scholarly journals Midbody remnant inheritance is regulated by the ESCRT subunit CHMP4C

2019 ◽  
Author(s):  
Javier Casares-Arias ◽  
María Ujué Gonzalez ◽  
Alvaro San Paulo ◽  
Leandro N. Ventimiglia ◽  
Jessica B. A. Sadler ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Apical Membrane ◽  
Ciliated Cells ◽  
Daughter Cells ◽  
Escrt Machinery ◽  
Cilium Formation ◽  
Functional Consequences ◽  
Development And Differentiation ◽  
The Relationship

AbstractThe inheritance of the midbody remnant (MBR) breaks the symmetry of the two daughter cells, with functional consequences for lumen and primary cilium formation by polarized epithelial cells, and also for development and differentiation. However, despite their importance, neither the relationship between the plasma membrane and the inherited MBR nor the mechanism of MBR inheritance is well known. Here, the analysis by correlative light and ultra-high-resolution scanning electron microscopy reveals a membranous stalk that physically connects the MBR to the apical membrane of epithelial cells. The stalk, which derives from the uncleaved side of the midbody, concentrates the ESCRT machinery. The ESCRT CHMP4C subunit enables MBR inheritance, and its depletion dramatically reduces the percentage of ciliated cells. We demonstrate: (1) that MBRs are physically connected to the plasma membrane, (2) how CHMP4C helps maintain the integrity of the connection, and (3) the functional importance of the connection.

scholarly journals Novel role for the midbody in primary ciliogenesis by polarized epithelial cells

2016 ◽  
Vol 214 (3) ◽  
pp. 259-273 ◽  
Author(s):  
Miguel Bernabé-Rubio ◽  
Germán Andrés ◽  
Javier Casares-Arias ◽  
Jaime Fernández-Barrera ◽  
Laura Rangel ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Population Based ◽  
Tissue Homeostasis ◽  
Apical Surface ◽  
Membrane Protrusion ◽  
Ciliated Cells ◽  
Daughter Cells ◽  
Cilium Formation ◽  
Vertebrate Tissue ◽  
Polarized Epithelial Cells

The primary cilium is a membrane protrusion that is crucial for vertebrate tissue homeostasis and development. Here, we investigated the uncharacterized process of primary ciliogenesis in polarized epithelial cells. We show that after cytokinesis, the midbody is inherited by one of the daughter cells as a remnant that initially locates peripherally at the apical surface of one of the daughter cells. The remnant then moves along the apical surface and, once proximal to the centrosome at the center of the apical surface, enables cilium formation. The physical removal of the remnant greatly impairs ciliogenesis. We developed a probabilistic cell population–based model that reproduces the experimental data. In addition, our model explains, solely in terms of cell area constraints, the various observed transitions of the midbody, the beginning of ciliogenesis, and the accumulation of ciliated cells. Our findings reveal a biological mechanism that links the three microtubule-based organelles—the midbody, the centrosome, and the cilium—in the same cellular process.

Purinergic control of apical plasma membrane PI(4,5)P2 levels sets ENaC activity in principal cells

2008 ◽  
Vol 294 (1) ◽  
pp. F38-F46 ◽  
Author(s):  
Oleh Pochynyuk ◽  
Vladislav Bugaj ◽  
Alain Vandewalle ◽  
James D. Stockand
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Purinergic Receptors ◽  
Apical Membrane ◽  
Purinergic Signaling ◽  
Apical Plasma Membrane ◽  
Resting Cells ◽  
Open Probability ◽  
Renal Epithelial Cells ◽  
Principal Cells

Activity of the epithelial sodium channel (ENaC) is limiting for Na+ reabsorption at the distal nephron. Phosphoinositides, such as phosphatidylinositol 4,5-biphosphate [PI(4,5)P2] modulate the activity of this channel. Activation of purinergic receptors triggers multiple events, including activation of PKC and PLC, with the latter depleting plasma membrane PI(4,5)P2. Here, we investigate regulation of ENaC in renal principal cells by purinergic receptors via PLC and PI(4,5)P2. Purinergic signaling rapidly decreases ENaC open probability and apical membrane PI(4,5)P2 levels with similar time courses. Moreover, inhibiting purinergic signaling with suramin rescues ENaC activity. The PLC inhibitor U73122, but not U73343, its inactive analog, recapitulates the action of suramin. In contrast, modulating PKC signaling failed to affect purinergic regulation of ENaC. Unexpectedly, inhibiting either purinergic receptors or PLC in resting cells dramatically increased ENaC activity above basal levels, indicating tonic activation of purinergic signaling in these polarized renal epithelial cells. Increased ENaC activity was associated with elevation of apical membrane PI(4,5)P2 levels. Subsequent treatment with ATP in the presence of inhibited purinergic signaling failed to decrease ENaC activity and apical membrane PI(4,5)P2 levels. Dwell-time analysis reveals that depletion of PI(4,5)P2 forces ENaC toward a closed state. In contrast, increasing PI(4,5)P2 levels above basal values locks the channel in an open state interrupted by brief closings. Thus our results suggest that purinergic control of apical membrane PI(4,5)P2 levels is a major regulator of ENaC activity in renal epithelial cells.

scholarly journals Local phosphocycling mediated by LOK/SLK restricts ezrin function to the apical aspect of epithelial cells

2012 ◽  
Vol 199 (6) ◽  
pp. 969-984 ◽  
Author(s):  
Raghuvir Viswanatha ◽  
Patrice Y. Ohouo ◽  
Marcus B. Smolka ◽  
Anthony Bretscher
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Dynamic Process ◽  
Ribonucleic Acid ◽  
Kinase Activity ◽  
Apical Membrane ◽  
Drug Resistant ◽  
Domain Specific ◽  
Local Inhibition ◽  
Apical Domain

In this paper, we describe how a dynamic regulatory process is necessary to restrict microvilli to the apical aspect of polarized epithelial cells. We found that local phosphocycling regulation of ezrin, a critical plasma membrane–cytoskeletal linker of microvilli, was required to restrict its function to the apical membrane. Proteomic approaches and ribonucleic acid interference knockdown identified lymphocyte-oriented kinase (LOK) and SLK as the relevant kinases. Using drug-resistant LOK and SLK variants showed that these kinases were sufficient to restrict ezrin function to the apical domain. Both kinases were enriched in microvilli and locally activated there. Unregulated kinase activity caused ezrin mislocalization toward the basolateral domain, whereas expression of the kinase regulatory regions of LOK or SLK resulted in local inhibition of ezrin phosphorylation by the endogenous kinases. Thus, the domain-specific presence of microvilli is a dynamic process requiring a localized kinase driving the phosphocycling of ezrin to continually bias its function to the apical membrane.

scholarly journals Evidence for Apical Endocytosis in Polarized Hepatic Cells: Phosphoinositide 3-Kinase Inhibitors Lead to the Lysosomal Accumulation of Resident Apical Plasma Membrane Proteins

1999 ◽  
Vol 145 (5) ◽  
pp. 1089-1102 ◽  
Author(s):  
Pamela L. Tuma ◽  
Catherine M. Finnegan ◽  
Ji-Hyun Yi ◽  
Ann L. Hubbard
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Epithelial Cells ◽  
Kinase Inhibitors ◽  
Apical Membrane ◽  
Membrane Dynamics ◽  
Endocytic Pathway ◽  
Apical Plasma Membrane ◽  
Plasma Membrane Proteins ◽  
Phosphoinositide 3 Kinase

The architectural complexity of the hepatocyte canalicular surface has prevented examination of apical membrane dynamics with methods used for other epithelial cells. By adopting a pharmacological approach, we have documented for the first time the internalization of membrane proteins from the hepatic apical surface. Treatment of hepatocytes or WIF-B cells with phosphoinositide 3-kinase inhibitors, wortmannin or LY294002, led to accumulation of the apical plasma membrane proteins, 5′-nucleotidase and aminopeptidase N in lysosomal vacuoles. By monitoring the trafficking of antibody-labeled molecules, we determined that the apical proteins in vacuoles came from the apical plasma membrane. Neither newly synthesized nor transcytosing apical proteins accumulated in vacuoles. In wortmannin-treated cells, transcytosing apical proteins traversed the subapical compartment (SAC), suggesting that this intermediate in the basolateral-to-apical transcytotic pathway remained functional. Ultrastructural analysis confirmed these results. However, apically internalized proteins did not travel through SAC en route to lysosomal vacuoles, indicating that SAC is not an intermediate in the apical endocytic pathway. Basolateral membrane protein distributions did not change in treated cells, uncovering another difference in endocytosis from the two domains. Similar effects were observed in polarized MDCK cells, suggesting conserved patterns of phosphoinositide 3-kinase regulation among epithelial cells. These results confirm a long-held but unproven assumption that lysosomes are the final destination of apical membrane proteins in hepatocytes. Significantly, they also confirm our hypothesis that SAC is not an apical endosome.

scholarly journals Mechanisms and functional features of polarized membrane traffic in epithelial and hepatic cells

1998 ◽  
Vol 336 (2) ◽  
pp. 257-269 ◽  
Author(s):  
Mirjam M. P. ZEGERS ◽  
Dick HOEKSTRA
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Transport Processes ◽  
Protein Kinase Activity ◽  
Transport Pathway ◽  
Hepatic Cells ◽  
Functional Features ◽  
Specific Lipid

Epithelial cells express plasma-membrane polarity in order to meet functional requirements that are imposed by their interaction with different extracellular environments. Thus apical and basolateral membrane domains are distinguished that are separated by tight junctions in order to maintain the specific lipid and protein composition of each domain. In hepatic cells, the plasma membrane is also polarized, containing a sinusoidal (basolateral) and a bile canalicular (apical)-membrane domain. Relevant to the biogenesis of these domains are issues concerning sorting, (co-)transport and regulation of transport of domain-specific membrane components. In epithelial cells, specific proteins and lipids, destined for the apical membrane, are sorted in the trans-Golgi network (TGN), which involves their sequestration into cholesterol/sphingolipid ‘rafts ’, followed by ‘direct ’ transport to the apical membrane. In hepatic cells, a direct apical transport pathway also exists, as revealed by transport of sphingolipids from TGN to the apical membrane. This is remarkable, since in these cells numerous apical membrane proteins are ‘indirectly ’ sorted, i.e. they are first transferred to the basolateral membrane prior to their subsequent transcytosis to the apical membrane. This raises intriguing questions as to the existence of specific lipid rafts in hepatocytes. As demonstrated in studies with HepG2 cells, it has become evident that, in hepatic cells, apical transport pathways can be regulated by protein kinase activity, which in turn modulates cell polarity. Finally, an important physiological function of hepatic cells is their involvement in intracellular transport and secretion of bile-specific lipids. Mechanisms of these transport processes, including the role of multidrug-resistant proteins in lipid translocation, will be discussed in the context of intracellular vesicular transport. Taken together, hepatic cell systems provide an important asset to studies aimed at elucidating mechanisms of sorting and trafficking of lipids (and proteins) in polarized cells in general.

Pseudomonas aeruginosainhibits endocytic recycling of CFTR in polarized human airway epithelial cells

2006 ◽  
Vol 290 (3) ◽  
pp. C862-C872 ◽  
Author(s):  
Agnieszka Swiatecka-Urban ◽  
Sophie Moreau-Marquis ◽  
Daniel P. MacEachran ◽  
John P. Connolly ◽  
Caitlin R. Stanton ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Apical Membrane ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Membrane Expression ◽  
Endocytic Recycling ◽  
Human Airway ◽  
Δf508 Cftr ◽  
Human Airway Epithelial Cells

The most common mutation in the CFTR gene in individuals with cystic fibrosis (CF), ΔF508, leads to the absence of CFTR Cl−channels in the apical plasma membrane, which in turn results in impairment of mucociliary clearance, the first line of defense against inhaled bacteria. Pseudomonas aeruginosa is particularly successful at colonizing and chronically infecting the lungs and is responsible for the majority of morbidity and mortality in patients with CF. Rescue of ΔF508-CFTR by reduced temperature or chemical means reveals that the protein is at least partially functional as a Cl−channel. Thus current research efforts have focused on identification of drugs that restore the presence of CFTR in the apical membrane to alleviate the symptoms of CF. Because little is known about the effects of P. aeruginosa on CFTR in the apical membrane, whether P. aeruginosa will affect the efficacy of new drugs designed to restore the plasma membrane expression of CFTR is unknown. Accordingly, the objective of the present study was to determine whether P. aeruginosa affects CFTR-mediated Cl−secretion in polarized human airway epithelial cells. We report herein that a cell-free filtrate of P. aeruginosa reduced CFTR-mediated transepithelial Cl−secretion by inhibiting the endocytic recycling of CFTR and thus the number of WT-CFTR and ΔF508-CFTR Cl−channels in the apical membrane in polarized human airway epithelial cells. These data suggest that chronic infection with P. aeruginosa may interfere with therapeutic strategies aimed at increasing the apical membrane expression of ΔF508-CFTR.

scholarly journals Apiconuclear Organization of Microtubules Does Not Specify Protein Delivery from the Trans-Golgi Network to Different Membrane Domains in Polarized Epithelial Cells

1998 ◽  
Vol 9 (3) ◽  
pp. 685-699 ◽  
Author(s):  
Kent K. Grindstaff ◽  
Robert L. Bacallao ◽  
W. James Nelson
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Golgi Complex ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Membrane Domains ◽  
Trans Golgi Network ◽  
G Cells ◽  
Polarized Epithelial Cells ◽  
Golgi Network

In nonpolarized epithelial cells, microtubules originate from a broad perinuclear region coincident with the distribution of the Golgi complex and extend outward to the cell periphery (perinuclear [PN] organization). During development of epithelial cell polarity, microtubules reorganize to form long cortical filaments parallel to the lateral membrane, a meshwork of randomly oriented short filaments beneath the apical membrane, and short filaments at the base of the cell; the Golgi becomes localized above the nucleus in the subapical membrane cytoplasm (apiconuclear [AN] organization). The AN-type organization of microtubules is thought to be specialized in polarized epithelial cells to facilitate vesicle trafficking between the trans-Golgi Network (TGN) and the plasma membrane. We describe two clones of MDCK cells, which have different microtubule distributions: clone II/G cells, which gradually reorganize a PN-type distribution of microtubules and the Golgi complex to an AN-type during development of polarity, and clone II/J cells which maintain a PN-type organization. Both cell clones, however, exhibit identical steady-state polarity of apical and basolateral proteins. During development of cell surface polarity, both clones rapidly establish direct targeting pathways for newly synthesized gp80 and gp135/170, and E-cadherin between the TGN and apical and basolateral membrane, respectively; this occurs before development of the AN-type microtubule/Golgi organization in clone II/G cells. Exposure of both clone II/G and II/J cells to low temperature and nocodazole disrupts >99% of microtubules, resulting in: 1) 25–50% decrease in delivery of newly synthesized gp135/170 and E-cadherin to the apical and basolateral membrane, respectively, in both clone II/G and II/J cells, but with little or no missorting to the opposite membrane domain during all stages of polarity development; 2) ∼40% decrease in delivery of newly synthesized gp80 to the apical membrane with significant missorting to the basolateral membrane in newly established cultures of clone II/G and II/J cells; and 3) variable and nonspecific delivery of newly synthesized gp80 to both membrane domains in fully polarized cultures. These results define several classes of proteins that differ in their dependence on intact microtubules for efficient and specific targeting between the Golgi and plasma membrane domains.

scholarly journals Functional Consequences of Dual Oxidase-Thyroperoxidase Interaction at the Plasma Membrane

2010 ◽  
Vol 95 (12) ◽  
pp. 5403-5411 ◽  
Author(s):  
Rodrigo Soares Fortunato ◽  
Elaine Cristina Lima de Souza ◽  
Rabii Ameziane-el Hassani ◽  
Myriem Boufraqech ◽  
Urbain Weyemi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Apical Membrane ◽  
Hek293 Cells ◽  
Enzyme Properties ◽  
Heterologous System ◽  
Thyroid Hormone Biosynthesis ◽  
Dual Oxidase ◽  
Functional Consequences ◽  
Normal Enzyme ◽  
Hormone Biosynthesis

Context: Thyroperoxidase (TPO) and dual oxidase (DUOX) are present at the apical membrane of thyrocytes, where TPO catalyzes thyroid hormone biosynthesis in the presence of H2O2 produced by DUOX. Both enzymes are colocalized and associated, but the consequences of this interaction remain obscure. Objective: The objective of this study was to evaluate the functional consequences of TPO-DUOX interaction at the plasma membrane. Design: The functional consequences of DUOX-TPO interaction were studied by measuring extracellular H2O2 concentration and TPO activity in a heterologous system. For this purpose, HEK293 cells were transiently transfected with a combination of human TPO with human DUOX1 or DUOX2 in the presence of their respective maturation factors, DUOXA1 or DUOXA2. The effect of human DUOX2 mutants in which cysteine residues in the N-terminal domain were replaced by glycines was also analyzed. Results: We observed that production of H2O2 decreases both TPO and DUOX activities. We show that TPO presents a catalase-like effect that protects DUOX from inhibition by H2O2. This catalase-like effect depends on the association between both enzymes, which probably occurs through the DUOX peroxidase-like domain because this effect was not observed with human DUOX2 mutants. Conclusion: The DUOX-TPO association at the plasma membrane is relevant for normal enzyme properties. Normally, TPO consumes H2O2 produced by DUOX, decreasing the availability of this substance at the apical membrane of thyrocytes and, in turn, probably decreasing the oxidative damage of macromolecules.

Cellular and subcellular localizations of annexins I, IV, and VI in lung epithelia

1996 ◽  
Vol 270 (5) ◽  
pp. L863-L871 ◽  
Author(s):  
N. Mayran ◽  
V. Traverso ◽  
S. Maroux ◽  
D. Massey-Harroche
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Lung Epithelial Cells ◽  
Type I ◽  
Type Ii ◽  
Ciliated Cells ◽  
Alveolar Cells ◽  
Annexin I ◽  
Type Ii Pneumocytes ◽  
Immunofluorescence Labeling

The cellular and subcellular localizations of annexins I, IV, and VI in the rabbit tracheal and alveolar epithelia were studied by performing immunofluorescence labeling on thin frozen sections of these tissues, using specific monoclonal antibodies. Annexin I was highly expressed by ciliated cells, where it was concentrated in the cilia but was also present along the basolateral domain of the plasma membrane and in the nuclei. It was also abundant in the cytoplasm of type II pneumocytes and alveolar macrophages. Either one or both of these alveolar cells might be capable of secreting a very small amount of annexin I, which was found to be associated with the surfactant layer. Annexin IV was synthesized by all the lung epithelial cells. It was associated with the plasma membrane basolateral domain in ciliated cells and with either the apical or basal domain of plasma membrane in type I pneumocytes, whereas it was cytoplasmic in goblet cells and type II pneumocytes. Annexin VI was expressed only by alveolar endothelial cells, where it was probably cytoplasmic. None of these three annexins seem to be expressed in the nondifferentiated tracheal basal epithelial cells.

scholarly journals Polarity protein distribution on the metaphase furrow regulates hexagon dominated plasma membrane organization in syncytial Drosophila embryos

2019 ◽  
Author(s):  
Bipasha Dey ◽  
Debasmita Mitra ◽  
Tirthasree Das ◽  
Aparna Sherlekar ◽  
Ramya Balaji ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Apical Membrane ◽  
Protein Complexes ◽  
Membrane Organization ◽  
Protein Distribution ◽  
Lateral Membrane ◽  
Nuclear Cycle ◽  
Complex Distribution ◽  
Drosophila Embryos

AbstractEpithelial cells have a polarised distribution of protein complexes on the lateral membrane and are present as a polygonal array dominated by hexagons. Metazoan embryogenesis enables the study of temporal formation of the polygonal array and mechanisms that regulate its distribution. The plasma membrane of the syncytial Drosophila blastoderm embryo is organized as a polygonal array during cortical division cycles with an apical membrane and lateral furrow in between adjacent nuclei. We find that polygonal plasma membrane organization arises in syncytial division cycle 11 and hexagon dominance occurs with increase in furrow length in cycle 12. This is coincident with DE-cadherin and Bazooka enrichment at edges and the septin, Peanut enrichment at vertices of the base of the furrow. DE-cadherin depletion leads to loss of hexagon dominance. Bazooka and Peanut depletion leads to a delay in occurrence of hexagon dominance from nuclear cycle 12 to 13. Hexagon dominance in Bazooka and Peanut mutants occurs with furrow extension and correlates with increase in DE-cadherin in syncytial cycle 13. We conclude that a change in polarity complex distribution leads to loss of furrow stability thereby changing the polygonal organization of the blastoderm embryo.Highlight Summary for TOCMetazoan embryogenesis starts with the formation of polygonal epithelial-like cells. We show that hexagon dominance in polygonal epithelial-like plasma membrane organization occurs in nuclear cycle 12 in the syncytial blastoderm Drosophila embryo. DE-cadherin and Bazooka distribution along the lateral furrow regulates this hexagon dominance.

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