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 Dual pulse-chase microscopy reveals early divergence in the biosynthetic trafficking of the Na,K-ATPase and E-cadherin

2015 ◽  
Vol 26 (24) ◽  
pp. 4401-4411 ◽  
Author(s):  
Glen A. Farr ◽  
Michael Hull ◽  
Emily H. Stoops ◽  
Rosalie Bateson ◽  
Michael J. Caplan
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Golgi Complex ◽  
Plasma Membranes ◽  
Secretory Pathway ◽  
Trans Golgi Network ◽  
Polarized Epithelial Cells ◽  
Golgi Network ◽  
E Cadherin

Recent evidence indicates that newly synthesized membrane proteins that share the same distributions in the plasma membranes of polarized epithelial cells can pursue a variety of distinct trafficking routes as they travel from the Golgi complex to their common destination at the cell surface. In most polarized epithelial cells, both the Na,K-ATPase and E-cadherin are localized to the basolateral domains of the plasma membrane. To examine the itineraries pursued by newly synthesized Na,K-ATPase and E-cadherin in polarized MDCK epithelial cells, we used the SNAP and CLIP labeling systems to fluorescently tag temporally defined cohorts of these proteins and observe their behaviors simultaneously as they traverse the secretory pathway. These experiments reveal that E-cadherin is delivered to the cell surface substantially faster than is the Na,K-ATPase. Furthermore, the surface delivery of newly synthesized E-cadherin to the plasma membrane was not prevented by the 19°C temperature block that inhibits the trafficking of most proteins, including the Na,K-ATPase, out of the trans-Golgi network. Consistent with these distinct behaviors, populations of newly synthesized E-cadherin and Na,K-ATPase become separated from one another within the trans-Golgi network, suggesting that they are sorted into different carrier vesicles that mediate their post-Golgi trafficking.

scholarly journals Epithelial polarity requires septin coupling of vesicle transport to polyglutamylated microtubules

2008 ◽  
Vol 180 (2) ◽  
pp. 295-303 ◽  
Author(s):  
Elias T. Spiliotis ◽  
Stephen J. Hunt ◽  
Qicong Hu ◽  
Makoto Kinoshita ◽  
W. James Nelson
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Basolateral Membrane ◽  
Vesicle Transport ◽  
Epithelial Polarity ◽  
Membrane Domains ◽  
Polarized Epithelia ◽  
Membrane Growth ◽  
Basolateral Plasma Membrane ◽  
Golgi Network

In epithelial cells, polarized growth and maintenance of apical and basolateral plasma membrane domains depend on protein sorting from the trans-Golgi network (TGN) and vesicle delivery to the plasma membrane. Septins are filamentous GTPases required for polarized membrane growth in budding yeast, but whether they function in epithelial polarity is unknown. Here, we show that in epithelial cells septin 2 (SEPT2) fibers colocalize with a subset of microtubule tracks composed of polyglutamylated (polyGlu) tubulin, and that vesicles containing apical or basolateral proteins exit the TGN along these SEPT2/polyGlu microtubule tracks. Tubulin-associated SEPT2 facilitates vesicle transport by maintaining polyGlu microtubule tracks and impeding tubulin binding of microtubule-associated protein 4 (MAP4). Significantly, this regulatory step is required for polarized, columnar-shaped epithelia biogenesis; upon SEPT2 depletion, cells become short and fibroblast-shaped due to intracellular accumulation of apical and basolateral membrane proteins, and loss of vertically oriented polyGlu microtubules. We suggest that septin coupling of the microtubule cytoskeleton to post-Golgi vesicle transport is required for the morphogenesis of polarized epithelia.

scholarly journals Exogenous MAL Reroutes Selected Hepatic Apical Proteins into the Direct Pathway in WIF-B Cells

2007 ◽  
Vol 18 (7) ◽  
pp. 2707-2715 ◽  
Author(s):  
Sai Prasad Ramnarayanan ◽  
Christina A. Cheng ◽  
Maria Bastaki ◽  
Pamela L. Tuma
Keyword(s):  
Plasma Membrane ◽  
B Cells ◽  
Epithelial Cells ◽  
Transmembrane Domain ◽  
Apical Membrane ◽  
Apical Plasma Membrane ◽  
Indirect Pathway ◽  
Trans Golgi Network ◽  
Direct Pathway ◽  
Golgi Network

Unlike simple epithelial cells that directly target newly synthesized glycophosphatidylinositol (GPI)-anchored and single transmembrane domain (TMD) proteins from the trans-Golgi network to the apical membrane, hepatocytes use an indirect pathway: proteins are delivered to the basolateral domain and then selectively internalized and transcytosed to the apical plasma membrane. Myelin and lymphocyte protein (MAL) and MAL2 have been identified as regulators of direct and indirect apical delivery, respectively. Hepatocytes lack endogenous MAL consistent with the absence of direct apical targeting. Does MAL expression reroute hepatic apical residents into the direct pathway? We found that MAL expression in WIF-B cells induced the formation of cholesterol and glycosphingolipid-enriched Golgi domains that contained GPI-anchored and single TMD apical proteins; polymeric IgA receptor (pIgA-R), polytopic apical, and basolateral resident distributions were excluded. Basolateral delivery of newly synthesized apical residents was decreased in MAL-expressing cells concomitant with increased apical delivery; pIgA-R and basolateral resident delivery was unchanged. These data suggest that MAL rerouted selected hepatic apical proteins into the direct pathway.

scholarly journals Sorting of Marburg Virus Surface Protein and Virus Release Take Place at Opposite Surfaces of Infected Polarized Epithelial Cells

2001 ◽  
Vol 75 (3) ◽  
pp. 1274-1283 ◽  
Author(s):  
Christian Sänger ◽  
Elke Mühlberger ◽  
Elena Ryabchikova ◽  
Larissa Kolesnikova ◽  
Hans-Dieter Klenk ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Surface Protein ◽  
Hemorrhagic Fever ◽  
Marburg Virus ◽  
Virus Surface ◽  
Mdckii Cells ◽  
Vesicular Stomatitis ◽  
Polarized Epithelial Cells

ABSTRACT Marburg virus, a filovirus, causes severe hemorrhagic fever with hitherto poorly understood molecular pathogenesis. We have investigated here the vectorial transport of the surface protein GP of Marburg virus in polarized epithelial cells. To this end, we established an MDCKII cell line that was able to express GP permanently (MDCK-GP). The functional integrity of GP expressed in these cells was analyzed using vesicular stomatitis virus pseudotypes. Further experiments revealed that GP is transported in MDCK-GP cells mainly to the apical membrane and is released exclusively into the culture medium facing the apical membrane. When MDCKII cells were infected with Marburg virus, the majority of GP was also transported to the apical membrane, suggesting that the protein contains an autonomous apical transport signal. Release of infectious progeny virions, however, took place exclusively at the basolateral membrane of the cells. Thus, vectorial budding of Marburg virus is presumably determined by factors other than the surface protein.

scholarly journals Sphingomyelin homeostasis is required to form functional enzymatic domains at the trans-Golgi network

2014 ◽  
Vol 206 (5) ◽  
pp. 609-618 ◽  
Author(s):  
Josse van Galen ◽  
Felix Campelo ◽  
Emma Martínez-Alonso ◽  
Margherita Scarpa ◽  
José Ángel Martínez-Menárguez ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Hela Cells ◽  
Transmembrane Proteins ◽  
Short Chain ◽  
Membrane Domains ◽  
Trans Golgi Network ◽  
And Function ◽  
Golgi Network ◽  
Specific Membrane

Do lipids such as sphingomyelin (SM) that are known to assemble into specific membrane domains play a role in the organization and function of transmembrane proteins? In this paper, we show that disruption of SM homeostasis at the trans-Golgi network (TGN) by treatment of HeLa cells with d-ceramide-C6, which was converted together with phosphatidylcholine to short-chain SM and diacylglycerol by SM synthase, led to the segregation of Golgi-resident proteins from each other. We found that TGN46, which cycles between the TGN and the plasma membrane, was not sialylated by a sialyltransferase at the TGN and that this enzyme and its substrate TGN46 could not physically interact with each other. Our results suggest that SM organizes transmembrane proteins into functional enzymatic domains at the TGN.

scholarly journals An Apical-Type Trafficking Pathway Is Present in Cultured Oligodendrocytes but the Sphingolipid-enriched Myelin Membrane Is the Target of a Basolateral-Type Pathway

1998 ◽  
Vol 9 (3) ◽  
pp. 599-609 ◽  
Author(s):  
Hans de Vries ◽  
Cobi Schrage ◽  
Dick Hoekstra
Keyword(s):  
Plasma Membrane ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Insoluble Fraction ◽  
Membrane Domains ◽  
Myelin Membrane ◽  
Influenza Virus Hemagglutinin ◽  
Polarized Cells ◽  
Vesicular Stomatitis ◽  
Triton X

Myelin sheets originate from distinct areas at the oligodendrocyte (OLG) plasma membrane and, as opposed to the latter, myelin membranes are relatively enriched in glycosphingolipids and cholesterol. The OLG plasma membrane can therefore be considered to consist of different membrane domains, as in polarized cells; the myelin sheet is reminiscent of an apical membrane domain and the OLG plasma membrane resembles the basolateral membrane. To reveal the potentially polarized membrane nature of OLG, the trafficking and sorting of two typical markers for apical and basolateral membranes, the viral proteins influenza virus–hemagglutinin (HA) and vesicular stomatitis virus–G protein (VSVG), respectively, were examined. We demonstrate that in OLG, HA and VSVG are differently sorted, which presumably occurs upon their trafficking through the Golgi. HA can be recovered in a Triton X-100-insoluble fraction, indicating an apical raft type of trafficking, whereas VSVG was only present in a Triton X-100-soluble fraction, consistent with its basolateral sorting. Hence, both an apical and a basolateral sorting mechanism appear to operate in OLG. Surprisingly, however, VSVG was found within the myelin sheets surrounding the cells, whereas HA was excluded from this domain. Therefore, despite its raft-like transport, HA does not reach a membrane that shows features typical of an apical membrane. This finding indicates either the uniqueness of the myelin membrane or the requirement of additional regulatory factors, absent in OLG, for apical delivery. These remarkable results emphasize that polarity and regulation of membrane transport in cultured OLG display features that are quite different from those in polarized cells.

scholarly journals Estimation of the amount of internalized ricin that reaches the trans-Golgi network

1988 ◽  
Vol 106 (2) ◽  
pp. 253-267 ◽  
Author(s):  
B van Deurs ◽  
K Sandvig ◽  
OW Petersen ◽  
S Olsnes ◽  
K Simons ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Golgi Complex ◽  
Endocytic Pathway ◽  
Membrane Glycoproteins ◽  
Double Labeling ◽  
Trans Golgi Network ◽  
A Chain ◽  
Golgi Network ◽  
Ricin A

We have used a protocol for internalization of ricin, a ligand binding to plasma membrane glycoproteins and glycolipids with terminal galactosyl residues, and infection with the vesicular stomatitis virus ts 045 mutant in BHK-21 cells to determine whether internalized plasma membrane molecules tagged by ricin reach distinct compartments of the biosynthetic-exocytic pathway. At 39.5 degrees C newly synthesized G protein of ts 045 was largely prevented from leaving the endoplasmic reticulum. At the same temperature ricin was endocytosed and reached, in addition to endosomes and lysosomes, elements of the Golgi complex. When the temperature was lowered to 19.5 degrees C, no more ricin was delivered to the Golgi complex, but now G protein accumulated in the Golgi stacks and the trans-Golgi network (TGN). Double-labeling immunogold cytochemistry on ultracryosections was used to detect G protein and ricin simultaneously. These data, combined with stereological and biochemical methods, showed that approximately 5% of the total amount of ricin within the cells, corresponding to 6-8 X 10(4) molecules per cell, colocalized with G protein in the Golgi complex after 60 min at 39.5 degrees C. Of this amount approximately 70-80% was present in the TGN. Since most of the ricin molecules remain bound to their binding sites at the low pH prevailing in compartments of the endocytic pathway, the results indicate that a fraction of the internalized plasma membrane molecules with terminal galactose are not recycled directly from endosomes or delivered to lysosomes, but are routed to the Golgi complex. Also, the results presented here, in combination with other recent studies on ricin internalization, suggest that translocation of the toxic ricin A-chain to the cytosol occurs in the TGN.

scholarly journals Phosphatidylinositol 3,4,5-trisphosphate Localization in Recycling Endosomes Is Necessary for AP-1B–dependent Sorting in Polarized Epithelial Cells

2010 ◽  
Vol 21 (1) ◽  
pp. 95-105 ◽  
Author(s):  
Ian C. Fields ◽  
Shelby M. King ◽  
Elina Shteyn ◽  
Richard S. Kang ◽  
Heike Fölsch
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Basolateral Membrane ◽  
Apical Plasma Membrane ◽  
Amino Acid Residues ◽  
Recycling Endosomes ◽  
C Terminus ◽  
Polarized Epithelial Cells ◽  
Clathrin Adaptor

Polarized epithelial cells coexpress two almost identical AP-1 clathrin adaptor complexes: the ubiquitously expressed AP-1A and the epithelial cell–specific AP-1B. The only difference between the two complexes is the incorporation of the respective medium subunits μ1A or μ1B, which are responsible for the different functions of AP-1A and AP-1B in TGN to endosome or endosome to basolateral membrane targeting, respectively. Here we demonstrate that the C-terminus of μ1B is important for AP-1B recruitment onto recycling endosomes. We define a patch of three amino acid residues in μ1B that are necessary for recruitment of AP-1B onto recycling endosomes containing phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3]. We found this lipid enriched in recycling endosomes of epithelial cells only when AP-1B is expressed. Interfering with PI(3,4,5)P3 formation leads to displacement of AP-1B from recycling endosomes and missorting of AP-1B–dependent cargo to the apical plasma membrane. In conclusion, PI(3,4,5)P3 formation in recycling endosomes is essential for AP-1B function.

scholarly journals A dileucine motif in the COOH-terminal domain of NKCC1 targets the cotransporter to the plasma membrane

2019 ◽  
Vol 316 (4) ◽  
pp. C545-C558 ◽  
Author(s):  
Rainelli Koumangoye ◽  
Salma Omer ◽  
Eric Delpire
Keyword(s):  
Epithelial Cells ◽  
Basolateral Membrane ◽  
Site Directed Mutagenesis ◽  
Heterozygous Mutation ◽  
Terminal Domain ◽  
Late Endosomes ◽  
Dileucine Motif ◽  
Polarized Epithelial Cells ◽  
Epithelial Dysfunction ◽  
Golgi Network

Na+-K+-2Cl− cotransporter-1 (NKCC1) mediates the electroneutral transport of Na+, K+, and Cl− and is normally localized to the basolateral membrane of polarized epithelial cells. We recently reported the first known solute carrier family 12 member 2 ( SLC12A2) mutation (we call NKCC1-DFX) that causes epithelial dysfunction in an undiagnosed disease program case. The heterozygous mutation leads to truncation of the COOH-terminal tail of the cotransporter, resulting in both mutant and wild-type cotransporters being mistrafficked to the apical membrane of polarized epithelial cells. Here we demonstrate by using consecutive truncations and site-directed mutagenesis of the COOH-terminal domain of NKCC1 that truncation of NKCC1 COOH domain uncouples the cotransporter from the lateral membrane. We identify a dileucine motif that, when mutated, leads to cotransporter accumulation in the cytoplasm and mistrafficking to the apical/subapical region of epithelial cells, thereby recapitulating the phenotype observed with the patient mutation. We show that truncation deletion and LL substitution mutants are trafficked out of the endoplasmic reticulum and trans-Golgi network but accumulate in early and late endosomes where they are degraded.

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.

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