Recycling Endosomes of Polarized Epithelial Cells Actively Sort Apical and Basolateral Cargos into Separate Subdomains

Thompson, Andrew; Neßler, R.; Wisco, Dolora; Anderson, Eric; Winckler, Bettina; Sheff, David

doi:10.1091/mbc.e05-09-0873

Cited by 67 publications

(65 citation statements)

References 65 publications

(78 reference statements)

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“…Thus, wild-type RFP-Rab8 was expressed in MDCK cells using adenovirus (infected 24 h after plating). Tfn was bound to its receptors on ice and internalized for 25 min to specifically label the RE (Sheff et al, 1999;Thompson et al, 2007). Cells expressing relatively low levels of RFP-Rab8 were selected for visualization, and the RFP signal was found to colocalize with that of the internalized Alexa-488 Tfn (see X-Z reconstruction, Figure 3A).…”

Section: Resultsmentioning

confidence: 99%

“…The DA-Rab8 construct was next applied to MDCKT monolayers 3 d after plating; this time point was chosen because the cells would be in the process of becoming polarized, but polarization would be completed before the construct was fully expressed (Thompson et al, 2007). Consistent with the infection efficiency of this virus, Ͼ90% of the cells infected 3 d after plating expressed DA-Rab8 (as assessed by visual evaluation of RFP in cells in random microscope fields; Supplementary Figure 1).…”

Section: Resultsmentioning

confidence: 99%

“…Sorting within the RE is a multistep process. Arriving ligands are first separated into distinct apical and basolateral subdomains within the RE, with ligands destined for each pathway thereby being concentrated (Thompson et al, 2007). Apical and basolateral transport vesicles then bud from these ligand-enriched domains, a process that results in high fidelity of cargo sorting, at least along the basolateral transport pathway (Sheff et al, 1999).…”

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confidence: 99%

“…When applied to MDCKT cells 24 h after plating (before the cells are polarized), the DA and the DN constructs each led to significant Tfn missorting, although the mis-sorting at these expression levels ( Figure 2, A and B) was not as great as that observed when wild-type Rab8 was expressed using adenovirus (Supplementary Figure 1). All Tfn recycling assays were performed The DA-Rab8 construct was next applied to MDCKT monolayers 3 d after plating; this time point was chosen because the cells would be in the process of becoming polarized, but polarization would be completed before the construct was fully expressed (Thompson et al, 2007). Consistent with the infection efficiency of this virus, Ͼ90% of the cells infected 3 d after plating expressed DA-Rab8 (as assessed by visual evaluation of RFP in cells in random microscope fields; Supplementary Figure 1).…”

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confidence: 99%

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Rab8 Regulates Basolateral Secretory, But Not Recycling, Traffic at the Recycling Endosome

Henry

Sheff

2008

MBoC

108

116

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Rab8 is a monomeric GTPase that regulates the delivery of newly synthesized proteins to the basolateral surface in polarized epithelial cells. Recent publications have demonstrated that basolateral proteins interacting with the 1-B clathrin adapter subunit pass through the recycling endosome (RE) en route from the TGN to the plasma membrane. Because Rab8 interacts with these basolateral proteins, these findings raise the question of whether Rab8 acts before, at, or after the RE. We find that Rab8 overexpression during the formation of polarity in MDCK cells, disrupts polarization of the cell, explaining how Rab8 mutants can disrupt basolateral endocytic and secretory traffic. However, once cells are polarized, Rab8 mutants cause mis-sorting of newly synthesized basolateral proteins such as VSV-G to the apical surface, but do not cause mis-sorting of membrane proteins already at the cell surface or in the endocytic recycling pathway. Enzymatic ablation of the RE also prevents traffic from the TGN from reaching the RE and similarly results in mis-sorting of newly synthesized VSV-G. We conclude that Rab8 regulates biosynthetic traffic through REs to the plasma membrane, but not trafficking of endocytic cargo through the RE. The data are consistent with a model in which Rab8 functions in regulating the delivery of TGN-derived cargo to REs. INTRODUCTIONThe Rab proteins are small monomeric GTPases of the Ras family. More than 60 known Rabs collectively regulate the flow of nearly all membrane traffic within the cell (Pfeffer, 1994;Gurkan et al., 2005;Jordens et al., 2005). Of these, mammalian Rab8 has been associated with the flow of newly synthesized proteins to the basolateral surface in polarized epithelial cells (Huber et al., 1993b;Moritz et al., 2001). In yeast, the Rab8 homolog, sec4, is also required for the targeting of membrane traffic to the plasma membrane (Guo et al., 1999). It has been suggested that Rab8 may be active in delivery of secretory cargo from the TGN to the plasma membrane. (Chen et al., 1993;Huber et al., 1993b). Studies of vesicular stomatitis virus protein G (VSV-G) delivery from the trans-Golgi network (TGN) have suggested that this process is regulated by Rab8, as dominant-negative (DN) mutants of Rab8 interfere with the delivery of basolateral cargo (Huber et al., 1993b). Rab8 is not localized to the TGN; rather it is localized at or near the recycling endosome (RE) in both polarized and nonpolarized cells (Hattula et al., 2006; Roland et al., 2007). The function of Rab8 at the RE is likely to regulate recycling of membrane traffic, although its role in polarized sorting is not clear. Rab8 is not required for the delivery of all basolateral proteins, but appears to be obligatory in the case of those that bear a tyrosine-dependent basolateral-sorting determinant capable of interacting with the clathrin adapter 1-B (Ang et al., 2003). The basolateral TGN-to-plasma membrane trafficking pathway is not as direct as once thought. Recent reports demonstrate clearly that 1-B-dependent basolater...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Rab8 Regulates Basolateral Secretory, But Not Recycling, Traffic at the Recycling Endosome

Henry

Sheff

2008

MBoC

108

116

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“…As with other endosomes, each type of recycling endosome is characterized by various resident membrane proteins, by the more transient populations of membrane-associated proteins, and by cohorts of cargo proteins that pass dynamically through these organelles (7). While endosomes are best known for handling protein cargo being internalized from and recycled back to the cell surface-such as transferrin-these endosomes are now also recognized as stations for exocytic cargo moving to the cell surface in secretory pathways (6)(7)(8)(9). The multiple trafficking pathways feeding into endosomes and the selective egress of proteins leaving endosomes require sophisticated mechanisms for protein sorting and handling.…”

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confidence: 99%

Automated organelle‐based colocalization in whole‐cell imaging

et al. 2009

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The use of fluorescence microscopy to investigate protein colocalization is an invaluable tool for understanding subcellular structures and their associated proteins. However, current techniques are largely limited to two-dimensional (2D) imaging and often require manual segmentation. Here, we present OBCOL, a methodology to automatically segment and quantify protein colocalization not within an image as a whole but on all individual punctuate organelles within a 3D multichannel image. A wide variety of colocalization statistics may then be calculated on the objects found, and features reported for each such as position, degree of overlap between channels, and number of component objects. OBCOL was validated on imaging of two fluorescent markers (Dextran, EGF) in 3D microscopy imaging. OBCOL's application was then exemplified by investigating the colocalization of three fluorescently tagged proteins (VAMP3, Rab11, and transferrin) on recycling endosomes in mammalian cells. The methodology showed for the first time the diversity of endosomes labeled with one or more of these proteins and quantitatively demonstrated the degree of overlap among these proteins in individual recycling endosomes. The consistent segregation of these markers provides novel evidence for the subcompartmentalization of recycling endosomes. OBCOL is a flexible methodology for 3D multifluorophore image analysis. This study clearly demonstrated its value for investigating subcellular structures and their constituent proteins. ' 2009 International Society for Advancement of Cytometry Key terms colocalization; confocal microscopy; fluorescent imaging; image analysis; image segmentation; organelle; cell biology COLOCALIZATION between fluorescently labeled molecules is one of the most widely used techniques to assess the degree of spatial coincidence, and hence potential interactions, among subcellular species such as proteins. To date, the analysis of colocalization has been largely qualitative via the ''dye-overlay'' method (1). This method images two proteins and their subcellular localizations in red and green channels and uses the merged image to visually assess the degree of colocalization. Pixel fluorograms or scattergrams in which a plot is made of the pair of intensities for each pixel are also often used to provide more detailed colocalization (2). Recently, there has been much interest in quantitative approaches to measure colocalization. Broadly, such methods can be divided into threshold based or intensity based. In threshold-based methods, a threshold is set for each channel. At its simplest, the percentage of above-threshold pixels in one channel that are in regions above the threshold in the other channel is recorded. For intensity-based approaches, pixel values are taken into account. For instance, Pearson's correlation coefficient calculates the correlation between pixel intensities in each channel, and Mander's coefficient (3) gives a ratio of intensities in one channel relative to above-threshold regions in the other....

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Golgi stress response and organelle zones

Sasaki

Yoshida

2019

FEBS Letters

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Organelles have been studied traditionally as single units, but a novel concept is now emerging: each organelle has distinct functional zones that regulate specific functions. The Golgi apparatus seems to have various zones, including zones for: glycosylphosphatidylinositol‐anchored proteins; proteoglycan, mucin and lipid glycosylation; transport of cholesterol and ceramides; protein degradation (Golgi membrane‐associated degradation); and signalling for apoptosis. The capacity for these specific functions and the size of the corresponding zones appear to be tightly regulated by the Golgi stress response to accommodate cellular demands. For instance, the proteoglycan and mucin zones seem to be separately augmented during the differentiation of chondrocytes and goblet cells, respectively. The mammalian Golgi stress response consists of several response pathways. The TFE3 pathway regulates the general function of the Golgi, such as structural maintenance, N‐glycosylation and vesicular transport, whereas the proteoglycan pathway increases the expression of glycosylation enzymes for proteoglycans. The CREB3 and HSP47 pathways regulate pro‐ and anti‐apoptotic functions, respectively. These observations indicate that the Golgi is a dynamic organelle, the capacity of which is upregulated according to cellular needs.

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Recycling Endosomes of Polarized Epithelial Cells Actively Sort Apical and Basolateral Cargos into Separate Subdomains

Cited by 67 publications

References 65 publications

Rab8 Regulates Basolateral Secretory, But Not Recycling, Traffic at the Recycling Endosome

Rab8 Regulates Basolateral Secretory, But Not Recycling, Traffic at the Recycling Endosome

Automated organelle‐based colocalization in whole‐cell imaging

Golgi stress response and organelle zones

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