Analysis of the signals for polarized transport of influenza virus (A/WSN/33) neuraminidase and human transferrin receptor, type II transmembrane proteins.

In polarized MDCK cells influenza virus (A/WSN/33) neuraminidase (NA) and human transferrin receptor (TR), type II glycoproteins, when expressed from cloned cDNAs, were transported and accumulated preferentially on the apical and basolateral surfaces, respectively. We have investigated the signals for polarized sorting by constructing chimeras between NA and TR and by making deletion mutants. NATR delta 90, which contains the cytoplasmic tail and transmembrane domain of NA and the ectodomain of TR, was found to be localized predominantly on the apical membrane, whereas TRNA delta 35, containing the cytoplasmic and transmembrane domains of TR and the ectodomain of NA, was expressed preferentially on the basolateral membrane. TR delta 57, a TR deletion mutant lacking 57 amino acids in the TR cytoplasmic tail, did not exhibit any polarized expression and was present on both apical and basolateral surfaces, whereas a deletion mutant (NA delta 28-35) lacking amino acid residues from 28 to 35 in the transmembrane domain of NA resulted in secretion of the NA ectodomain predominantly from the apical side. These results taken together indicate that the cytoplasmic tail of TR was sufficient for basolateral transport, but influenza virus NA possesses two sorting signals, one in the cytoplasmic or transmembrane domain and the other within the ectodomain, both of which are independently able to transport the protein to the apical plasma membrane.     

The internalization signal and the phosphorylation site of transferrin receptor are distinct from the main basolateral sorting information.

Wild-type human transferrin receptor (hTfR), like endogenous canine receptor, is expressed almost exclusively (97%) at the basolateral membrane of transfected Madin-Darbey canine kidney (MDCK) cells. We investigated the role of two distinct features of the hTfR cytoplasmic domain, namely the endocytic signal and the unique phosphorylation site, in polarized cell surface delivery. Basolateral location was not altered by point mutation of Ser24-->Ala24, indicating that phosphorylation is not involved in vectorial sorting of hTfR. The steady state distribution of hTfR was partially affected by a deletion of 36 cytoplasmic residues encompassing the internalization sequence. However, 80% of the receptors were still basolateral. As assessed by pulse-chase experiments in combination with biotinylation, newly synthesized wild-type and deletion mutant receptors were directly sorted to the domain of their steady state residency. Although both receptors could bind human transferrin, endocytosis of the deletion mutant was strongly impaired at either surface. These data indicate that the predominant basolateral targeting signal of hTfR is independent of the internalization sequence.     

Transmembrane domain of influenza virus neuraminidase, a type II protein, possesses an apical sorting signal in polarized MDCK cells.

The influenza virus neuraminidase (NA), a type II transmembrane protein, is directly transported to the apical plasma membrane in polarized MDCK cells. By using deletion mutants and chimeric constructs of influenza virus NA with the human transferrin receptor, a type II basolateral transmembrane protein, we investigated the location of the apical sorting signal of influenza virus NA. When these mutant and chimeric proteins were expressed in stably transfected polarized MDCK cells, the transmembrane domain of NA, and not the cytoplasmic tail, provided a determinant for apical targeting in polarized MDCK cells and this transmembrane signal was sufficient for sorting and transport of the ectodomain of a reporter protein (transferrin receptor) directly to the apical plasma membrane of polarized MDCK cells. In addition, by using differential detergent extraction, we demonstrated that influenza virus NA and the chimeras which were transported to the apical plasma membrane also became insoluble in Triton X-100 but soluble in octylglucoside after extraction from MDCK cells during exocytic transport. These data indicate that the transmembrane domain of NA provides the determinant(s) both for apical transport and for association with Triton X-100-insoluble lipids.     

Characterization of a di-leucine-based signal in the cytoplasmic tail of the nucleotide-pyrophosphatase NPP1 that mediates basolateral targeting but not endocytosis

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.     

The Cytoplasmic Tail of Rhodopsin Acts as a Novel Apical Sorting Signal in Polarized MDCK Cells

All basolateral sorting signals described to date reside in the cytoplasmic domain of proteins, whereas apical targeting motifs have been found to be lumenal. In this report, we demonstrate that wild-type rhodopsin is targeted to the apical plasma membrane via the TGN upon expression in polarized epithelial MDCK cells. Truncated rhodopsin with a deletion of 32 COOH-terminal residues shows a nonpolar steady-state distribution. Addition of the COOH-terminal 39 residues of rhodopsin redirects the basolateral membrane protein CD7 to the apical membrane. Fusion of rhodopsin''s cytoplasmic tail to a cytosolic protein glutathione S-transferase (GST) also targets this fusion protein (GST–Rho39Tr) to the apical membrane. The targeting of GST–Rho39Tr requires both the terminal 39 amino acids and the palmitoylation membrane anchor signal provided by the rhodopsin sequence. The apical transport of GST–Rho39Tr can be reversibly blocked at the Golgi complex by low temperature and can be altered by brefeldin A treatment. This indicates that the membrane-associated GST–Rho39Tr protein may be sorted along a yet unidentified pathway that is similar to the secretory pathway in polarized MDCK cells. We conclude that the COOH-terminal tail of rhodopsin contains a novel cytoplasmic apical sorting determinant. This finding further indicates that cytoplasmic sorting machinery may exist in MDCK cells for some apically targeted proteins, analogous to that described for basolaterally targeted proteins.     

Hierarchy of sorting signals in chimeras of intestinal lactase-phlorizin hydrolase and the influenza virus hemagglutinin

Lactase-phlorizin hydrolase (LPH) is an apical protein in intestinal cells. The location of sorting signals in LPH was investigated by preparing a series of mutants that lacked the LPH cytoplasmic domain or had the cytoplasmic domain of LPH replaced by sequences that comprised basolateral targeting signals and overlapping internalization signals of various potency. These signals are mutants of the cytoplasmic domain of the influenza hemagglutinin (HA), which have been shown to be dominant in targeting HA to the basolateral membrane. The LPH-HA chimeras were expressed in Madin-Darby canine kidney (MDCK) and colon carcinoma (Caco-2) cells, and their transport to the cell surface was analyzed. All of the LPH mutants were targeted correctly to the apical membrane. Furthermore, the LPH-HA chimeras were internalized, indicating that the HA tails were available to interact with the cytoplasmic components of clathrin-coated pits. The introduction of a strong basolateral sorting signal into LPH was not sufficient to override the strong apical signals of the LPH external domain or transmembrane domains. These results show that basolateral sorting signals are not always dominant over apical sorting signals in proteins that contain each and suggest that sorting of basolateral from apical proteins occurs within a common compartment where competition for sorting signals can occur.     

Rat liver dipeptidylpeptidase IV contains competing apical and basolateral targeting information.

Madin-Darby canine kidney (MDCK) cells deliver endogenous apical and basolateral proteins directly to the appropriate domains. We are investigating the molecular signals on a model plasma membrane hydrolase, dipeptidylpeptidase IV (DPPIV). Most newly synthesized rat liver DPPIV is delivered directly to the apical surface of transfected MDCK cells; however, about 20% is delivered first to the basolateral surface and reaches the apical surface via transcytosis (Casanova, J. E., Mishumi, Y., Ikehara, Y., Hubbard, A. L., and Mostov, K. E. (1991) J. Biol. Chem. 266, 24428-24432). A soluble form of DPPIV (solDPPIV) containing only the lumenal domain of the protein was efficiently transported and secreted by stably transfected MDCK cells. If this domain contains apical sorting information, we would expect 80% of the soluble protein to be secreted apically. Surprisingly, 95% of the secreted solDPPIV was found in the apical medium. The high efficiency of apical secretion suggested that the transmembrane domain and cytoplasmic tail of DPPIV might contain competing basolateral targeting information. To test this hypothesis, we investigated the trafficking of a chimera in which the cytoplasmic tail and transmembrane domains of DPPIV were joined to lysozyme, an exogenous protein which should not contain sorting information. This protein was delivered predominantly to the basolateral surface. Our results suggest that the lumenal domain of DPPIV carries dominant apical sorting information while the transmembrane domain and cytoplasmic tail of the molecule contains competing basolateral sorting information.     

Interaction with mLin-7 alters the targeting of endocytosed transmembrane proteins in mammalian epithelial cells

To investigate the targeting mechanism for proteins bound to the mammalian Lin-7 (mLin-7) PDZ domain, we created receptor protein chimeras composed of the carboxyl-terminal amino acids of LET-23 fused to truncated nerve growth factor receptor/P75. mLin-7 bound to the chimera with a wild-type LET-23 carboxyl-terminal tail (P75t-Let23WT), but not a mutant tail (P75t-Let23MUT). In Madin-Darby canine kidney (MDCK) cells, P75t-Let23WT localized to the basolateral plasma membrane domain, whereas P75t-Let23MUT remained apical. Furthermore, mutant mLin-7 constructs acted as dominant interfering proteins and inhibited the basolateral localization of P75t-Let23WT. The mechanisms for this differential localization were examined further, and, initially, we found that P75t-Let23WT and P75t-Let23MUT were delivered equally to the apical and basolateral plasma membrane domains. Although basolateral retention of P75t-Let23WT, but not P75t-Let23MUT, was observed, the greatest difference in receptor localization was seen in the rapid trafficking of P75t-Let23WT to the basolateral plasma membrane domain after endocytosis, whereas P75t-Let23MUT was degraded in lysosomes, indicating that mLin-7 binding can alter the fate of endocytosed proteins. Altogether, these data support a model for basolateral protein targeting in mammalian epithelial cells dependent on protein-protein interactions with mLin-7, and also suggest a dynamic role for mLin-7 in endosomal sorting.     

Polarized sorting of the polymeric immunoglobulin receptor in the exocytotic and endocytotic pathways is controlled by the same amino acids.

Polarized epithelial cells can sort plasma membrane proteins to the apical or basolateral domain either by direct targeting from the trans-Golgi network (TGN) or by targeting to one surface, followed by endocytosis and transcytosis to the opposite surface. In Madin-Darby canine kidney (MDCK) cells, targeting of the polymeric immunoglobulin receptor (pIgR) to the basolateral surface is controlled by a sorting signal residing in the membrane proximal 17 amino acids of the cytoplasmic domain of this receptor. We have recently found that individual mutations at any of three residues in this signal, His656, Arg657 and Val660, substantially decrease targeting from the TGN to the basolateral surface and correspondingly increase targeting from the TGN to the apical surface. Here we report that these mutations decrease the recycling of basolaterally endocytosed pIgR to that surface, and correspondingly increase its transcytosis to the apical surface. This effect occurred in mutant pIgRs that either contained the full-length cytoplasmic domain or were truncated to contain only the 17-residue basolateral targeting signal, and was independent of phosphorylation of pIgR at Ser664. Our results indicate that polarized sorting of the pIgR in the endocytotic and exocytotic pathways are controlled by the same amino acids.     

A novel cellular phenotype for familial hypercholesterolemia due to a defect in polarized targeting of LDL receptor

Basolateral targeting of membrane proteins in polarized epithelial cells typically requires cytoplasmic domain sorting signals. In the familial hypercholesterolemia (FH)-Turku LDL receptor allele, a mutation of glycine 823 residue affects the signal required for basolateral targeting in MDCK cells. We show that the mutant receptor is mistargeted to the apical surface in both MDCK and hepatic epithelial cells, resulting in reduced endocytosis of LDL from the basolateral/sinusoidal surface. Consequently, virally encoded mutant receptor fails to mediate cholesterol clearance in LDL receptor-deficient mice, suggesting that a defect in polarized LDL receptor expression in hepatocytes underlies the hypercholesterolemia in patients harboring this allele. This evidence directly links the pathogenesis of a human disease to defects in basolateral targeting signals, providing a genetic confirmation of these signals in maintaining epithelial cell polarity.     

Naked2 acts as a cargo recognition and targeting protein to ensure proper delivery and fusion of TGF-alpha containing exocytic vesicles at the lower lateral membrane of polarized MDCK cells

Transforming growth factor-alpha (TGF-alpha) is the major autocrine EGF receptor ligand in vivo. In polarized epithelial cells, proTGF-alpha is synthesized and then delivered to the basolateral cell surface. We previously reported that Naked2 interacts with basolateral sorting determinants in the cytoplasmic tail of a Golgi-processed form of TGF-alpha and that TGF-alpha is not detected at the basolateral surface of Madin-Darby canine kidney (MDCK) cells expressing myristoylation-deficient (G2A) Naked2. By high-resolution microscopy, we now show that wild-type, but not G2A, Naked2-associated vesicles fuse at the plasma membrane. We further demonstrate that Naked2-associated vesicles are delivered to the lower lateral membrane of polarized MDCK cells independent of mu1B adaptin. We identify a basolateral targeting segment within Naked2; residues 1-173 redirect NHERF-1 from the apical cytoplasm to the basolateral membrane, and internal deletion of residues 37-104 results in apical mislocalization of Naked2 and TGF-alpha. Short hairpin RNA knockdown of Naked2 leads to a dramatic reduction in the 16-kDa cell surface isoform of TGF-alpha and increased cytosolic TGF-alpha immunoreactivity. We propose that Naked2 acts as a cargo recognition and targeting (CaRT) protein to ensure proper delivery, tethering, and fusion of TGF-alpha-containing vesicles to a distinct region at the basolateral surface of polarized epithelial cells.     

Importance of the carboxyl-terminal FTSL motif of membrane cofactor protein for basolateral sorting and endocytosis. Positive and negative modulation by signals inside and outside the cytoplasmic tail.

Membrane cofactor protein (MCP), a widely distributed complement regulatory protein, is expressed on the basolateral surface of polarized epithelial cells, and it is not endocytosed. The carboxyl-terminal tetrapeptide (FTSL) is required for polarized surface expression. The ability of this tetrapeptide to serve as an autonomous sorting signal has been analyzed by adding this sequence motif to the C terminus of an apical membrane protein, the influenza A virus hemagglutinin (HA). The recombinant protein HA-FTSL retained the apical localization of the parental HA protein. Substitution of the complete cytoplasmic tail of MCP for the cytoplasmic tail of HA resulted in the targeting of the chimeric protein (HA/MCP) to the basolateral surface suggesting that the carboxyl-terminal FTSL motif is a weak sorting signal that requires additional targeting information from the membrane-proximal part of the cytoplasmic tail of MCP for redirecting an apical protein to the basolateral membrane domain. In contrast to the native HA, the HA-FTSL protein was subject to endocytosis. The basolateral HA/MCP was also found to be internalized and thus differed from the basolateral MCP. This result suggests that the carboxyl-terminal FTSL motif serves as an internalization signal and that in native MCP sorting information outside the cytoplasmic tail counteracts this endocytosis signal. Substitution of a tyrosine for the phenylalanine dramatically increased the internalization with most of the HA-YTSL protein being present intracellularly. Our results are consistent with the view that the interplay of multiple sorting signals and the modification of a well known targeting signal (YTSL) by amino acid exchange (FTSL) determine the constitutive expression of MCP on the basolateral surface of polarized epithelial cells.     

Analysis of transport and targeting of syndecan-1: effect of cytoplasmic tail deletions.

Madin-Darby canine kidney (MDCK) cells and Chinese hamster ovary (CHO) cells were transfected with wild-type and cytoplasmic deletion mutants of mouse syndecan-1 to study the requirements for transport and polarized expression of this proteoglycan. Expression in MDCK cells revealed that wild-type syndecan-1 is directed to the basolateral surface via a brefeldin A-insensitive route. A deletion of the last 12 amino acids of the syndecan-1 cytoplasmic tail (CT22) was sufficient to result in the appearance of mutant proteoglycans at both the basolateral and apical cell surfaces. Treatment with brefeldin A was able to prevent apical transport of the mutants. We thus propose that the C-terminal part of the cytoplasmic tail is required for steady-state basolateral distribution of syndecan-1. In CHO cells a deletion of the last 25 or 33 amino acids of the 34-residue cytoplasmic domain (CT9 and CT1, respectively) resulted in partial retention of the mutants in the endoplasmic reticulum (ER). A deletion mutant lacking the last 12 amino acids (CT22) was not retained. Interestingly, the unglycosylated core proteins of the CT9 and CT1 mutants showed a significantly lower apparent molecular weight when analyzed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis than wild-type syndecan-1. However, when CHO transfectants expressing the CT1 mutant were incubated with brefeldin A, causing fusion of the ER and Golgi, CT1 ran with an almost equally high apparent molecular weight as the wild-type molecule. This would suggest that syndecan-1 undergoes extensive posttranslational modifications or forms an SDS-resistant dimer/complex after transit from the ER.     

A single amino acid change in the cytoplasmic domain alters the polarized delivery of influenza virus hemagglutinin

In the polarized kidney cell line MDCK, the influenza virus hemagglutinin (HA) has been well characterized as a model for apically sorted membrane glycoproteins. Previous work from our laboratory has shown that a single amino acid change in the cytoplasmic sequence of HA converts it from a protein that is excluded from coated pits to one that is efficiently internalized. Using trypsin or antibodies to mark protein on the surface, we have shown in MDCK cells that HA containing this mutation is no longer transported to the apical surface but instead is delivered directly to the basolateral plasma membrane. We propose that a cytoplasmic feature similar to an endocytosis signal can cause exclusive basolateral delivery.     

The cytoplasmic tail of lysosomal acid phosphatase contains overlapping but distinct signals for basolateral sorting and rapid internalization in polarized MDCK cells.

Lysosomal acid phosphatase (LAP) is synthesized as a type I membrane glycoprotein and targeted to lysosomes via the plasma membrane. Its cytoplasmic tail harbours a tyrosine-containing signal for rapid internalization. Expression in Madine-Darby canine kidney cells results in direct sorting to the basolateral cell surface, rapid endocytosis and delivery to lysosomes. In contrast, a deletion mutant lacking the cytoplasmic tail is delivered to the apical plasma membrane where it accumulates before it is slowly internalized. A chimeric protein, in which the cytoplasmic tail of LAP is fused to the extracytoplasmic and transmembrane domain of the apically sorted haemagglutinin, is sorted to the basolateral plasma membrane. A series of truncation and substitution mutants in the cytoplasmic tail was constructed and comparison of their polarized sorting and internalization revealed that the determinants for basolateral sorting and rapid internalization reside in the same segment of the cytoplasmic tail. The cytoplasmic factors decoding these signals, however, tolerate distinct mutations indicating that different receptors are involved in sorting at the trans-Golgi network and at the plasma membrane.     

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

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.     

Identification of an apical sorting determinant in the cytoplasmic tail of megalin

Megalin is the main endocytic receptor ofthe proximal tubule and is responsible for reabsorption of manyfiltered proteins. In contrast to other members of the low-densitylipoprotein (LDL) receptor gene family, it is expressed on the apicalplasma membrane (PM) of polarized epithelial cells. To identifymegalin's apical sorting signal, we generated deletion mutants andchimeric minireceptors composed of complementary regions of megalin andLDL receptor-related protein (LRP) and assessed the distribution of themutants in Madin-Darby canine kidney (MDCK) cells by immunofluorescenceand cell surface biotinylation. Megalin and LRP minireceptors are correctly targeted to the apical and basolateral PM, respectively, ofMDCK cells. We found that the information that directs apical sortingis present in the cytoplasmic tail (CT) of megalin, which containsthree NPXY motifs, YXXØ, SH3, and dileucine motifs, and a PDZ-bindingmotif at its COOH terminus. Deletion analysis established that aminoacids 107-136 of the megalin-CT containing the second NPXY-likemotif are critical for apical sorting and targeting, whereas theregions containing the first and third NPXY motifs are required forefficient endocytosis. We conclude that the megalin-CT contains a novelapical sorting determinant and that cytoplasmic sorting machineryexists in MDCK cells for some apical transmembrane proteins.

     

Bidirectional transepithelial IgG transport by a strongly polarized basolateral membrane Fcgamma-receptor

The human MHC class I-related neonatal Fc receptor, hFcRn, mediates bidirectional transport of IgG across mucosal barriers. Here, we find that at steady state hFcRn distributes predominantly to an apical intracellular compartment and almost exclusively to the basolateral cell surface of polarized epithelial cells. It moves only transiently to the apical membrane. Ligand binding does not redistribute the steady state location of the receptor. Removal of the cytoplasmic tail that contains di-leucine and tryptophan-based endocytosis motifs or incubation at low temperature (18 degrees C) redistributes the receptor apically. The rates of endocytosis of the full-length hFcRn from the apical or basolateral membrane domains, however, are equal. Thus, the strong cell surface polarity displayed by hFcRn results from dominant basolateral sorting by motifs in the cytoplasmic tail that nonetheless allows for a cycle of bidirectional transcytosis.     

Transport of vesicular stomatitis virus G protein to the cell surface is signal mediated in polarized and nonpolarized cells

Current model propose that in nonpolarized cells, transport of plasma membrane proteins to the surface occurs by default. In contrast, compelling evidence indicates that in polarized epithelial cells, plasma membrane proteins are sorted in the TGN into at least two vectorial routes to apical and basolateral surface domains. Since both apical and basolateral proteins are also normally expressed by both polarized and nonpolarized cells, we explored here whether recently described basolateral sorting signals in the cytoplasmic domain of basolateral proteins are recognized and used for post TGN transport by nonpolarized cells. To this end, we compared the inhibitory effect of basolateral signal peptides on the cytosol-stimulated release of two basolateral and one apical marker in semi-intact fibroblasts (3T3), pituitary (GH3), and epithelial (MDCK) cells. A basolateral signal peptide (VSVGp) corresponding to the 29-amino acid cytoplasmic tail of vesicular stomatitis virus G protein (VSVG) inhibited with identical potency the vesicular release of VSVG from the TGN of all three cell lines. On the other hand, the VSVG peptide did not inhibit the vesicular release of HA in MDCK cells not of two polypeptide hormones (growth hormone and prolactin) in GH3 cells, whereas in 3T3 cells (influenza) hemagglutinin was inhibited, albeit with a 3x lower potency than VSVG. The results support the existence of a basolateral-like, signal-mediated constitutive pathway from TGN to plasma membrane in all three cell types, and suggest that an apical-like pathway may be present in fibroblast. The data support cargo protein involvement, not bulk flow, in the formation of post-TGN vesicles and predict the involvement of distinct cytosolic factors in the assembly of apical and basolateral transport vesicles.