent cells.


Dissection of keratin network formation, turnover and reorganization in living murine embryos.

Schwarz N, Windoffer R, Magin TM, Leube RE - Sci Rep (2015)

Detection of Krt8-YFP by fluorescence microscopy in living intestinal mucosa.(a–a′′) Two different z planes (a, a′) from a stack of 19 focal planes (maximum intensity projection of all planes in a′′) are shown to depict the subapical dense network in the enterocytes and the laterally restricted filaments below (see also corresponding animation in Supplementary Movie 2). (b–b′) Images taken from a high-resolution stack of 13 focal planes showing an oblique surface views of the dense subapical network (b) and the network below that is restricted to the cortical juxtamembraneous domain of adjacent intestinal epithelial cells (b′; complete image series in Supplementary Movie 3). lu, lumen. Scale bars, 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4355630&req=5

f3: Detection of Krt8-YFP by fluorescence microscopy in living intestinal mucosa.(a–a′′) Two different z planes (a, a′) from a stack of 19 focal planes (maximum intensity projection of all planes in a′′) are shown to depict the subapical dense network in the enterocytes and the laterally restricted filaments below (see also corresponding animation in Supplementary Movie 2). (b–b′) Images taken from a high-resolution stack of 13 focal planes showing an oblique surface views of the dense subapical network (b) and the network below that is restricted to the cortical juxtamembraneous domain of adjacent intestinal epithelial cells (b′; complete image series in Supplementary Movie 3). lu, lumen. Scale bars, 10 μm.
Mentions: To test the suitability of the Krt8-YFP reporter for in vivo imaging, we examined freshly prepared, non-fixed intestinal mucosa. The resulting fluorescence images (Fig. 3a–b′; Supplementary Movies 2, 3) revealed details that were clearly superior to those obtainable by standard immunofluorescence techniques. Examples include the dense subapical network and the clearly distinguishable filament bundles below the lateral membranes of adjacent cells.

Bottom Line: Epithelial functions are fundamentally determined by cytoskeletal keratin network organization.However, our understanding of keratin network plasticity is only based on analyses of cultured cells overexpressing fluorescently tagged keratins.This mouse model will help to further dissect keratin network dynamics in its native tissue context during physiological and also pathological events.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cellular Anatomy, RWTH Aachen University, Aachen, Germany.

ABSTRACT
Epithelial functions are fundamentally determined by cytoskeletal keratin network organization. However, our understanding of keratin network plasticity is only based on analyses of cultured cells overexpressing fluorescently tagged keratins. In order to learn how keratin network organization is affected by various signals in functional epithelial tissues in vivo, we generated a knock-in mouse that produces fluorescence-tagged keratin 8. Homozygous keratin 8-YFP knock-in mice develop normally and show the expected expression of the fluorescent keratin network both in fixed and in vital tissues. In developing embryos, we observe for the first time de novo keratin network biogenesis in close proximity to desmosomal adhesion sites, keratin turnover in interphase cells and keratin rearrangements in dividing cells at subcellular resolution during formation of the first epithelial tissue. This mouse model will help to further dissect keratin network dynamics in its native tissue context during physiological and also pathological events.

No MeSH data available.


Related in: MedlinePlus