Recycling endosomes of polarized epithelial cells actively sort apical and basolateral cargos into separate subdomains

The plasma membranes of epithelial cells plasma membranes contain distinct apical and basolateral domains that are critical for their polarized functions. However, both domains are continuously internalized, with proteins and lipids from each intermixing in supranuclear recycling endosomes (REs). To maintain polarity, REs must faithfully recycle membrane proteins back to the correct plasma membrane domains. We examined sorting within REs and found that apical and basolateral proteins were laterally segregated into subdomains of individual REs. Subdomains were absent in unpolarized cells and developed along with polarization. Subdomains were formed by an active sorting process within REs, which precedes the formation of AP-1B-dependent basolateral transport vesicles. Both the formation of subdomains and the fidelity of basolateral trafficking were dependent on PI3 kinase activity. This suggests that subdomain and transport vesicle formation occur as separate sorting steps and that both processes may contribute to sorting fidelity.     

Membrane domains and polarized trafficking of sphingolipids

The plasma membrane of polarized cells consists of distinct domains, the apical and basolateral membrane, that are characterized by a distinct lipid and protein content. Apical protein transport is largely mediated by (glyco)sphingolipid--cholesterol enriched membrane microdomains, so called rafts. In addition changes in the direction of polarized sphingolipid transport appear instrumental in cell polarity development. Knowledge is therefore required of the mechanisms that mediate sphingolipid sorting and the complexity of the trafficking pathways that are involved in polarized transport of both sphingolipids and proteins. Here we summarize specific biophysical properties that underly mechanisms relevant to sphingolipid sorting, cargo recruitment and polarized trafficking, and discuss the central role of a subapical compartment, SAC or common endosome (CE), as a major intracellular site involved in polarized sorting of sphingolipids, and in development and maintenance of membrane polarity.     

Myosin VI is required for sorting of AP-1B-dependent cargo to the basolateral domain in polarized MDCK cells

In polarized epithelial cells, newly synthesized membrane proteins are delivered on specific pathways to either the apical or basolateral domains, depending on the sorting motifs present in these proteins. Because myosin VI has been shown to facilitate secretory traffic in nonpolarized cells, we investigated its role in biosynthetic trafficking pathways in polarized MDCK cells. We observed that a specific splice isoform of myosin VI with no insert in the tail domain is required for the polarized transport of tyrosine motif containing basolateral membrane proteins. Sorting of other basolateral or apical cargo, however, does not involve myosin VI. Site-directed mutagenesis indicates that a functional complex consisting of myosin VI, optineurin, and probably the GTPase Rab8 plays a role in the basolateral delivery of membrane proteins, whose sorting is mediated by the clathrin adaptor protein complex (AP) AP-1B. Our results suggest that myosin VI is a crucial component in the AP-1B-dependent biosynthetic sorting pathway to the basolateral surface in polarized epithelial cells.     

Detergent insoluble microdomains are not involved in transcytosis of polymeric Ig receptor in FRT and MDCK cells

In polarized epithelial cells, sorting of proteins and lipids to the apical or basolateral domain of the plasma membrane can occur via direct or indirect (transcytotic) pathways from the trans Golgi network (TGN). The 'rafts' hypothesis postulates that the key event for direct apical sorting of some transmembrane proteins and the majority of GPI-anchored proteins depends on their association with glycosphingolipid and cholesterol enriched microdomains (rafts). However, the mechanism of indirect sorting to the apical membrane is not clear. The polyimmunoglobulin receptor (pIgR) is one of the best studied proteins that follow the transcytotic pathway. It is normally delivered from the TGN to the basolateral surface of polarized Madin–Darby Canine Kidney (MDCK) cells from where it transports dIgA or dIgM to the apical surface. We have studied the intracellular trafficking of pIgR in Fischer rat thyroid cells (FRT), and have investigated the sorting machinery involved in transcytosis of this receptor in both FRT and MDCK cells. We found that, in contrast with MDCK cells, a significant amount (∼30%) of pIgR reaches the apical surface by a direct pathway. Furthermore, in both cell lines it does not associate with Triton X-100-insoluble microdomains, suggesting that at least in these cells 'rafts' are not involved in basolateral to apical transcytosis.     

On the biogenesis of the myelin sheath: cognate polarized trafficking pathways in oligodendrocytes

Oligodendrocytes, the myelinating cells of the central nervous system, are capable of transporting vast quantities of proteins and of lipids, in particular galactosphingolipids, to the myelin sheath. The sheath is continuous with the plasma membrane of the oligodendrocyte, but the composition of both membrane domains differs substantially. Given its high glycosphingolipid and cholesterol content the myelin sheath bears similarity to the lipid composition of the apical domain of a polarized cell. The question thus arises whether myelin components, like typical apical membrane proteins are transported by an apical-like trafficking mechanism to the sheath, involving a 'raft'-mediated mechanism. Indeed, the evidence indicates the presence of cognate apical and basolateral pathways in oligodendrocytes. However, all major myelin proteins do not participate in this pathway, and remarkably apical-like trafficking seems to be restricted to the oligodendrocyte cell body. In this review, we summarize the evidence on the existence of different trafficking pathways in the oligodendrocyte, and discuss possible mechanisms separating the oligodendrocyte's membrane domains.     

Protein trafficking mechanisms associated with neurite outgrowth and polarized sorting in neurons.

Neuronal differentiation in vitro and in vivo involves coordinated changes in the cellular cytoskeleton and protein trafficking processes. I review here recent progress in our understanding of the membrane trafficking aspects of neurite outgrowth of neurons in culture and selective microtubule-based polarized sorting in fully polarized neurons, focusing on the involvement of some key molecules. Early neurite outgrowth appears to involve the protein trafficking machineries that are responsible for constitutive trans-Golgi network (TGN) to plasma membrane exocytosis, utilizing transport carrier generation mechanisms, SNARE proteins, Rab proteins and tethering mechanisms that are also found in non-neuronal cells. This vectorial TGN-plasma membrane traffic is directed towards several neurites, but can be switch to concentrate on the growth of a single axon. In a mature neuron, polarized targeting to the specific axonal and dendritic domains appears to involve selective microtubule-based mechanisms, utilizing motor proteins capable of distinguishing microtubule tracks to different destinations. The apparent gaps in our knowledge of these related protein transport processes will be highlighted.     

N-glycans mediate apical recycling of the sialomucin endolyn in polarized MDCK cells

Apical and basolateral proteins are maintained within distinct membrane subdomains in polarized epithelial cells by biosynthetic and postendocytic sorting processes. Sorting of basolateral proteins in these processes has been well studied; however, the sorting signals and mechanisms that direct proteins to the apical surface are less well understood. We previously demonstrated that an N-glycan-dependent sorting signal directs the sialomucin endolyn to the apical surface in polarized Madin-Darby canine kidney cells. Terminal processing of a subset of endolyn's N-glycans is key for polarized biosynthetic delivery to the apical membrane. Endolyn is subsequently internalized, and via a cytoplasmic tyrosine-based sorting motif is targeted to lysosomes from where it constitutively cycles to the cell surface. Here, we examine the polarized sorting of endolyn along the postendocytic pathway in polarized cells. Our results suggest that similar N-glycan sorting determinants are required for apical delivery of endolyn along both the biosynthetic and the postendocytic pathways.     

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

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.     

肝细胞极性与膜蛋白分选的分子机制

肝细胞是高度特化的极性上皮细胞,细胞质膜蛋白的分选和极性转运对于肝细胞极性的建立与维持至关重要.首先,膜蛋白在内质网中合成,随后经高尔基体加工修饰,再由反面高尔基体进一步分选,最后通过膜泡运输等不同的机制分别转运到胆汁腔面或窦状隙面,行使其特殊的功能.近些年来,细胞内负责转运的细胞器和主要的分选信号已逐步被揭示.特别是循环内体也被证明参与了胆汁腔面和窦状隙面膜蛋白的极性分选和转运.肝细胞的极性一旦遭到破坏,将会引起胆汁分泌障碍以及其他肝脏功能的损伤,从而可能导致肝脏糖脂代谢紊乱,甚至丧失正常的生理功能.因此,深入研究肝脏细胞极性的形成与维持机制,将为多种肝脏疾病的预防和治疗寻找到新的方向和靶点,具有重要的理论和临床实践意义.     

Polarized sorting and trafficking in epithelial cells

The polarized distribution of proteins and lipids at the surface membrane of epithelial cells results in the formation of an apical and a basolateral domain, which are separated by tight junctions. The generation and maintenance of epithelial polarity require elaborate mechanisms that guarantee correct sorting and vectorial delivery of cargo molecules. This dynamic process involves the interaction of sorting signals with sorting machineries and the formation of transport carriers. Here we review the recent advances in the field of polarized sorting in epithelial cells. We especially highlight the role of lipid rafts in apical sorting.     

Sphingolipid trafficking and protein sorting in epithelial cells

Sphingolipids represent a minor, but highly dynamic subclass of lipids in all eukaryotic cells. They are involved in functions that range from structural protection to signal transduction and protein sorting, and participate in lipid raft assembly. In polarized epithelial cells, which display an asymmetric apical and basolateral membrane surface, rafts have been proposed as a sorting principle for apical resident proteins, following their biosynthesis. However, raft-mediated trafficking is ubiquitous in cells. Also, sphingolipids per se, which are strongly enriched in the apical domain, are subject to sorting in polarity development. Next to the trans Golgi network, a subapical compartment called SAC or common endosome appears instrumental in regulating these sorting events.     

SNARE protein trafficking in polarized MDCK cells

A key feature of polarized epithelial cells is the ability to maintain the specific biochemical composition of the apical and basolateral plasma membrane domains. This polarity is generated and maintained by the continuous sorting of apical and basolateral components in the secretory and endocytic pathways. Soluble N-ethyl maleimide-sensitive factor attachment protein receptors (SNARE) proteins of vesicle-associated membrane protein (VAMP) and syntaxin families have been suggested to play a role in the biosynthetic transport to the apical and basolateral plasma membranes of polarized cells, where they likely mediate membrane fusion. To investigate the involvement of SNARE proteins in membrane trafficking to the apical and basolateral plasma membrane in the endocytic pathway we have monitored the recycling of various VAMP and syntaxin molecules between intracellular compartments and the two plasma membrane domains in Madin–Darby canine kidney (MDCK) cells. Here we show that VAMP8/endobrevin cycles through the apical but not through the basolateral plasma membrane. Furthermore, we found that VAMP8 localizes to apical endosomal membranes in nephric tubule epithelium and in MDCK cells. This asymmetry in localization and cycling behavior suggests that VAMP8/endobrevin may play a role in apical endosomal trafficking in polarized epithelium cells.     

Apical membrane proteins are transported in distinct vesicular carriers

The function of polarized epithelial cells and neurons is achieved through intracellular sorting mechanisms that recognize classes of proteins in the trans-Golgi network (TGN) and deliver them into separate vesicles for transport to the correct surface domain. Some proteins are delivered to the apical membrane after their association with membrane detergent-insoluble glycophosphatidylinositol/cholesterol (DIG) membrane microdomains [1], while some do not associate with DIGs [2-4]. However, it is not clear if this represents transport by two different pathways or if it can be explained by differences in the affinity of individual proteins for DIGs. Here, we investigate the different trafficking mechanisms of two apically sorted proteins, the DIG-associated sucrase-isomaltase (SI) and lactase-phlorizin hydrolase, which uses a DIG-independent pathway [5]. These proteins were tagged with YFP or CFP, and their trafficking in live cells was visualized using confocal laser microscopy. We demonstrate that each protein is localized to distinct subdomains in the same transport vesicle. A striking triangular pattern of concentration of the DIG-associated SI in subvesicular domains was observed. The original vesicles partition into smaller carriers containing either sucrase-isomaltase or lactase-phlorizin hydrolase, but not both, demonstrating for the first time a post-TGN segregation step and transport of apical proteins in different vesicular carriers.     

Retromer-Mediated Protein Sorting and Vesicular Trafficking

Retromer is an evolutionarily conserved multimeric protein complex that mediates intracellular transport of various vesicular cargoes and functions in a wide variety of cellular processes including polarized trafficking,developmental signaling and lysosome biogenesis.Through its interaction with the Rab GTPases and their effectors,membrane lipids,molecular motors,the endocytic machinery and actin nucleation promoting factors,retromer regulates sorting and trafficking of transmembrane proteins from endosomes to the trans-Golgi network(TGN) and the plasma membrane.In this review.I highlight recent progress in the understanding of relromer-medialed protein sorting and vesicle trafficking and discuss how retromer contributes to a diverse set of developmental,physiological and pathological processes.     

Rab27 effector Slp2-a transports the apical signaling molecule podocalyxin to the apical surface of MDCK II cells and regulates claudin-2 expression

Most cells in tissues are polarized and usually have two distinct plasma membrane domains-an apical membrane and a basolateral membrane, which are the result of polarized trafficking of proteins and lipids. However, the mechanism underlying the cell polarization is not fully understood. In this study, we investigated the involvement of synaptotagmin-like protein 2-a (Slp2-a), an effector molecule for the small GTPase Rab27, in polarized trafficking by using Madin-Darby canine kidney II cells as a model of polarized cells. The results show that the level of Slp2-a expression in MDCK II cells increases greatly as the cells become polarized and that its expression is specifically localized at the apical membrane. The results also reveal that Slp2-a is required for targeting of the signaling molecule podocalyxin to the apical membrane in a Rab27A-dependent manner. In addition, ezrin, a downstream target of podocalyxin, and ERK1/2 are activated in Slp2-a-knockdown cells, and their activation results in a dramatic reduction in the amount of the tight junction protein claudin-2. Because both Slp2-a and claudin-2 are highly expressed in mouse renal proximal tubules, Slp2-a is likely to regulate claudin-2 expression through trafficking of podocalyxin to the apical surface in mouse renal tubule epithelial cells.     

Selective roles for cholesterol and actin in compartmentalization of different proteins in the Golgi and plasma membrane of polarized cells

To determine the roles of cholesterol and the actin cytoskeleton in apical and basolateral protein organization and sorting, we have performed comprehensive confocal fluorescence recovery after photobleaching analyses of apical and basolateral and raft- and non-raft-associated proteins, both at the plasma membrane and in the Golgi apparatus of polarized MDCK cells. We show that at both the apical and basolateral plasma membrane domains, raft-associated proteins diffuse faster than non-raft-associated proteins and that, different from the latter, they become restricted upon depletion of cholesterol. Furthermore, only transmembrane apical proteins are restricted by the actin network. This indicates that cholesterol-dependent domains exist both at the apical and basolateral membranes of polarized cells and that the actin cytoskeleton has a predominant role in the organization of transmembrane proteins independent of their association with rafts at the apical membrane. In the Golgi apparatus apical proteins appear to be segregated from the basolateral ones in a compartment that is sensitive both to cholesterol depletion and actin rearrangements. Furthermore, consistent with the role of actin rearrangements in apical protein sorting, we found that apical proteins exhibit a differential sensitivity to actin depolymerization in the Golgi of polarized and nonpolarized cells.     

Protein trafficking in the exocytic pathway of polarized epithelial cells

Ten years ago, we knew much about the function of polarized epithelia from the work of physiologists, but, as cell biologists, our understanding of how these cells were constructed was poor. We knew proteins were sorted and targeted to different plasma membrane domains and that, in some cells, the Golgi was the site of sorting, but we did not know the mechanisms involved. Between 1991 and the present, significant advances were made in defining sorting motifs for apical and basal-lateral proteins, describing the sorting machinery in the trans-Golgi network (TGN) and plasma membrane, and in understanding how cells specify delivery of transport vesicles to different membrane domains. The challenge now is to extend this knowledge to defining molecular mechanisms in detail in vitro and comprehending the development of complex epithelial structures in vivo.     

On the biogenesis of the myelin sheath: Cognate polarized trafficking pathways in oligodendrocytes

Oligodendrocytes, the myelinating cells of the central nervous system, are capable of transporting vast quantities of proteins and of lipids, in particular galactosphingolipids, to the myelin sheath. The sheath is continuous with the plasma membrane of the oligodendrocyte, but the composition of both membrane domains differs substantially. Given its high glycosphingolipid and cholesterol content the myelin sheath bears similarity to the lipid composition of the apical domain of a polarized cell. The question thus arises whether myelin components, like typical apical membrane proteins are transported by an apical-like trafficking mechanism to the sheath, involving a raft-mediated mechanism. Indeed, the evidence indicates the presence of cognate apical and basolateral pathways in oligodendrocytes. However, all major myelin proteins do not participate in this pathway, and remarkably apical-like trafficking seems to be restricted to the oligodendrocyte cell body. In this review, we summarize the evidence on the existence of different trafficking pathways in the oligodendrocyte, and discuss possible mechanisms separating the oligodendrocyte's membrane domains.     

Remodeling the cell surface distribution of membrane proteins during the development of epithelial cell polarity

The development of polarized epithelial cells from unpolarized precursor cells follows induction of cell-cell contacts and requires resorting of proteins into different membrane domains. We show that in MDCK cells the distributions of two membrane proteins, Dg-1 and E-cadherin, become restricted to the basal-lateral membrane domain within 8 h of cell-cell contact. During this time, however, 60-80% of newly synthesized Dg-1 and E-cadherin is delivered directly to the forming apical membrane and then rapidly removed, while the remainder is delivered to the basal-lateral membrane and has a longer residence time. Direct delivery of greater than 95% of these proteins from the Golgi complex to the basal-lateral membrane occurs greater than 48 h later. In contrast, we show that two apical proteins are efficiently delivered and restricted to the apical cell surface within 2 h after cell-cell contact. These results provide insight into mechanisms involved in the development of epithelial cell surface polarity, and the establishment of protein sorting pathways in polarized cells.     

Delivery of raft-associated, GPI-anchored proteins to the apical surface of polarized MDCK cells by a transcytotic pathway

Epithelial cell polarity depends on mechanisms for targeting proteins to different plasma membrane domains. Here, we dissect the pathway for apical delivery of several raft-associated, glycosyl phosphatidylinositol (GPI)-anchored proteins in polarized MDCK cells using live-cell imaging and selective inhibition of apical or basolateral exocytosis. Rather than trafficking directly from the trans-Golgi network (TGN) to the apical plasma membrane as previously thought, the GPI-anchored proteins followed an indirect, transcytotic route. They first exited the TGN in membrane-bound carriers that also contained basolateral cargo, although the two cargoes were laterally segregated. The carriers were then targeted to and fused with a zone of lateral plasma membrane adjacent to tight junctions that is known to contain the exocyst. Thereafter, the GPI-anchored proteins, but not basolateral cargo, were rapidly internalized, together with endocytic tracer, into clathrin-free transport intermediates that transcytosed to the apical plasma membrane. Thus, apical sorting of these GPI-anchored proteins occurs at the plasma membrane, rather than at the TGN.