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Title: Deep nuclear invaginations are linked to cytoskeletal filaments – integrated bioimaging of epithelial cells in 3D culture

Abstract

The importance of context in regulation of gene expression is now an accepted principle; yet the mechanism by which the microenvironment communicates with the nucleus and chromatin in healthy tissues is poorly understood. A functional role for nuclear and cytoskeletal architecture is suggested by the phenotypic differences observed between epithelial and mesenchymal cells. Capitalizing on recent advances in cryogenic techniques, volume electron microscopy and super-resolution light microscopy, we studied human mammary epithelial cells in three-dimensional (3D) cultures forming growtharrested acini. Intriguingly, we found deep nuclear invaginations and tunnels traversing the nucleus, encasing cytoskeletal actin and/or intermediate filaments, which connect to the outer nuclear envelope. Also, the cytoskeleton is connected both to other cells through desmosome adhesion complexes and to the extracellular matrix through hemidesmosomes. This finding supports a physical and/or mechanical link from the desmosomes and hemidesmosomes to the nucleus, which had previously been hypothesized but now is visualized for the first time. These unique structures, including the nuclear invaginations and the cytoskeletal connectivity to the cell nucleus, are consistent with a dynamic reciprocity between the nucleus and the outside of epithelial cells and tissues.

Authors:
ORCiD logo [1];  [2];  [3];  [2]; ORCiD logo [4];  [4];  [3];  [5];  [6];  [2];  [7]; ORCiD logo [4]
  1. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division; Oregon Health and Science Univ., Portland, OR (United States). Dept. of Biomedical Engineering
  2. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division
  3. Univ. of California, Berkeley, CA (United States). Dept. of Chemistry
  4. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division
  5. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Univ. of Sao Paulo (Brazil). Dept. of Biochemistry, Inst. of Chemistry
  6. Oregon Health and Science Univ., Portland, OR (United States). Dept. of Biomedical Engineering
  7. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division; Univ. fo California, Berkeley, CA (United States). Dept. of Chemistry
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23); National Institutes of Health (NIH)
OSTI Identifier:
1337799
Alternate Identifier(s):
OSTI ID: 1379655
Grant/Contract Number:
AC02-05CH11231; P01GM051487; R01CA064789; U54CA143836I
Resource Type:
Journal Article: Published Article
Journal Name:
Journal of Cell Science
Additional Journal Information:
Journal Volume: 130; Journal Issue: 1; Journal ID: ISSN 0021-9533
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; integrated bioimaging; cytoskeleton; mechanotransduction; human mammary epithelial cells; nucleoplasmic reticulum; extracellular matrix

Citation Formats

Jorgens, Danielle M., Inman, Jamie L., Wojcik, Michal, Robertson, Claire, Palsdottir, Hildur, Tsai, Wen-Ting, Huang, Haina, Bruni-Cardoso, Alexandre, López, Claudia S., Bissell, Mina J., Xu, Ke, and Auer, Manfred. Deep nuclear invaginations are linked to cytoskeletal filaments – integrated bioimaging of epithelial cells in 3D culture. United States: N. p., 2016. Web. doi:10.1242/jcs.190967.
Jorgens, Danielle M., Inman, Jamie L., Wojcik, Michal, Robertson, Claire, Palsdottir, Hildur, Tsai, Wen-Ting, Huang, Haina, Bruni-Cardoso, Alexandre, López, Claudia S., Bissell, Mina J., Xu, Ke, & Auer, Manfred. Deep nuclear invaginations are linked to cytoskeletal filaments – integrated bioimaging of epithelial cells in 3D culture. United States. doi:10.1242/jcs.190967.
Jorgens, Danielle M., Inman, Jamie L., Wojcik, Michal, Robertson, Claire, Palsdottir, Hildur, Tsai, Wen-Ting, Huang, Haina, Bruni-Cardoso, Alexandre, López, Claudia S., Bissell, Mina J., Xu, Ke, and Auer, Manfred. Fri . "Deep nuclear invaginations are linked to cytoskeletal filaments – integrated bioimaging of epithelial cells in 3D culture". United States. doi:10.1242/jcs.190967.
@article{osti_1337799,
title = {Deep nuclear invaginations are linked to cytoskeletal filaments – integrated bioimaging of epithelial cells in 3D culture},
author = {Jorgens, Danielle M. and Inman, Jamie L. and Wojcik, Michal and Robertson, Claire and Palsdottir, Hildur and Tsai, Wen-Ting and Huang, Haina and Bruni-Cardoso, Alexandre and López, Claudia S. and Bissell, Mina J. and Xu, Ke and Auer, Manfred},
abstractNote = {The importance of context in regulation of gene expression is now an accepted principle; yet the mechanism by which the microenvironment communicates with the nucleus and chromatin in healthy tissues is poorly understood. A functional role for nuclear and cytoskeletal architecture is suggested by the phenotypic differences observed between epithelial and mesenchymal cells. Capitalizing on recent advances in cryogenic techniques, volume electron microscopy and super-resolution light microscopy, we studied human mammary epithelial cells in three-dimensional (3D) cultures forming growtharrested acini. Intriguingly, we found deep nuclear invaginations and tunnels traversing the nucleus, encasing cytoskeletal actin and/or intermediate filaments, which connect to the outer nuclear envelope. Also, the cytoskeleton is connected both to other cells through desmosome adhesion complexes and to the extracellular matrix through hemidesmosomes. This finding supports a physical and/or mechanical link from the desmosomes and hemidesmosomes to the nucleus, which had previously been hypothesized but now is visualized for the first time. These unique structures, including the nuclear invaginations and the cytoskeletal connectivity to the cell nucleus, are consistent with a dynamic reciprocity between the nucleus and the outside of epithelial cells and tissues.},
doi = {10.1242/jcs.190967},
journal = {Journal of Cell Science},
number = 1,
volume = 130,
place = {United States},
year = {Fri Aug 05 00:00:00 EDT 2016},
month = {Fri Aug 05 00:00:00 EDT 2016}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record at 10.1242/jcs.190967

Citation Metrics:
Cited by: 7works
Citation information provided by
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  • The importance of context in regulation of gene expression is now an accepted principle; yet the mechanism by which the microenvironment communicates with the nucleus and chromatin in healthy tissues is poorly understood. A functional role for nuclear and cytoskeletal architecture is suggested by the phenotypic differences observed between epithelial and mesenchymal cells. Capitalizing on recent advances in cryogenic techniques, volume electron microscopy and super-resolution light microscopy, we studied human mammary epithelial cells in three-dimensional (3D) cultures forming growtharrested acini. Intriguingly, we found deep nuclear invaginations and tunnels traversing the nucleus, encasing cytoskeletal actin and/or intermediate filaments, which connect tomore » the outer nuclear envelope. Also, the cytoskeleton is connected both to other cells through desmosome adhesion complexes and to the extracellular matrix through hemidesmosomes. This finding supports a physical and/or mechanical link from the desmosomes and hemidesmosomes to the nucleus, which had previously been hypothesized but now is visualized for the first time. These unique structures, including the nuclear invaginations and the cytoskeletal connectivity to the cell nucleus, are consistent with a dynamic reciprocity between the nucleus and the outside of epithelial cells and tissues.« less
  • Highlights: Black-Right-Pointing-Pointer Vitreous induces morphological changes and cytoskeletal rearrangements in RPE cells. Black-Right-Pointing-Pointer Rac1 is activated in vitreous-transformed RPE cells. Black-Right-Pointing-Pointer Rac inhibition prevents morphological changes in vitreous-transformed RPE cells. Black-Right-Pointing-Pointer Rac inhibition suppresses cytoskeletal rearrangements in vitreous-transformed RPE cells. Black-Right-Pointing-Pointer The vitreous-induced effects are mediated by a Rac1 GTPase/LIMK1/cofilin pathway. -- Abstract: Proliferative vitreoretinopathy (PVR) is mainly caused by retinal pigment epithelial (RPE) cell migration, invasion, proliferation and transformation into fibroblast-like cells that produce the extracellular matrix (ECM). The vitreous humor is known to play an important role in PVR. An epithelial-to-mesenchymal transdifferentiation (EMT) of human RPE cells inducedmore » by 25% vitreous treatment has been linked to stimulation of the mesenchymal phenotype, migration and invasion. Here, we characterized the effects of the vitreous on the cell morphology and cytoskeleton in human RPE cells. The signaling pathway that mediates these effects was investigated. Serum-starved RPE cells were incubated with 25% vitreous, and the morphological changes were examined by phase-contrast microscopy. Filamentous actin (F-actin) was examined by immunofluorescence and confocal microscopy. Protein phosphorylation of AKT, ERK1/2, Smad2/3, LIM kinase (LIMK) 1 and cofilin was analyzed by Western blot analysis. Vitreous treatment induced cytoskeletal rearrangements, activated Rac1 and enhanced the phosphorylation of AKT, ERK1/2 and Smad2/3. When the cells were treated with a Rac activation-specific inhibitor, the cytoskeletal rearrangements were prevented, and the phosphorylation of Smad2/3 was blocked. Vitreous treatment also enhanced the phosphorylation of LIMK1 and cofilin and the Rac inhibitor blocked this effect. We propose that vitreous-transformed human RPE cells undergo cytoskeletal rearrangements via Rac1 GTPase-dependent pathways that modulate LIMK1 and cofilin activity. The TGF{beta}-like activity of the vitreous may participate in this effect. Actin polymerization causes the cytoskeletal rearrangements that lead to the plasticity of vitreous-transformed RPE cells in PVR.« less
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  • We present a multi-technique study on in vitro epithelial–mesenchymal transition (EMT) in human MCF-7 cells cultured on electrospun scaffolds of poly(L-lactic acid) (PLA), with random and aligned fiber orientations. Our aim is to investigate the morphological and genetic characteristics induced by extracellular matrix in tumor cells cultured in different 3D environments, and at different time points. Cell vitality was assessed with AlamarBlue at days 1, 3, 5 and 7. Scanning electron microscopy was performed at culture days 3 and 7. Immunohistochemistry (for E-cadherin, β-catenin, cytokeratins, nucleophosmin, tubulin, Ki-67 and vimentin), immunofluorescence (for F-actin) western blot (for E-cadherin, β-catenin and vimentin)more » and transmission electron microscopy were carried out at day 7. An EMT gene array followed by PCR analysis confirmed the regulation of selected genes. At day 7, scanning electron microscopy on aligned-PLA revealed spindle-shaped cells gathered in buds and ribbon-like structures, with a higher nucleolar/nuclear ratio and a loss in E-cadherin and β-catenin at immunohistochemistry and western blot. An up-regulation of SMAD2, TGF-β2, TFPI2 and SOX10 was found in aligned-PLA compared to random-PLA cultured cells. The topography of the extracellular matrix has a role in tumor EMT, and a more aggressive phenotype characterizes MCF-7 cells cultured on aligned-PLA scaffold. -- Highlights: • After 7 culture days an aligned-PLA scaffold induces a spindle shape to MCF-7 cells. • Despite these changes, the aligned MCF-7 cells keep an epithelial phenotype. • The extracellular environment alone influences the E-cadherin/β-catenin axis. • The extracellular environment can promote the epithelial–mesenchymal transition.« less
  • Epithelial–mesenchymal transition (EMT) acts as a facilitator of metastatic dissemination in the invasive margin of malignant tumors where active tumor–stromal crosstalks take place. Co-cultures of cancer cells with cancer-associated fibroblasts (CAFs) are often used as in vitro models of EMT. We established a tumor–fibroblast proximity co-culture using HT-29 tumor spheroids (TSs) with CCD-18co fibroblasts. When co-cultured with TSs, CCD-18co appeared activated, and proliferative activity as well as cell migration increased. Expression of fibronectin increased whereas laminin and type I collagen decreased in TSs co-cultured with fibroblasts compared to TSs alone, closely resembling the margin of in vivo xenograft tissue. Activemore » TGFβ1 in culture media significantly increased in TS co-cultures but not in 2D co-cultures of cancer cells–fibroblasts, indicating that 3D context-associated factors from TSs may be crucial to crosstalks between cancer cells and fibroblasts. We also observed in TSs co-cultured with fibroblasts increased expression of α-SMA, EGFR and CTGF; reduced expression of membranous β-catenin and E-cadherin, together suggesting an EMT-like changes similar to a marginal region of xenograft tissue in vivo. Overall, our in vitro TS–fibroblast proximity co-culture mimics the EMT-state of the invasive margin of in vivo tumors in early metastasis. - Highlights: • An adjacent co-culture of tumor spheroids and fibroblasts is presented as EMT model. • Activation of fibroblasts and increased cell migration were shown in co-culture. • Expression of EMT-related factors in co-culture was similar to that in tumor tissue. • Crosstalk between spheroids and fibroblasts was demonstrated by secretome analysis.« less