High-tailing it to the apical surface. Focus on “Apical targeting of the P2Y4 receptor is directed by hydrophobic and basic residues in the cytoplasmic tail”

2013 ◽  
Vol 304 (3) ◽  
pp. C226-C227
Author(s):  
Ora A. Weisz
Keyword(s):  
Cytoplasmic Tail ◽  
Apical Surface ◽  
P2y4 Receptor

Sorting of rat liver and ileal sodium-dependent bile acid transporters in polarized epithelial cells

1998 ◽  
Vol 275 (5) ◽  
pp. G1045-G1055 ◽  
Author(s):  
An-Qiang Sun ◽  
Meenakshisundaram Ananthanarayanan ◽  
Carol J. Soroka ◽  
Sundararajah Thevananther ◽  
Benjamin L. Shneider ◽  
...  
Keyword(s):  
Bile Acid ◽  
Amino Acid ◽  
Mdck Cells ◽  
Cytoplasmic Tail ◽  
Apical Surface ◽  
Acid Transport ◽  
Transport Activity ◽  
Wild Type ◽  
Bile Acid Transport ◽  
Apical Domain

The rat ileal apical Na+-dependent bile acid transporter (ASBT) and the liver Na+-taurocholate cotransporting polypeptide (Ntcp) are members of a new family of anion transporters. These transport proteins share limited sequence homology and almost identical predicted secondary structures but are localized to the apical surface of ileal enterocytes and the sinusoidal surface of hepatocytes, respectively. Stably transfected Madin-Darby canine kidney (MDCK) cells appropriately localized wild-type ASBT and Ntcp apically and basolaterally as assessed by functional activity and immunocytochemical localization studies. Truncated and chimeric transporters were used to determine the functional importance of the cytoplasmic tail in bile acid transport activity and membrane localization. Two cDNAs were created encoding a truncated transporter in which the 56-amino-acid COOH-terminal tail of Ntcp was removed or substituted with an eight-amino-acid epitope FLAG. For both mutants there was some loss of fidelity in basolateral sorting in that ∼75% of each protein was delivered to the basolateral surface compared with ∼90% of the wild-type Ntcp protein. In contrast, deletion of the cytoplasmic tail of ASBT led to complete loss of transport activity and sorting to the apical membrane. An Ntcp chimera in which the 56-amino-acid COOH-terminal tail of Ntcp was replaced with the 40-amino-acid cytoplasmic tail of ASBT was largely redirected (82.4 ± 3.9%) to the apical domain of stably transfected MDCK cells, based on polarity of bile acid transport activity and localization by confocal immunofluorescence microscopy. These results indicate that a predominant signal for sorting of the Ntcp protein to the basolateral domain is located in a region outside of the cytoplasmic tail. These studies have further shown that a novel apical sorting signal is localized to the cytoplasmic tail of ASBT and that it is transferable and capable of redirecting a protein normally sorted to the basolateral surface to the apical domain of MDCK cells.

scholarly journals A Critical Phenylalanine Residue in the Respiratory Syncytial Virus Fusion Protein Cytoplasmic Tail Mediates Assembly of Internal Viral Proteins into Viral Filaments and Particles

mBio ◽  
2012 ◽  
Vol 3 (1) ◽  
Author(s):  
Fyza Y. Shaikh ◽  
Reagan G. Cox ◽  
Aaron W. Lifland ◽  
Anne L. Hotard ◽  
John V. Williams ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Cell Surface ◽  
Fusion Protein ◽  
Cytoplasmic Tail ◽  
Viral Proteins ◽  
General Strategy ◽  
Apical Surface ◽  
Filament Formation ◽  
F Protein ◽  
Syncytial Virus

ABSTRACTRespiratory syncytial virus (RSV) is a single-stranded RNA virus in theParamyxoviridaefamily that assembles into filamentous structures at the apical surface of polarized epithelial cells. These filaments contain viral genomic RNA and structural proteins, including the fusion (F) protein, matrix (M) protein, nucleoprotein (N), and phosphoprotein (P), while excluding F-actin. It is known that the F protein cytoplasmic tail (FCT) is necessary for filament formation, but the mechanism by which the FCT mediates assembly into filaments is not clear. We hypothesized that the FCT is necessary for interactions with other viral proteins in order to form filaments. In order to test this idea, we expressed the F protein with cytoplasmic tail (CT) truncations or specific point mutations and determined the abilities of these variant F proteins to form filaments independent of viral infection when coexpressed with M, N, and P. Deletion of the terminal three FCT residues (amino acids Phe-Ser-Asn) or mutation of the Phe residue resulted in a loss of filament formation but did not affect F-protein expression or trafficking to the cell surface. Filament formation could be restored by addition of residues Phe-Ser-Asn to an FCT deletion mutant and was unaffected by mutations to Ser or Asn residues. Second, deletion of residues Phe-Ser-Asn or mutation of the Phe residue resulted in a loss of M, N, and P incorporation into virus-like particles. These data suggest that a C-terminal Phe residue in the FCT mediates assembly through incorporation of internal virion proteins into virus filaments at the cell surface.IMPORTANCERespiratory syncytial virus (RSV) is a leading cause of bronchiolitis and pneumonia in infants and the elderly worldwide. There is no licensed RSV vaccine and only limited therapeutics for use in infected patients. Many aspects of the RSV life cycle have been studied, but the mechanisms that drive RSV assembly at the cell surface are not well understood. This study provides evidence that a specific residue in the RSV fusion protein cytoplasmic tail coordinates assembly into viral filaments by mediating the incorporation of internal virion proteins. Understanding the mechanisms that drive RSV assembly could lead to targeted development of novel antiviral drugs. Moreover, since RSV exits infected cells in an ESCRT (endosomal sorting complexes required for transport)-independent manner, these studies may contribute new knowledge about a general strategy by which ESCRT-independent viruses mediate outward bud formation using viral protein-mediated mechanisms during assembly and budding.

scholarly journals Characterization of a Di-leucine–based Signal in the Cytoplasmic Tail of the Nucleotide-pyrophosphatase NPP1 That Mediates Basolateral Targeting but not Endocytosis

2001 ◽  
Vol 12 (10) ◽  
pp. 3004-3015 ◽  
Author(s):  
Valérie Bello ◽  
James W. Goding ◽  
Vicki Greengrass ◽  
Adnan Sali ◽  
Valentina Dubljevic ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cytoplasmic Tail ◽  
Full Length ◽  
Apical Surface ◽  
Short Sequence ◽  
Conserved Sequence ◽  
Nucleotide Pyrophosphatase ◽  
Targeting Signal ◽  
Basolateral Targeting

Enzymes of the nucleotide pyrophosphatase/phosphodiesterase (NPPase) family are expressed at opposite surfaces in polarized epithelial cells. We investigated the targeting signal of NPP1, which is exclusively expressed at the basolateral surface. Full-length NPP1 and different constructs and mutants were transfected into the polarized MDCK cell line. Expression of the proteins was analyzed by confocal microscopy and surface biotinylation. The basolateral signal of NPP1 was identified as a di-leucine motif located in the cytoplasmic tail. Mutation of either or both leucines largely redirected NPP1 to the apical surface. Furthermore, addition of the conserved sequence AAASLLAP redirected the apical nucleotide pyrophosphatase/phosphodiesterase NPP3 to the basolateral surface. Full-length NPP1 was not significantly internalized. However, when the cytoplasmic tail was deleted upstream the di-leucine motif or when the six upstream flanking amino acids were deleted, the protein was mainly found intracellularly. Endocytosis experiments indicated that these mutants were endocytosed from the basolateral surface. These results identify the basolateral signal of NPP1 as a short sequence including a di-leucine motif that is dominant over apical determinants and point to the importance of surrounding amino acids in determining whether the signal will function as a basolateral signal only or as an endocytotic signal as well.

scholarly journals The cytoplasmic tail of CD44 is required for basolateral localization in epithelial MDCK cells but does not mediate association with the detergent-insoluble cytoskeleton of fibroblasts.

1993 ◽  
Vol 121 (6) ◽  
pp. 1299-1310 ◽  
Author(s):  
S J Neame ◽  
C M Isacke
Keyword(s):  
Epithelial Cells ◽  
Mdck Cells ◽  
Constitutive Expression ◽  
Cytoplasmic Tail ◽  
Apical Surface ◽  
Wild Type ◽  
Coated Pits ◽  
Triton X ◽  
Cortical Cytoskeleton ◽  
Human Specific

A number of recent reports on the trafficking of receptor proteins in MDCK epithelial cells have provided evidence that delivery to the basolateral domain requires a specific targeting sequence and that deletion of this sequence results in constitutive expression on the apical surface. To date, these studies have concentrated on receptors which are competent for internalization via the clathrin coated pits. We have examined the localization of a resident plasma membrane protein by transfecting human CD44 into MDCK cells. Using human specific and cross-species reactive antibodies, we show that in MDCK cells both the endogenous and transfected wild-type CD44 are found on the basolateral surface where they are restricted to the lateral domain. Deletion of the CD44 cytoplasmic tail reduces the half life of this mutant protein and causes it to be expressed both on the apical surface and to a significant extent within the cell. We have also used biochemical and morphological analysis to investigate the interaction of CD44 with the cytoskeleton in detergent extracted cells. Strikingly different extraction results were obtained between epithelial and fibroblast cells. However, there is no difference in the Triton X-100 solubility of the transfected wild-type and tail-less CD44 in fibroblasts and both forms of the protein remain associated with the cortical cytoskeleton after Triton X-100 extraction. These results demonstrate that the sequence present in the cytoplasmic domain of CD44 responsible for its distribution in epithelial cells is functionally and spatially separate from the ability of this protein to associate with the cytoskeleton.

scholarly journals Basolateral protein transport in streptolysin O-permeabilized MDCK cells.

1994 ◽  
Vol 125 (5) ◽  
pp. 1025-1035 ◽  
Author(s):  
S W Pimplikar ◽  
E Ikonen ◽  
K Simons
Keyword(s):  
Protein Transport ◽  
Mdck Cells ◽  
Cytoplasmic Tail ◽  
Cell System ◽  
Apical Surface ◽  
Tail Domain ◽  
Permeabilized Cell ◽  
Interacting Protein ◽  
Streptolysin O

We have reconstituted polarized protein transport in streptolysin O-permeabilized MDCK cells from the TGN to the basolateral surface and to the apical surface. These transport steps are dependent on temperature, energy and exogenously supplied cytosol. Using this in vitro system we show that a whole tail peptide (WT peptide) corresponding to the cytoplasmic tail of a basolaterally sorted protein, the vesicular stomatitis virus glycoprotein (VSV G) inhibits the TGN to basolateral transport but does not affect any other transport step. Inhibition of VSV G transport to basolateral surface by WT peptide did not result in missorting of the protein to the apical surface. Mutation of the single tyrosine residue in the WT peptide reduced its inhibitory potency four- to fivefold. These results suggest that the VSV G tail physically interacts with a component of the sorting machinery. Using a cross-linking approach, we have identified proteins that associate with the cytoplasmic tail domain of VSV G. One of these polypeptides, Tin-2 (Tail interacting protein-2), associates with VSV G in the TGN, the site of protein sorting, but not in the ER nor at the cell surface. Tin-2 does not associate with apically targeted hemagglutinin. WT peptide that inhibited the basolateral transport of VSV G also inhibited the association of Tin-2 with VSV G. Together, these properties make Tin-2 a candidate basolateral sorter. The results demonstrate the usefulness of the SLO-permeabilized cell system in dissecting the sorting machinery.

A Study on the Fine Structure of the Lateral Line Organ of the Sea Eel

1973 ◽  
Vol 31 ◽  
pp. 648-649
Author(s):  
K. Hama
Keyword(s):  
Fine Structure ◽  
Electron Microscope ◽  
Lateral Line ◽  
Low Frequency ◽  
Sensory Cells ◽  
Apical Surface ◽  
Voltage Electron ◽  
Sensory Epithelia ◽  
Low Frequency Vibration ◽  
Transmission Electron

The lateral line organs of the sea eel consist of canal and pit organs which are different in function. The former is a low frequency vibration detector whereas the latter functions as an ion receptor as well as a mechano receptor.The fine structure of the sensory epithelia of both organs were studied by means of ordinary transmission electron microscope, high voltage electron microscope and of surface scanning electron microscope.The sensory cells of the canal organ are polarized in front-caudal direction and those of the pit organ are polarized in dorso-ventral direction. The sensory epithelia of both organs have thinner surface coats compared to the surrounding ordinary epithelial cells, which have very thick fuzzy coatings on the apical surface.

Organization of tight junctions in the choroid plexus epithelium

1978 ◽  
Vol 36 (2) ◽  
pp. 336-337
Author(s):  
B. Van Deurs ◽  
J. K. Koehler
Keyword(s):  
Choroid Plexus ◽  
Present Report ◽  
Freeze Fracture ◽  
Barrier Properties ◽  
Cell Junctions ◽  
Structural Basis ◽  
Apical Surface ◽  
Choroid Plexus Epithelium ◽  
Solid Nitrogen ◽  
Special Composition

The choroid plexus epithelium constitutes a blood-cerebrospinal fluid (CSF) barrier, and is involved in regulation of the special composition of the CSF. The epithelium is provided with an ouabain-sensitive Na/K-pump located at the apical surface, actively pumping ions into the CSF. The choroid plexus epithelium has been described as “leaky” with a low transepithelial resistance, and a passive transepithelial flux following a paracellular route (intercellular spaces and cell junctions) also takes place. The present report describes the structural basis for these “barrier” properties of the choroid plexus epithelium as revealed by freeze fracture.Choroid plexus from the lateral, third and fourth ventricles of rats were used. The tissue was fixed in glutaraldehyde and stored in 30% glycerol. Freezing was performed either in liquid nitrogen-cooled Freon 22, or directly in a mixture of liquid and solid nitrogen prepared in a special vacuum chamber. The latter method was always used, and considered necessary, when preparations of complementary (double) replicas were made.

Ultrastructural autoradiography of L6 skeletal-muscle cells exposed to a tritiated macrolide antibiotic (LY237216) in vitro

1992 ◽  
Vol 50 (1) ◽  
pp. 612-613
Author(s):  
S.L. White ◽  
C.B. Jensen ◽  
D.D. Giera ◽  
D.A. Laska ◽  
M.N. Novilla ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Quantitative Analysis ◽  
Macrolide Antibiotic ◽  
Circle Method ◽  
Apical Surface ◽  
Polycarbonate Membrane ◽  
L6 Cells ◽  
Low Viscosity ◽  
Erythromycin A

In vitro exposure to LY237216 (9-Deoxo-11-deoxy-9,11-{imino[2-(2-methoxyethoxy)ethylidene]-oxy}-(9S)-erythromycin), a macrolide antibiotic, was found to induce cytoplasmic vacuolation in L6 skeletal muscle myoblast cultures (White, S.L., unpubl). The present study was done to determine, by autoradiographic quantitative analysis, the subcellular distribution of 3H-LY237216 in L6 cells.L6 cells (ATCC, CRL 1458) were cultured to confluency on polycarbonate membrane filters (Millipore Corp., Bedford, MA) in M-199 medium (GIBCO® Labs) with 10% fetal bovine serum. The cells were exposed from the apical surface for 1-hour to unlabelled-compound (0 μCi/ml) or 50 (μCi/ml of 3H-LY237216 at a compound concentration of 0.25 mg/ml. Following a rapid rinse in compound-free growth medium, the cells were slam-frozen against a liquid nitrogen cooled, polished copper block in a CF-100 cryofixation unit (LifeCell Corp., The Woodlands, TX). Specimens were dried in the MDD-C Molecular Distillation Drier (LifeCell Corp.), vapor osmicated and embedded in Spurrs low viscosity resin. Ultrathin sections collected on formvar coated stainless steel grids were counter-stained, then individually mounted on corks. A monolayer of Ilford L4 nuclear emulsion (Polysciences, Inc., Warrington, PA) was placed on the sections, utilizing a modified “loop method”. The emulsions were exposed for 7-weeks in a light-tight box at 4°C. Autoradiographs were developed in Microdol-X developer and examined on a Philips EM410LS transmission electron microscope. Quantitative analysis of compound localization employed the point and circle approach of Williams; incorporating the probability circle method of Salpeter and McHenry.

Further studies on the ultrastructure of the cochlea

1990 ◽  
Vol 48 (3) ◽  
pp. 440-441
Author(s):  
Zhixian Wang ◽  
Pinjin Zhu ◽  
Jianhe Sun ◽  
Xuezheng Song
Keyword(s):  
Hair Cells ◽  
Military Medicine ◽  
Outer Hair Cells ◽  
Apical Surface ◽  
Epithelial Surface ◽  
Hearing Research ◽  
New Findings ◽  
Fine Structures ◽  
Temporal Bones ◽  
Accurate Observation

Hearing research is important not only for clinical, professional and military medicine, but also for toxicology, gerontology and genetics. Ultrastructure of the cochlea attracts much attention of electron microscopists, (1―3) but the research lags far behind that of the other parts of the organnism. On the basis of careful microdissection, technical improvment and accurate observation, we have got some new findings which have not been reported in the literature.We collected four cochleas from human corpses. Temporal bones dissected 1 h after death and cochleas perfused with fixatives 4 h after death were good enough in terms of preservation of fine structures. SEM:The apical surface of OHCs (Outer hair cells) and DTs (Deiters cells) is narrower than that of IPs (Inner pillar cells). The mosaic configuration of the reticular membrane is not typical. The stereocilia of IHCs (Inner hair cells) are not uniform and some kinocilia could be seen on the OHCs in adults. The epithelial surface of RM (Reissner’s membrane) is not smooth and no mesh could be seen on the mesothelial surface of RM. TEM.

The Organization of Actin Filaments in the Stereocilia of the Bird Cochlea

1980 ◽  
Vol 38 ◽  
pp. 554-555
Author(s):  
E.J. Battles ◽  
D. DeRosier ◽  
J.C. Saunders ◽  
L.G. Tilney
Keyword(s):  
Hair Cell ◽  
Diffraction Pattern ◽  
Sea Urchin ◽  
Actin Filaments ◽  
Optical Diffraction ◽  
Dense Material ◽  
Apical Surface ◽  
Structural Rigidity ◽  
High Degree ◽  
Degree Of Order

Extending from the apical surface of each hair cell of the chick cochlea are from 75 to 200 microvilli or stereocllia and one true cllium, the kinocilium. The stereocllia are arranged in rows of progressively increasing length (Fig. 1). Within each tapering sterocilium is a bundle of actin filaments with over 900 filaments near the tip yet only approximately 25 at the base where filaments are enmeshed in a dense material (Fig. 1); from here some of the filaments enter the apical surface of the cell (cuticular plate) as a rootlet. Examination of longitudinal sections of the stereocilia (Fig. 2) show that the filaments are aligned parallel to each other and show considerable order. Examination of an optical diffraction pattern of this bundle (Fig. 4) reveal that the actin filaments are packed such that the crossover points of adjacent actin filaments are inregister. A prominent reflection at 125Å−1 demonstrates that the filaments are cjossbridged by a macromolecular bridge situated at an average of 125Å−1 intervals (Fig. 4) in transverse sections the filaments appear hexagonally packed although there are regions where the filaments are less ordered (Fig. 3). In images processed in the computer to remove, noise and enhance detail periodic nature of the bridge can be clearly seen (see arrows Fig. 5). This image resembles that of an actin paracrystal formed from sea urchin extract composed of bundles of actin filaments crossbridged by a second protein. Thus the actin filaments in the bird stereocilia by being cross-bridged and packed with a high degree of order and produces a structure with considerable structural rigidity. Embryos were studied at various stages in development in an attempt to determine how the stereocilia form and how does the actin packing develops. These stages will be discussed.

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