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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 immunoblotting and fluorescence microscopy in tissues of adult knock-in mice.(a) Immunoblots of cytoskeletal extracts that were prepared from lung, liver, kidney and intestine of wild-type (wt) and homozygous Krt8-YFP mice (Krt8-YFP). Blots were successively incubated with anti-keratin 8 antibodies (top), anti-vimentin antibodies (middle) and anti-actin antibodies (bottom). Note the increase in the size of keratin 8 due to the YFP tag and the complete absence of wild-type keratin 8 in the homozygous Krt8-YFP knock-in mouse samples. Full-length blots are presented in Supplementary Figure 2. (b–e) Fluorescence microscopy of cryosections of liver (b), trachea (c) and intestine (d, e) obtained from homozygous Krt8-YFP mice. Note the exclusive expression of the transgene in the cytoplasm of epithelial cells but not in the nucleus (nu) nor in connective tissue of the lamina propria (lp; c) and submucosa (sm; d). Note also the cell type-specific network organization with a pancytoplasmic distribution in adluminal cells of the tracheal epithelium (c; ep, epithelium) and in glandular epithelial cells (c; gl, gland), the accumulation around bile canaliculi in the liver (b) and subapical enrichment in intestinal epithelial cells (d, e; ep, epithelium). lu, lumen. Scale bars, 10 μm in (b, e) and 100 μm in (c, d).
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f2: Detection of Krt8-YFP by immunoblotting and fluorescence microscopy in tissues of adult knock-in mice.(a) Immunoblots of cytoskeletal extracts that were prepared from lung, liver, kidney and intestine of wild-type (wt) and homozygous Krt8-YFP mice (Krt8-YFP). Blots were successively incubated with anti-keratin 8 antibodies (top), anti-vimentin antibodies (middle) and anti-actin antibodies (bottom). Note the increase in the size of keratin 8 due to the YFP tag and the complete absence of wild-type keratin 8 in the homozygous Krt8-YFP knock-in mouse samples. Full-length blots are presented in Supplementary Figure 2. (b–e) Fluorescence microscopy of cryosections of liver (b), trachea (c) and intestine (d, e) obtained from homozygous Krt8-YFP mice. Note the exclusive expression of the transgene in the cytoplasm of epithelial cells but not in the nucleus (nu) nor in connective tissue of the lamina propria (lp; c) and submucosa (sm; d). Note also the cell type-specific network organization with a pancytoplasmic distribution in adluminal cells of the tracheal epithelium (c; ep, epithelium) and in glandular epithelial cells (c; gl, gland), the accumulation around bile canaliculi in the liver (b) and subapical enrichment in intestinal epithelial cells (d, e; ep, epithelium). lu, lumen. Scale bars, 10 μm in (b, e) and 100 μm in (c, d).

Mentions: According to previous reports on keratin 8 expression45 Krt8-YFP is expected to be produced in simple and complex epithelia. Cytoskeletal extracts were therefore prepared from lung, liver, kidney, and intestine of adult knock-in mice to detect Krt8-YFP by immunoblotting. Fig. 2a shows that Krt8-YFP fusion proteins with the expected molecular mass of approximately 82 kDa are present in these tissues of homozygous knock-in mice at levels comparable to those observed for wild-type keratin 8 in control animals. We next examined Krt8-YFP fluorescence in cryosections of liver, trachea and intestine (Fig. 2b–e). Krt8-YFP was restricted to the cell types known to express keratin 84. In addition, network organization such as accumulation around bile canaliculi in hepatocytes and subapical location in enterocytes was identical to that reported for the wild-type situation212223.


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 immunoblotting and fluorescence microscopy in tissues of adult knock-in mice.(a) Immunoblots of cytoskeletal extracts that were prepared from lung, liver, kidney and intestine of wild-type (wt) and homozygous Krt8-YFP mice (Krt8-YFP). Blots were successively incubated with anti-keratin 8 antibodies (top), anti-vimentin antibodies (middle) and anti-actin antibodies (bottom). Note the increase in the size of keratin 8 due to the YFP tag and the complete absence of wild-type keratin 8 in the homozygous Krt8-YFP knock-in mouse samples. Full-length blots are presented in Supplementary Figure 2. (b–e) Fluorescence microscopy of cryosections of liver (b), trachea (c) and intestine (d, e) obtained from homozygous Krt8-YFP mice. Note the exclusive expression of the transgene in the cytoplasm of epithelial cells but not in the nucleus (nu) nor in connective tissue of the lamina propria (lp; c) and submucosa (sm; d). Note also the cell type-specific network organization with a pancytoplasmic distribution in adluminal cells of the tracheal epithelium (c; ep, epithelium) and in glandular epithelial cells (c; gl, gland), the accumulation around bile canaliculi in the liver (b) and subapical enrichment in intestinal epithelial cells (d, e; ep, epithelium). lu, lumen. Scale bars, 10 μm in (b, e) and 100 μm in (c, d).
© Copyright Policy - open-access
Related In: Results  -  Collection

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f2: Detection of Krt8-YFP by immunoblotting and fluorescence microscopy in tissues of adult knock-in mice.(a) Immunoblots of cytoskeletal extracts that were prepared from lung, liver, kidney and intestine of wild-type (wt) and homozygous Krt8-YFP mice (Krt8-YFP). Blots were successively incubated with anti-keratin 8 antibodies (top), anti-vimentin antibodies (middle) and anti-actin antibodies (bottom). Note the increase in the size of keratin 8 due to the YFP tag and the complete absence of wild-type keratin 8 in the homozygous Krt8-YFP knock-in mouse samples. Full-length blots are presented in Supplementary Figure 2. (b–e) Fluorescence microscopy of cryosections of liver (b), trachea (c) and intestine (d, e) obtained from homozygous Krt8-YFP mice. Note the exclusive expression of the transgene in the cytoplasm of epithelial cells but not in the nucleus (nu) nor in connective tissue of the lamina propria (lp; c) and submucosa (sm; d). Note also the cell type-specific network organization with a pancytoplasmic distribution in adluminal cells of the tracheal epithelium (c; ep, epithelium) and in glandular epithelial cells (c; gl, gland), the accumulation around bile canaliculi in the liver (b) and subapical enrichment in intestinal epithelial cells (d, e; ep, epithelium). lu, lumen. Scale bars, 10 μm in (b, e) and 100 μm in (c, d).
Mentions: According to previous reports on keratin 8 expression45 Krt8-YFP is expected to be produced in simple and complex epithelia. Cytoskeletal extracts were therefore prepared from lung, liver, kidney, and intestine of adult knock-in mice to detect Krt8-YFP by immunoblotting. Fig. 2a shows that Krt8-YFP fusion proteins with the expected molecular mass of approximately 82 kDa are present in these tissues of homozygous knock-in mice at levels comparable to those observed for wild-type keratin 8 in control animals. We next examined Krt8-YFP fluorescence in cryosections of liver, trachea and intestine (Fig. 2b–e). Krt8-YFP was restricted to the cell types known to express keratin 84. In addition, network organization such as accumulation around bile canaliculi in hepatocytes and subapical location in enterocytes was identical to that reported for the wild-type situation212223.

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