scholarly journals Multi-dimensional and spatiotemporal correlative imaging at the plasma membrane of live cells to determine the continuum nano-to-micro scale lipid adaptation and collective motion

2021 ◽  
Author(s):  
Miguel Bernabé-Rubio ◽  
Minerva Bosch-Fortea ◽  
Miguel A. Alonso ◽  
Jorge Bernardino de la Serna
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Primary Cilium ◽  
Cell Biology ◽  
Spatial Scales ◽  
Apical Surface ◽  
Ciliary Membrane ◽  
Correlative Imaging ◽  
Polarized Epithelial Cells ◽  
Membrane Patch

AbstractThe primary cilium is a specialized plasma membrane protrusion with important receptors for signalling pathways. In polarized epithelial cells, the primary cilium assembles after the midbody remnant (MBR) encounters the centrosome at the apical surface. The membrane surrounding the MBR, namely remnant associated membrane patch (RAMP) once situated next to the centrosome, releases some of its lipid components to form a centrosome-associated membrane patch (CAMP) from which the ciliary membrane stems. The RAMP undergoes a spatiotemporal membrane refinement during the formation of the CAMP, which becomes highly enriched in condensed membranes with low lateral mobility. To better understand this process, we have developed a correlative imaging approach that yields quantitative information about the lipid lateral packing, its mobility and collective assembly at the plasma membrane at different spatial scales over time. Our work paves the way towards a quantitative understanding of lipid collective assembly at the plasma membrane spatiotemporally as a functional determinant in cell biology and its direct correlation with the membrane physicochemical state. These findings allowed us to gain a deeper insight into the mechanisms behind the biogenesis of the ciliary membrane of polarized epithelial cells.

scholarly journals The ciliary membrane of polarized epithelial cells stems from a midbody remnant-associated membrane patch with condensed nanodomains

2019 ◽  
Author(s):  
Miguel Bernabé-Rubio ◽  
Minerva Bosch-Fortea ◽  
Esther García ◽  
Jorge Bernardino de la Serna ◽  
Miguel A. Alonso
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Primary Cilium ◽  
Mdck Cells ◽  
Cell Types ◽  
Apical Surface ◽  
Cellular Behavior ◽  
Ciliary Membrane ◽  
Lipid Dynamics

AbstractThe primary cilium is a specialized plasma membrane protrusion that harbors receptors involved in important signaling pathways. Despite its central role in regulating cellular behavior, the biogenesis of the primary cilium is not fully understood. In fact, the source of the ciliary membrane remains a mystery in cell types that assemble their primary cilium entirely at the cell surface, such as polarized renal epithelial cells. After cytokinesis, the remnant of the midbody of these cells moves to the center of the apical surface, where it licenses the centrosome for ciliogenesis through an unidentified mechanism. Here, to investigate the origin of the ciliary membrane and the role of the midbody remnant, we analyzed membrane compaction and lipid dynamics at the microscale and nanoscale in living renal epithelial MDCK cells. We found that a specialized patch made of condensed membranes with restricted lipid lateral mobility surrounds the midbody remnant. This patch accompanies the remnant on its journey towards the centrosome and, once the two structures have met, the remnant delivers part of membranes of the patch to build the ciliary membrane. In this way, we have determined the origin of the ciliary membrane and the contribution of the midbody remnant to primary cilium formation in cells whose primary cilium is assembled at the plasma membrane.

scholarly journals Par3 functions in the biogenesis of the primary cilium in polarized epithelial cells

2007 ◽  
Vol 179 (6) ◽  
pp. 1133-1140 ◽  
Author(s):  
Jeff Sfakianos ◽  
Akashi Togawa ◽  
Sandra Maday ◽  
Mike Hull ◽  
Marc Pypaert ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Primary Cilium ◽  
Basolateral Membrane ◽  
Intraflagellar Transport ◽  
Binding Motif ◽  
Apical Surface ◽  
Anterograde Transport ◽  
Ciliary Membrane ◽  
Microtubule Motor ◽  
Dependent Transport

Par3 is a PDZ protein important for the formation of junctional complexes in epithelial cells. We have identified an additional role for Par3 in membrane biogenesis. Although Par3 was not required for maintaining polarized apical or basolateral membrane domains, at the apical surface, Par3 was absolutely essential for the growth and elongation of the primary cilium. The activity reflected its ability to interact with kinesin-2, the microtubule motor responsible for anterograde transport of intraflagellar transport particles to the tip of the growing cilium. The Par3 binding partners Par6 and atypical protein kinase C interacted with the ciliary membrane component Crumbs3 and we show that the PDZ binding motif of Crumbs3 was necessary for its targeting to the ciliary membrane. Thus, the Par complex likely serves as an adaptor that couples the vectorial movement of at least a subset of membrane proteins to microtubule-dependent transport during ciliogenesis.

scholarly journals The periciliary ring in polarized epithelial cells is a hot spot for delivery of the apical protein gp135

2015 ◽  
Vol 211 (2) ◽  
pp. 287-294 ◽  
Author(s):  
Emily H. Stoops ◽  
Michael Hull ◽  
Christina Olesen ◽  
Kavita Mistry ◽  
Jennifer L. Harder ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Primary Cilium ◽  
Protein Delivery ◽  
Hot Spot ◽  
Basolateral Membrane ◽  
Protein A ◽  
Surface Proteins ◽  
Apical Surface ◽  
Directed Movement ◽  
Polarized Epithelial Cells

In polarized epithelial cells, newly synthesized cell surface proteins travel in carrier vesicles from the trans Golgi network to the apical or basolateral plasma membrane. Despite extensive research on polarized trafficking, the sites of protein delivery are not fully characterized. Here we use the SNAP tag system to examine the site of delivery of the apical glycoprotein gp135. We show that a cohort of gp135 is delivered to a ring surrounding the base of the primary cilium, followed by microtubule-dependent radial movement away from the cilium. Delivery to the periciliary ring was specific to newly synthesized and not recycling protein. A subset of this newly delivered protein traverses the basolateral membrane en route to the apical membrane. Crumbs3a, another apical protein, was not delivered to the periciliary region, instead making its initial apical appearance in a pattern that resembled its steady-state distribution. Our results demonstrate a surprising “hot spot” for gp135 protein delivery at the base of the primary cilium and suggest the existence of a novel microtubule-based directed movement of a subset of apical surface proteins.

scholarly journals Rab8 Regulates Basolateral Secretory, But Not Recycling, Traffic at the Recycling Endosome

2008 ◽  
Vol 19 (5) ◽  
pp. 2059-2068 ◽  
Author(s):  
Lauren Henry ◽  
David R. Sheff
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Mdck Cells ◽  
Apical Surface ◽  
Recycling Endosome ◽  
Recycling Pathway ◽  
Polarized Epithelial Cells ◽  
Pass Through

Rab8 is a monomeric GTPase that regulates the delivery of newly synthesized proteins to the basolateral surface in polarized epithelial cells. Recent publications have demonstrated that basolateral proteins interacting with the μ1-B clathrin adapter subunit pass through the recycling endosome (RE) en route from the TGN to the plasma membrane. Because Rab8 interacts with these basolateral proteins, these findings raise the question of whether Rab8 acts before, at, or after the RE. We find that Rab8 overexpression during the formation of polarity in MDCK cells, disrupts polarization of the cell, explaining how Rab8 mutants can disrupt basolateral endocytic and secretory traffic. However, once cells are polarized, Rab8 mutants cause mis-sorting of newly synthesized basolateral proteins such as VSV-G to the apical surface, but do not cause mis-sorting of membrane proteins already at the cell surface or in the endocytic recycling pathway. Enzymatic ablation of the RE also prevents traffic from the TGN from reaching the RE and similarly results in mis-sorting of newly synthesized VSV-G. We conclude that Rab8 regulates biosynthetic traffic through REs to the plasma membrane, but not trafficking of endocytic cargo through the RE. The data are consistent with a model in which Rab8 functions in regulating the delivery of TGN-derived cargo to REs.

scholarly journals Calcium levels in the Golgi complex regulate clustering and apical sorting of GPI-APs in polarized epithelial cells

2021 ◽  
Vol 118 (33) ◽  
pp. e2014709118
Author(s):  
Stéphanie Lebreton ◽  
Simona Paladino ◽  
Dandan Liu ◽  
Maria Nitti ◽  
Julia von Blume ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Golgi Complex ◽  
Calcium Binding ◽  
Calcium Concentration ◽  
Apical Surface ◽  
Vital Functions ◽  
Molecular Machinery ◽  
Polarized Epithelial Cells ◽  
Apical Sorting

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are lipid-associated luminal secretory cargoes selectively sorted to the apical surface of the epithelia where they reside and play diverse vital functions. Cholesterol-dependent clustering of GPI-APs in the Golgi is the key step driving their apical sorting and their further plasma membrane organization and activity; however, the specific machinery involved in this Golgi event is still poorly understood. In this study, we show that the formation of GPI-AP homoclusters (made of single GPI-AP species) in the Golgi relies directly on the levels of calcium within cisternae. We further demonstrate that the TGN calcium/manganese pump, SPCA1, which regulates the calcium concentration within the Golgi, and Cab45, a calcium-binding luminal Golgi resident protein, are essential for the formation of GPI-AP homoclusters in the Golgi and for their subsequent apical sorting. Down-regulation of SPCA1 or Cab45 in polarized epithelial cells impairs the oligomerization of GPI-APs in the Golgi complex and leads to their missorting to the basolateral surface. Overall, our data reveal an unexpected role for calcium in the mechanism of GPI-AP apical sorting in polarized epithelial cells and identify the molecular machinery involved in the clustering of GPI-APs in the Golgi.

scholarly journals Sensitivity of Polarized Epithelial Cells to the Pore-Forming Toxin Aerolysin

2003 ◽  
Vol 71 (2) ◽  
pp. 739-746 ◽  
Author(s):  
Laurence Abrami ◽  
Marc Fivaz ◽  
Pierre-Etienne Glauser ◽  
Nakaba Sugimoto ◽  
Chiara Zurzolo ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Cell Line ◽  
Mammalian Cells ◽  
Mdck Cells ◽  
Lectin Binding ◽  
Membrane Surface ◽  
Apical Surface ◽  
Apical Side ◽  
Polarized Epithelial Cells

ABSTRACT Aerolysin is one of the major virulence factors produced by Aeromonas hydrophila, a human pathogen that produces deep wound infection and gastroenteritis. The toxin interacts with target mammalian cells by binding to the glycan core of glycosylphosphatidyl inositol (GPI)-anchored proteins and subsequently forms a pore in the plasma membrane. Since epithelial cells of the intestine are the primary targets of aerolysin, we investigated its effect on three types of polarized epithelial cells: Caco-2 cells, derived from human intestine; MDCK cells, a well-characterized cell line in terms of protein targeting; and FRT cells, an unusual cell line in that it targets its GPI-anchored proteins to the basolateral plasma membrane in contrast to other epithelial cells, which target them almost exclusively to the apical surface. Surprisingly, we found that all three cell types were sensitive to the toxin from both the apical and the basolateral sides. Apical sensitivity was always higher, even for FRT cells. In contrast, FRT cells were more sensitive from the basolateral than from the apical side to the related toxin Clostridium septicum alpha-toxin, which also binds to GPI-anchored proteins but lacks the lectin binding domain found in aerolysin. These observations are consistent with the notion that a shuttling mechanism involving low-affinity interactions with surface sugars allows aerolysin to gradually move toward the membrane surface, where it can finally encounter the glycan cores of GPI-anchored proteins.

scholarly journals Mumps Virus Is Released from the Apical Surface of Polarized Epithelial Cells, and the Release Is Facilitated by a Rab11-Mediated Transport System

2015 ◽  
Vol 89 (23) ◽  
pp. 12026-12034 ◽  
Author(s):  
Hiroshi Katoh ◽  
Yuichiro Nakatsu ◽  
Toru Kubota ◽  
Masafumi Sakata ◽  
Makoto Takeda ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Measles Virus ◽  
Intracellular Transport ◽  
Systemic Infection ◽  
Mumps Virus ◽  
Progeny Virus ◽  
Apical Surface ◽  
Virus Release ◽  
Polarized Cells ◽  
Polarized Epithelial Cells

ABSTRACTMumps virus (MuV) is an airborne virus that causes a systemic infection in patients.In vivo, the epithelium is a major replication site of MuV, and thus, the mode of MuV infection of epithelial cells is a subject of interest. Our data in the present study showed that MuV entered polarized epithelial cells via both the apical and basolateral surfaces, while progeny viruses were predominantly released from the apical surface. In polarized cells, intracellular transport of viral ribonucleoprotein (vRNP) complexes was dependent on Rab11-positive endosomes, and vRNP complexes were transported to the apical membrane. Expression of a dominant negative form of Rab11 (Rab11S25N) reduced the progeny virus release in polarized cells but not in nonpolarized cells. Although in this way these effects were correlated with cell polarity, Rab11S25N did not modulate the direction of virus release from the apical surface. Therefore, our data suggested that Rab11 is not a regulator of selective apical release of MuV, although it acts as an activator of virus release from polarized epithelial cells. In addition, our data and previous studies on Sendai virus, respiratory syncytial virus, and measles virus suggested that selective apical release from epithelial cells is used by many paramyxoviruses, even though they cause either a systemic infection or a local respiratory infection.IMPORTANCEMumps virus (MuV) is the etiological agent of mumps and causes a systemic infection. However, the precise mechanism by which MuV breaks through the epithelial barriers and achieves a systemic infection remains unclear. In the present study, we show that the entry of MuV is bipolar, while the release is predominantly from the apical surface in polarized epithelial cells. In addition, the release of progeny virus was facilitated by a Rab11-positive recycling endosome and microtubule network. Our data provide important insights into the mechanism of transmission and pathogenesis of MuV.

scholarly journals Apical Scaffolding Protein NHERF2 Modulates the Localization of Alternatively Spliced Plasma Membrane Ca2+Pump 2B Variants in Polarized Epithelial Cells

2010 ◽  
Vol 285 (41) ◽  
pp. 31704-31712 ◽  
Author(s):  
Rita Padányi ◽  
Yuning Xiong ◽  
Géza Antalffy ◽  
Krisztina Lór ◽  
Katalin Pászty ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Scaffolding Protein ◽  
Alternatively Spliced ◽  
Polarized Epithelial Cells

scholarly journals Polarized Expression of CD74 by Gastric Epithelial Cells

2005 ◽  
Vol 53 (12) ◽  
pp. 1481-1489 ◽  
Author(s):  
Carlos A. Barrera ◽  
Ellen J. Beswick ◽  
Johanna C. Sierra ◽  
David Bland ◽  
Rosario Espejo ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Mhc Class Ii ◽  
Cell Biology ◽  
Constitutive Expression ◽  
Class Ii ◽  
Protein Isoforms ◽  
Apical Surface ◽  
Gastric Epithelial Cells ◽  
Mhc Molecules ◽  
Class Ii Mhc

CD74 is known as the major histocompatibility complex (MHC) class II-associated invariant chain (Ii) that regulates the cell biology and functions of MHC class II molecules. Class II MHC and Ii expression was believed to be restricted to classical antigen-presenting cells (APC); however, during inflammation, other cell types, including mucosal epithelial cells, have also been reported to express class II MHC molecules. Given the importance of Ii in the biology of class II MHC, we sought to examine the expression of Ii by gastric epithelial cells (GEC) to determine whether class II MHC molecules in these nonconventional APC cells were under the control of Ii and to further support the role that these cells may play in local immune and inflammatory responses during Helicobacter pylori infection. Thus we examined the expression of Ii on GEC from human biopsy samples and then confirmed this observation using independent methods on several GEC lines. The mRNA for Ii was detected by RT-PCR, and the various protein isoforms were also detected. Interestingly, these cells have a high level expression of surface Ii, which is polarized to the apical surface. These studies are the first to demonstrate the constitutive expression of Ii by human GEC.

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