The kinesin KIF16B mediates apical transcytosis of transferrin receptor in AP-1B-deficient epithelia
2013; Springer Nature; Volume: 32; Issue: 15 Linguagem: Inglês
10.1038/emboj.2013.130
ISSN1460-2075
AutoresAndrés E. Perez Bay, Ryan Schreiner, Francesca Mazzoni, José María Carvajal-González, Diego Gravotta, Emilie Perret, Gullermo Lehmann Mantaras, Yuan‐Shan Zhu, Enrique Rodriguez‐Boulan,
Tópico(s)Microtubule and mitosis dynamics
ResumoArticle7 June 2013free access The kinesin KIF16B mediates apical transcytosis of transferrin receptor in AP-1B-deficient epithelia Andres E Perez Bay Andres E Perez Bay Department of Ophthalmology, Margaret Dyson Vision Research Institute, Weill Cornell Medical College, New York, NY, USA Search for more papers by this author Ryan Schreiner Ryan Schreiner Department of Ophthalmology, Margaret Dyson Vision Research Institute, Weill Cornell Medical College, New York, NY, USA Search for more papers by this author Francesca Mazzoni Francesca Mazzoni Department of Ophthalmology, Margaret Dyson Vision Research Institute, Weill Cornell Medical College, New York, NY, USA Search for more papers by this author Jose M Carvajal-Gonzalez Jose M Carvajal-Gonzalez Department of Ophthalmology, Margaret Dyson Vision Research Institute, Weill Cornell Medical College, New York, NY, USA Search for more papers by this author Diego Gravotta Diego Gravotta Department of Ophthalmology, Margaret Dyson Vision Research Institute, Weill Cornell Medical College, New York, NY, USA Search for more papers by this author Emilie Perret Emilie Perret Department of Ophthalmology, Margaret Dyson Vision Research Institute, Weill Cornell Medical College, New York, NY, USA Search for more papers by this author Gullermo Lehmann Mantaras Gullermo Lehmann Mantaras Department of Ophthalmology, Margaret Dyson Vision Research Institute, Weill Cornell Medical College, New York, NY, USA Search for more papers by this author Yuan-Shan Zhu Yuan-Shan Zhu Department of Medicine/Endocrinology, Weill Cornell Medical College, New York, NY, USA Search for more papers by this author Enrique J Rodriguez-Boulan Corresponding Author Enrique J Rodriguez-Boulan Department of Ophthalmology, Margaret Dyson Vision Research Institute, Weill Cornell Medical College, New York, NY, USA Department of Cell and Developmental Biology, Weill Cornell Medical College, New York, NY, USA Search for more papers by this author Andres E Perez Bay Andres E Perez Bay Department of Ophthalmology, Margaret Dyson Vision Research Institute, Weill Cornell Medical College, New York, NY, USA Search for more papers by this author Ryan Schreiner Ryan Schreiner Department of Ophthalmology, Margaret Dyson Vision Research Institute, Weill Cornell Medical College, New York, NY, USA Search for more papers by this author Francesca Mazzoni Francesca Mazzoni Department of Ophthalmology, Margaret Dyson Vision Research Institute, Weill Cornell Medical College, New York, NY, USA Search for more papers by this author Jose M Carvajal-Gonzalez Jose M Carvajal-Gonzalez Department of Ophthalmology, Margaret Dyson Vision Research Institute, Weill Cornell Medical College, New York, NY, USA Search for more papers by this author Diego Gravotta Diego Gravotta Department of Ophthalmology, Margaret Dyson Vision Research Institute, Weill Cornell Medical College, New York, NY, USA Search for more papers by this author Emilie Perret Emilie Perret Department of Ophthalmology, Margaret Dyson Vision Research Institute, Weill Cornell Medical College, New York, NY, USA Search for more papers by this author Gullermo Lehmann Mantaras Gullermo Lehmann Mantaras Department of Ophthalmology, Margaret Dyson Vision Research Institute, Weill Cornell Medical College, New York, NY, USA Search for more papers by this author Yuan-Shan Zhu Yuan-Shan Zhu Department of Medicine/Endocrinology, Weill Cornell Medical College, New York, NY, USA Search for more papers by this author Enrique J Rodriguez-Boulan Corresponding Author Enrique J Rodriguez-Boulan Department of Ophthalmology, Margaret Dyson Vision Research Institute, Weill Cornell Medical College, New York, NY, USA Department of Cell and Developmental Biology, Weill Cornell Medical College, New York, NY, USA Search for more papers by this author Author Information Andres E Perez Bay1, Ryan Schreiner1, Francesca Mazzoni1, Jose M Carvajal-Gonzalez1, Diego Gravotta1, Emilie Perret1, Gullermo Lehmann Mantaras1, Yuan-Shan Zhu2 and Enrique J Rodriguez-Boulan 1,3 1Department of Ophthalmology, Margaret Dyson Vision Research Institute, Weill Cornell Medical College, New York, NY, USA 2Department of Medicine/Endocrinology, Weill Cornell Medical College, New York, NY, USA 3Department of Cell and Developmental Biology, Weill Cornell Medical College, New York, NY, USA *Corresponding author. Department of Ophthalmology, Margaret Dyson Vision Research Institute, Weill Cornell Medical College, 1300 York Avenue, LC300, New York, NY 10065, USA. Tel.:+1 212 746 2272; Fax:+1 212 746 8101; E-mail: [email protected] The EMBO Journal (2013)32:2125-2139https://doi.org/10.1038/emboj.2013.130 There is a Have you seen? (July 2013) associated with this Article. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Polarized epithelial cells take up nutrients from the blood through receptors that are endocytosed and recycle back to the basolateral plasma membrane (PM) utilizing the epithelial-specific clathrin adaptor AP-1B. Some native epithelia lack AP-1B and therefore recycle cognate basolateral receptors to the apical PM, where they carry out important functions for the host organ. Here, we report a novel transcytotic pathway employed by AP-1B-deficient epithelia to relocate AP-1B cargo, such as transferrin receptor (TfR), to the apical PM. Lack of AP-1B inhibited basolateral recycling of TfR from common recycling endosomes (CRE), the site of function of AP-1B, and promoted its transfer to apical recycling endosomes (ARE) mediated by the plus-end kinesin KIF16B and non-centrosomal microtubules, and its delivery to the apical membrane mediated by the small GTPase rab11a. Hence, our experiments suggest that the apical recycling pathway of epithelial cells is functionally equivalent to the rab11a-dependent TfR recycling pathway of non-polarized cells. They define a transcytotic pathway important for the physiology of native AP-1B-deficient epithelia and report the first microtubule motor involved in transcytosis. Introduction Endocytosis and recycling of plasma membrane (PM) receptors play a key role in the uptake of many critical cell nutrients and in the relay of extracellular signals (Maxfield and McGraw, 2004). In non-polarized cells (e.g., fibroblasts), most nutrient receptors (e.g., transferrin receptor (TfR) and low-density lipoprotein receptor (LDLR)) traffic through a single major recycling pathway that involves sequential internalization into peripheral sorting endosomes (SE) and juxtanuclear recycling endosomes (RE) (Hopkins, 1983; Yamashiro et al, 1984; Maxfield and McGraw, 2004). In contrast, in epithelial cells, which display polarized apical and basolateral PM domains and receive most of their nutrients and signals from the blood, most endocytic receptors reside at the basolateral PM and must be recycled back to this PM domain after endocytosis (Perret et al, 2005; Rodriguez-Boulan et al, 2005). In these cells, most nutrient receptors are sequentially internalized into peripheral basolateral sorting endosomes (BSE) and juxtanuclear common recycling endosomes (CRE), where they mix with apical receptors internalized via separate apical sorting endosomes (ASE) (Figure 1A) (Hughson and Hopkins, 1990; Golachowska et al, 2010). Whereas in non-polarized cells recycling of nutrient receptors such as TfR along the major recycling route is regulated by the small GTPase rab11a, localized at RE (Ullrich et al, 1996; Ren et al, 1998) (see Supplementary Table 1), in epithelial cells recycling of nutrient receptors from CRE to the basolateral PM does not require rab11a (Wang et al, 2000). Remarkably, in epithelial cells rab11a resides at a more apically localized endosomal compartment inaccessible to TfR, the apical recycling endosomes (ARE) (Hunziker et al, 1990; Apodaca et al, 1994; Barroso and Sztul, 1994; Casanova et al, 1999; Golachowska et al, 2010), where it regulates instead the transcytotic route of polymeric IgA receptor (pIgR) and the biosynthetic route of a subset of apical PM proteins (Wang et al, 2000; Cresawn et al, 2007; Weisz and Rodriguez-Boulan, 2009). Figure 1.AP-1B KD MDCK cells display microtubule- and rab11a-dependent transcytosis of basolateral TfR. (A) Model of a WT MDCK cell displaying endosomal compartments and the endosomal itinerary of pIgR, TfR and LDLR postendocytic pathways. (B) A SulfoTag-Tf assay. Tf labelled with biotin and the luminophore SulfoTag (SulfoTag-Tf) provides a signal 10 × higher than 125I-Tf, allowing to detect 2 × 10−15 mol per 12 mm filter (the estimated amount of endogenous dog TfR present at the apical membrane of confluent MDCK cells). (C) MT mediate apical transcytosis of TfR. Values of apical transcytosis, basolateral recycling and the intracellular pool of SulfoTag-Tf (60 min) in control WT, nocodazole-treated WT, control AP-1B KD and nocodazole-treated AP-1B KD MDCK cells. (D) Dominant-negative rab11a inhibits apical transcytosis of TfR. A MDCK cell line was generated that stably expresses dominant-negative rab11a fused to monomeric Cherry fluorescent protein (mCh-DN-rab11a) under the control of a tetracycline-repressible promotor. Western blot analysis showed that expression of mCh-DN-rab11a increased four-fold by removing doxycycline for 12 h. (E) RNA levels of μ1B and GAPDH in WT, stable AP-1B KD, transient luciferase KD and transient AP-1B KD MDCK cells. (F) Values of apical transcytosis of SulfoTag-Tf (75 min) in WT and transient AP-1B KD MDCK cells with or without 12-h expression of mCh-DN-rab11a. *P<0.05, **P<0.001. Bars represent mean±standard error. Ap, apical; BL, basolateral; IC, intracellular. Download figure Download PowerPoint Because in epithelial cells basolateral and apical endocytic receptors are mixed at CRE, they must be sorted by specific signals before they return to their respective surface. Sorting is mediated by apical and basolateral sorting signals structurally similar to those that sort them in the biosynthetic route (Rodriguez-Boulan et al, 2005; Weisz and Rodriguez-Boulan, 2009). Sorting of basolateral recycling receptors at CRE is mediated by a sorting machinery that includes clathrin and the epithelial-specific clathrin adaptor AP-1B (Folsch et al, 1999; Ohno et al, 1999; Gan et al, 2002; Folsch et al, 2003; Gravotta et al, 2007; Deborde et al, 2008). Thus, the expression of AP-1B by epithelial cells appears to promote the incorporation of nutrient receptors to a recycling route to the PM that avoids interaction with rab11a. AP-1B is widely expressed by epithelial cells. However, some epithelial cell lines, e.g., LLC-PK1 (Roush et al, 1998; Folsch et al, 1999) and some native epithelia, e.g., retinal pigment epithelium (RPE) and kidney proximal tubule, lack AP-1B (Diaz et al, 2009; Schreiner et al, 2010), which promotes apical expression of many basolateral proteins that are normally sorted by AP-1B (Roush et al, 1998; Folsch et al, 1999). Importantly, the apical localizationof basolateral proteins has important physiological consequences for AP-1B-deficient native epithelia; for example, apical TfR in RPE is thought to regulate iron levels in the retina (Garcia-Castineiras, 2010). Unfortunately, the mechanisms and pathways that mediate apical localization of basolateral proteins in AP-1B-deficient epithelia are not known. Here, using ultrasensitive biochemical and microscopic recycling assays, we show that AP-1B-deficient epithelia incorporate two basolateral nutrient receptors, TfR and LDLR into an alternative rab11a-dependent recycling route to the apical membrane that, like the transcytotic pathway of pIgR, involves microtubule (MT)-mediated trafficking from CRE to ARE. This pathway requires non-centrosomal MTs and the plus-end kinesin KIF16B. The requirement for this kinesin is specific, as it is not shared by pIgR. Other experiments show that expression of AP-1B in fibroblastic CHO cells generates a recycling pathway for TfR independent of rab11a. In summary, our results illustrate for the first time the case of a cargo protein (TfR) that can use both the rab11a-dependent general recycling route of non-polarized cells and the rab11a-dependent apical recycling route of epithelial cells, directly supporting the concept that the apical recycling pathway of epithelial cells is equivalent to the general recycling route of non-polarized cells. They demonstrate that the epithelial-specific adaptor AP-1B can generate signal-dependent recycling routes for TfR in both polarized and non-polarized cells and report the first MT motor involved in transcytosis across epithelia. Results Ultrasensitive assay to measure TfR recycling and transcytosis Most studies on TfR recycling and transcytosis with 125I-Tf (transferrin) in MDCK cells were carried out overexpressing the human TfR because of the low amount of Tf transcytosed to the apical PM (Odorizzi and Trowbridge, 1997; Brown et al, 2000; Gravotta et al, 2007). Indeed, apical transcytosis of endogenous TfR requires long incubation times (i.e.,4–6 h) to allow accumulation of 125I-Tf in the apical medium (Fuller and Simons, 1986). To stay as close as possible to the physiological situation, we carried out our studies focusing on the endogenous (dog) TfR. Hence, we developed an ultrasensitive method to measure TfR recycling, that utilizes as a ligand dog Tf double-labelled with biotin and the luminophore SulfoTag (SulfoTag-Tf). This method proved to be 10 times more sensitive than 125I-Tf (Supplementary Figure S1A,B and Materials and methods). Titration curves of SulfoTag-Tf and 125I-Tf showed that only the former can detect 2 × 10−15 mol of Tf, which is the estimated amount of apical TfR present in confluent MDCK monolayers grown on a 12-mm Transwell filter (Figure 1B) (Fuller and Simons, 1986). Using this method, we showed that wild-type (WT) MDCK cells recycle most of the basolaterally internalized SulfoTag-Tf to the basolateral membrane (85%) and transcytose a small amount of SulfoTag-Tf to the apical PM (4%) (Figure 1C). MDCK cells lacking AP-1B by stable knockdown of its medium subunit μ1B (AP-1B KD), which exhibit normal levels of transepithelial electrical resistance (Gravotta et al, 2007), display 4 × higher apical transcytosis of basolaterally internalized SulfoTag-Tf (16% versus 4%, P<0.001) (Figure 1C, left) and lower basolateral recycling (71% versus 85%, P<0.001), compared to WT MDCK cells (Figure 1C, middle) (see recycling kinetics in Supplementary Figure S1C). Apical transcytosis of TfR is microtubule- and rab11a-dependent MTs play important roles in apical biosynthetic trafficking of PM proteins (Rodriguez-Boulan et al, 2005; Weisz and Rodriguez-Boulan, 2009) and transcytosis of pIgR (Hunziker et al, 1990; Apodaca et al, 1994). Hence, we studied whether apical transcytosis of TfR is MT dependent. Indeed, depolymerization of MTs by cold and nocodazole significantly inhibited apical transcytosis of TfR in AP-1B KD MDCK cells (from 16% to 8%, P<0.001) (Figure 1C left) and increased basolateral recycling (from 71% to 77%, P<0.05) (Figure 1C middle). As transcytosis of pIgR also depends on rab11a, we studied whether this was also the case for the transcytosis of endogenous TfR in AP-1B KD MDCK cells. To this end, we generated a tetracycline-repressible MDCK cell line stably expressing a GTP binding-deficient mutant of rab11a (S25N) tagged with monomeric Cherry fluorescent protein (mCh-DN-rab11a). This approach allows efficient overexpression of mCh-DN-rab11a only after the monolayer polarity is established, thus circumventing the disrupting effects of this protein during polarity development (Datta et al, 2011). In the presence of doxycycline, mCh-DN-rab11a expression was much lower than that of endogenous rab11a, whereas removal of the drug induced a 4 × increase ofmCh-DN-rab11a expression to a level comparable to the endogenous protein, thus avoiding non-specific effects associated with massive overexpression (Figure 1D). We transiently knocked down μ1B in these cells using electroporation of siRNA. RT-PCR analysis showed that transient knockdown of μ1B-KD was effective, albeit less efficient than in permanently transfected MDCK cells (Figure 1E), resulting in a smaller but statistically significant apical transcytosis of SulfoTag-Tf (Figure 1F). Importantly, expression of mCh-DN-rab11a caused a significant reduction of apical transcytosis of SulfoTag-Tf in transiently AP-1B KD MDCK cells (from 7% to 5%, P 10 times stronger signal than the routinely used Alexa488-Tf in the relevant concentration range (10−14 to ∼10−12 mol per well), as revealed by spectrofluorometry analysis (Supplementary Figure S2A). The specificity of 594-Tf for dog TfR was confirmed by colocalization experiments with anti-TfR antibody (Supplementary Figure S2B) and by competition experiments with unlabelled Tf (not shown). AP-1B KD MDCK cells incubated basolaterally with 594-Tf for 15 min displayed strong colocalization of 594-Tf with immunostained rab11a at ARE, which typically appeared as a bright spot under the apical PM (Figure 2A, column 3). This colocalization was not observed in AP-1B KD MDCK cells pretreated with nocodazole/cold (Figure 2A, column 4). In contrast, basolaterally internalized 594-Tf was not detected in ARE in WT MDCK cells in the absence or presence of nocodazole/cold treatment (Figure 2A, columns 1 and 2). As previously reported, MT disruption dispersed ARE in MDCK cells, but did not inhibit endocytosis of TfR (Apodaca et al, 1994; Daro et al, 1996; Casanova et al, 1999; Babbey et al, 2006). Quantitative analysis using the Mander's colocalization coefficient (see Materials and methods) showed that the percentage of rab11a colocalizing with 594-Tf increased from 0±0.1% in WT MDCK to 34±3% in AP-1B KD (P<0.001) and was reduced to 0.3±0.2% by nocodazole/cold treatment (P<0.001) (Figure 2A′ top). Accordingly, the percentage of 594-Tf colocalizing with rab11a increased from 0±0.1% in WT MDCK cells to 7±0.7% (P<0.001) in AP-1B KD MDCK cells and was reduced to 1.4±0.3% (P<0.001) after nocodazole/cold treatment (Figure 2A′ bottom). That the subapical rab11a-positive spot was indeed ARE was confirmed by the displacement of this spot to the apicolateral junction upon treatment with the MT-stabilizing drug taxol (Supplementary Figure S3A), a typical behaviour of ARE (Casanova et al, 1999; Lapierre et al, 2003). Notably, taxol treatment did not prevent transport of 594-Tf to the laterally displaced ARE in AP-1B KD MDCK cells (Supplementary Figure S3A,A′). Taken together, these results demonstrate that AP-1B KD MDCK cells carry out MT-mediated transcytosis of basolateral TfR to ARE. Figure 2.Microtubules mediate transport of basolateral TfR to both ARE and ASE in AP-1B KD MDCK cells. (A) MDCK cells were incubated from the basolateral surface with fluorescent 594-Tf for 15 min and immunostained with anti-rab11a. From left to right, columns show: control WT, nocodazole-treated WT, control AP-1B KD and nocodazole-treated AP-1B KD MDCK cells. (A′) Cells from experiments represented in (a) were quantified for the percentage of pixels of rab11a colocalizing with 594-Tf (top) and the percentage of pixels of 594-Tf colocalizing with rab11a (bottom). Circles correspond to individual cells obtained from different experiments and red lines indicate the median value. (B) MDCK cells were incubated with fluorescent 594-Tf as in (a) and stained for ASE with incubation of 488-WGA from the apical surface for 5 min at 37°C and washed with NADG at 4°C. (B′) Cells from experiments represented in (b) were quantified for the percentage of pixels of 488-WGA colocalizing with 594-Tf (top) and the percentage of pixels of 594-Tf colocalizing with 488-WGA (bottom). **P<0.001. Red line represents the median. Scale bar, 10 μm. Download figure Download PowerPoint Apical sorting endosomes (ASE) appear as multiple small dots located immediately under the apical membrane upon incubation for 5 min with apical 488-WGA (Leung et al,2000; Cresawn et al, 2007) (Figure 2B). Similar experiments to those performed for ARE, indicated that AP-1B KD MDCK cells transport significantly more basolaterally internalized 594-Tf than WT MDCK cells to ASE (Figure 2B, columns 1 and 3). Transport of basolaterally internalized 594-Tf to ASE was inhibited by nocodazole/cold treatment (Figure 2B,columns 3 and 4). Quantitative analysis showed that the percentage of 488-WGA colocalizing with 594-Tf increased from 1±0.1% in WT MDCK cells to 14±1% (P<0.001) in AP-1B KD MDCK cells and was reduced to 5±1% (P<0.001) after nocodazole/cold treatment (Figure 2B′ top). Accordingly, the percentage of 594-Tf colocalizing with 488-WGA increased from 0.6±0.1% in WT MDCK cells to 7±0.8% (P<0.001)in AP-1B KD MDCK cells and was reduced to 3.6±0.4% (P<0.001) by nocodazole/cold treatment (Figure 2B′ bottom). Control experiments indicated that basolaterally internalized 594-Tf also reached ASE in the presence of the iron chelator deferoxamine added to the apical medium (Supplementary Figure S3B,B′), ruling out the possibility that 594-Tf was delivered to the apical membrane and subsequently re-internalized into ASE. The amount of 594-Tf transported to ARE and ASE did not increase with longer incubation times (e.g., 30 min) indicating that the transport of basolateral 594-Tf to ARE and ASE reached steady state after ∼15 min (Supplementary Figure S3C,C′ and D,D′). The experiments reported in this section demonstrate that the transcytotic route of TfR in AP-1B KD MDCK cells involves MT-mediated transport to both classes of apical endosomes, ARE and ASE. In contrast, pIgR displayed an extensive colocalization with ARE, as previously reported (Apodaca et al, 1994; Brown et al, 2000), but colocalized poorly with ASE (Supplementary Figure S4D,D′). Our experiments demonstrate that the transcytotic pathways of TfR in AP-1B KD MDCK cells and pIgR involve different endosomal itineraries and unravel a novel role for ASE in apical transcytosis. LDL receptor undergoes apical transcytosis to ARE in AP-1B KD MDCK We next studied whether the apical transcytotic route utilized by TfR in AP-1B KD MDCK cells is also utilized by other AP-1B cargoes. Like TfR, LDLR is basolateral in WT MDCK and non-polar in AP-1B KD MDCK cells and LLC-PK1 cells (Folsch et al, 1999; Gan et al, 2002; Gravotta et al, 2007). In contrast, the bicarbonate transporter NCB1 localizes basolaterally in kidney proximal tubule cells indicating that its basolateral localization mechanism is independent of AP-1B (Li et al, 2007). For these experiments, we studied the expression of basolateral proteins tagged with GFP. TfR–GFP colocalized poorly with rab11a in WT MDCK cells and extensively in AP-1B KD MDCK cells (Figure 3A), confirming the results obtained with endogenous TfR. LDLR–GFP also extensively increased its colocalization with rab11a in AP-1B KD MDCK cells (Figure 3B). In contrast, NBC1–GFP colocalized poorly with rab11a in both WT and AP-1B KD MDCK cells. Quantitative analysis showed that the percentage of rab11a colocalizing with TfR–GFP increased from 4±1% in WT MDCK to 42±7% in AP-1B KD (P<0.001). The percentage of rab11a colocalizing with LDLR–GFP also increased from 6±2% to 50±8% (P<0.001), whereas the percentage of rab11a colocalizing with NBC1–GFP did not increase (6±4% and 5±3%, NS). Accordingly, the percentage of TfR–GFP colocalizing with rab11a increased from 2±0.8% to 13±2% (P<0.001). The percentage of LDLR–GFP colocalizing with rab11a also increased from 5±1% to 24±5% (P<0.001), whereas the percentage of NBC1–GFP colocalizing with rab11a did not increase (4±3% and 3±1%, NS) (Figure 3A′). Figure 3.LDLR undergoes apical transcytosis to ARE in AP-1B KD MDCK. MDCK cells were transiently transfected with TfR–GFP (A) LDLR (B) or NBC1 (C) and immunostained for rab11a. (A′, B′, C′) Cells from experiments represented in (a), (b) and (c) were quantified for the percentage of pixels of rab11a colocalizing with the cargo (left) and the percentage of pixels of the cargo colocalizing with rab11a (right). NS, no significance. **P<0.001. Red line represents the median. Scale bar, 10 μm. Download figure Download PowerPoint These results demonstrate that AP-1B cargoes undergo trancytosis via ARE in AP-1B KD MDCK cells, whereas AP-1B independent cargos remain strictly basolateral and avoid ARE in AP-1B KD MDCK cells. Transport of basolateral TfR to ARE requires the plus-end microtubule motor KIF16B To date, the MT motor(s) that mediate transcytosis across epithelia remain unknown. We used a candidate approach to search for MT motors involved in the transcytosis of TfR. KIF5B mediates transport of the apical protein p75-neurotrophin receptor (p75) in MDCK cells (Jaulin et al, 2007). KIF16B was discovered in a search for kinesins that bind the endosomal lipid PI3P and found to mediate TfR recycling in fibroblastic cells (Hoepfner et al, 2005). Transfection of a truncated, motor-less version of KIF5B (DN-KIF5B-CFP) that blocks biosynthetic trafficking of p75 did not prevent transport of basolaterally internalized 594-Tf to ARE in AP-1B KD MDCK cells (Figure 4B). In contrast, transfection of a motor-less form of KIF16B (DN-KIF16B-YFP) that inhibits TfR recycling in HeLa cells (Hoepfner et al, 2005) prevented transport of basolaterally internalized 594-Tf to ARE in AP-1B KD MDCK cells (Figure 4A). Quantitative analysis showed that the percentage of rab11a colocalizing with 594-Tf increased from 0.3±0.2% in untransfected WT MDCK cells to 32±3% (P<0.001) in untransfected AP-1B KD MDCK cells and was reduced to 12±3% (P<0.001) by the expression of DN-KIF16B-YFP, but not by the expression of DN-KIF5B-CFP (Figure 4C, left). Accordingly, the percentage of 594-Tf colocalizing with rab11a increased from 0±0.1% in untransfected WT MDCK cells to 8±1% (P<0.001) in untransfected AP-1B KD MDCK cells and was reduced to 2±1% (P<0.05) by expression of DN-KIF16B-YFP, but not by DN-KIF5B-CFP in AP-1B KD MDCK cells (Figure 4C, right). Figure 4.Transport of basolateral TfR to ARE requires the plus-end microtubule motor KIF16B. (A, B) WT and AP-1B KD MDCK cells transiently transfected with DN-KIF16B-YFP (a) or DN-KIF5B-CFP (b) were incubated from the basolateral surface with fluorescent 594-Tf for 15 min and immunostained with anti-rab11a. Transfected and non-transfected cells can be identified in the bottom panel by the signal of DN-KIF16B-YFP or DN-KIF5B-CFP. (C) Cells from experiments represented in (a) and (b) were quantified for the percentage of pixels of rab11a colocalizing with 594-Tf (left) and the percentage of pixels of 594-Tf colocalizing with rab11a (right). (D) WT and AP-1B KD MDCK cells nucleofected with either luciferase or KIF16B siRNA were incubated from the basolateral surface with fluorescent 594-Tf for 15 min and immunostained with anti-rab11a. (D′) Cells from experiments represented in (d) were quantified for the percentage of pixels of rab11a colocalizing with 594-Tf (left) and the percentage of pixels of 594-Tf colocalizing with rab11a (right). (D′′) RNA levels of KIF16B and GAPDH in AP-1B KD cells nucleofected with luciferase or KIF16B siRNA. NS, no significance, *P<0.05, ** P<0.001. Red line represents the median. Scale bar, 10 μm. Download figure Download PowerPoint Because antibodies against canine KIF16B are not available, we investigated the expression of KIF16B in MDCK cells by RT-PCR analysis using two different sets of primers. These experiments showed that KIF16B is endogenously expressed by both WT and AP-1B KD MDCK cells; furthermore, the sequence of the amplified product blasted exclusively with the canine KIF16B mRNA (Supplementary Figure S5). We next investigated whether knockdown of endogenous KIF16B in MDCK cells inhibits the apical transcytosis of basolaterally internalized TfR. Treatment of both WT and AP-1B KD MDCK cells with a pool of four siRNAs against KIF16B decreased its expression by 78% (Figure 4D′′ and Supplementary Figure S5C). KD of KIF16B in AP-1B KD MDCK cells caused a significant reduction in the colocalization of 594-Tf with immunostained rab11a at ARE after incubation with basolateral 594-Tf for 15 min (Figure 4D, columns 3 and 4), although this reduction was less pronounced than that observed after treatment with DN-KIF16B-YFP. In contrast, 594-Tf was virtually not detected in ARE in WT MDCK cells transfected with luciferase or KIF16B siRNA (Figure 4D, columns 1 and 2). Quantitation of these experiments showed that the percentage of rab11a colocalizing with 594-Tf increased from 3±0.4% in WT-Luc KD to 29±
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