Faculty Opinions recommendation of Vectorial insertion of apical and basolateral membrane proteins in polarized epithelial cells revealed by quantitative 3D live cell imaging.

2006 ◽  
Author(s):  
Heike Fölsch
Keyword(s):  
Membrane Proteins ◽  
Epithelial Cells ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Basolateral Membrane ◽  
Live Cell ◽  
Polarized Epithelial Cells

scholarly journals Vectorial insertion of apical and basolateral membrane proteins in polarized epithelial cells revealed by quantitative 3D live cell imaging

2006 ◽  
Vol 172 (7) ◽  
pp. 1035-1044 ◽  
Author(s):  
Wei Hua ◽  
David Sheff ◽  
Derek Toomre ◽  
Ira Mellman
Keyword(s):  
Membrane Proteins ◽  
Epithelial Cells ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Basolateral Membrane ◽  
Live Cell ◽  
Junctional Complex ◽  
Apical Surface

Although epithelial cells are known to exhibit a polarized distribution of membrane components, the pathways responsible for delivering membrane proteins to their appropriate domains remain unclear. Using an optimized approach to three-dimensional live cell imaging, we have visualized the transport of newly synthesized apical and basolateral membrane proteins in fully polarized filter-grown Madin–Darby canine kidney cells. We performed a detailed quantitative kinetic analysis of trans-Golgi network (TGN) exit, passage through transport intermediates, and arrival at the plasma membrane using cyan/yellow fluorescent protein–tagged glycosylphosphatidylinositol-anchored protein and vesicular stomatitis virus glycoprotein as apical and basolateral reporters, respectively. For both pathways, exit from the TGN was rate limiting. Furthermore, apical and basolateral proteins were targeted directly to their respective membranes, resolving current confusion as to whether sorting occurs on the secretory pathway or only after endocytosis. However, a transcytotic protein did reach the apical surface after a prior appearance basolaterally. Finally, newly synthesized proteins appeared to be delivered to the entire lateral or apical surface, suggesting—contrary to expectations—that there is not a restricted site for vesicle docking or fusion adjacent to the junctional complex.

scholarly journals Live cell imaging reveals focal adhesions mechanoresponses in mammary epithelial cells under sustained equibiaxial stress

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Lorena Sigaut ◽  
Catalina von Bilderling ◽  
Micaela Bianchi ◽  
Juan Eduardo Burdisso ◽  
Laura Gastaldi ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Mammary Epithelial Cells ◽  
Focal Adhesions ◽  
Mammary Epithelial ◽  
Live Cell

scholarly journals Quantitative live cell imaging of nanoparticle translocation into/across lung alveolar epithelial cells

2012 ◽  
Vol 26 (S1) ◽  
Author(s):  
Arnold Sipos ◽  
Farnoosh Fazlollahi ◽  
Yong Ho Kim ◽  
Robert H. Chow ◽  
Zea Borok ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Alveolar Epithelial Cells ◽  
Alveolar Epithelial

Visualization of transcytosis in MDCK cells using laser scanning confocal microscopy

1994 ◽  
Vol 52 ◽  
pp. 220-221
Author(s):  
Greg Martin ◽  
Rohit Cariappa ◽  
Ann L. Hubbard
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Epithelial Cells ◽  
Laser Scanning ◽  
Mdck Cells ◽  
Transmembrane Protein ◽  
Basolateral Membrane ◽  
Apical Plasma Membrane ◽  
Plasma Membrane Proteins ◽  
Polarized Epithelial Cells

The plasma membrane of polarized epithelial cells is composed of two structurally and functionally distinct domains -- the apical and basolateral -- that also differ in molecular composition. The routes followed by integral membrane proteins from their site of synthesis to their site of function varies between different kinds of epithelia. Madin-Darby canine kidney (MDCK) cells deliver plasma membrane proteins directly to the correct domain, while polarized hepatocytes deliver all newly synthesized plasma membrane proteins initially to the basolateral membrane, then retrieve and redirect the apical membrane proteins. We are studying the targeting signals and delivery routes of DPPIV, a single transmembrane protein whose destination is the apical domain in polarized epithelial cells.DPPIV transfected into MDCK cells is delivered to the basolateral plasma membrane after long (13hr) treatment with Brefeldin A (BFA). After BFA’s removal these molecules are retrieved from the basolateral membrane and transcytosed to the apical plasma membrane. This protocol provides a useful model for studies of the indirect route of protein sorting in polarized epithelial cells, since DPPIV at the basolateral surface can be labeled with specific antibody and then subsequently followed in living cells.

scholarly journals Membrane proteins follow multiple pathways to the basolateral cell surface in polarized epithelial cells

2009 ◽  
Vol 186 (2) ◽  
pp. 269-282 ◽  
Author(s):  
Glen A. Farr ◽  
Michael Hull ◽  
Ira Mellman ◽  
Michael J. Caplan
Keyword(s):  
Membrane Proteins ◽  
Epithelial Cells ◽  
Basolateral Membrane ◽  
Small Gtpases ◽  
Recycling Endosomes ◽  
Multiple Routes ◽  
Sorting Process ◽  
Polarized Epithelial Cells ◽  
Pass Through ◽  
Golgi Network

Newly synthesized apical and basolateral membrane proteins are sorted from one another in polarized epithelial cells. The trans-Golgi network participates in this sorting process, but some basolateral proteins travel from the Golgi to recycling endosomes (REs) before their surface delivery. Using a novel system for pulse–chase microscopy, we have visualized the postsynthetic route pursued by a newly synthesized cohort of Na,K-ATPase. We find that the basolateral delivery of newly synthesized Na,K-ATPase occurs via a pathway distinct from that pursued by the vesicular stomatitis virus G protein (VSV-G). Na,K-ATPase surface delivery occurs at a faster rate than that observed for VSV-G. The Na,K-ATPase does not pass through the RE compartment en route to the plasma membrane, and Na,K-ATPase trafficking is not regulated by the same small GTPases as other basolateral proteins. Finally, Na,K-ATPase and VSV-G travel in separate post-Golgi transport intermediates, demonstrating directly that multiple routes exist for transport from the Golgi to the basolateral membrane in polarized epithelial cells.

scholarly journals Correlative light and scanning electron microscopy (CLSEM) for analysis of bacterial infection of polarized epithelial cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Carina Kommnick ◽  
Andrea Lepper ◽  
Michael Hensel
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Cell Monolayers ◽  
Epithelial Cell Monolayers ◽  
Polarized Epithelial Cells ◽  
Scanning Electron

AbstractInfection of mammalian host cells by bacterial pathogens is a highly dynamic process and microscopy is instrumental to reveal the cellular and molecular details of host-pathogen interactions. Correlative light and electron microscopy (CLEM) combines the advantages of three-dimensional live cell imaging with ultrastructural analysis. The analyses of adhesion to, and invasion of polarized epithelial cells by pathogens often deploys scanning electron microscopy (SEM), since surface structures of the apical brush border can be analyzed in detail. Most available CLEM approaches focus on relocalization of separated single cells in different imaging modalities, but are not readily applicable to polarized epithelial cell monolayers, since orientation marks on substrate are overgrown during differentiation. To address this problem, we developed a simple and convenient workflow for correlative light and scanning electron microscopy (CLSEM), using gold mesh grids as carrier for growth of epithelial cell monolayers, and for imaging infection. The approach allows fast live cell imaging of bacterial infection of polarized cells with subsequent analyses by SEM. As examples for CLSEM applications, we investigated trigger invasion by Salmonella enterica, zipper invasion by Listeria monocytogenes, and the enterocyte attachment and effacement phenotype of enteropathogenic Escherichia coli. Our study demonstrates the versatile use of gold mesh grids for CLSEM of the interaction of bacterial pathogens with the apical side of polarized epithelial cells.

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