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Plasmid, virus and cell cultureFor CRISPR mediated gene knockouts, the sgRNA sequences were cloned into the CRISPR plasmid pLentiCRISPRv2(Addgene, MA, USA #52961) as previously described [13Genome-scale CRISPR screening for modifiers of cellular LDL uptake.]. Virus particles were then prepared by co-transfection of cloned sgRNA together with pCMV-VSV-G (Addgene #8454) and (Addgene #12260) into HEK293T cells with Lipofectamine LTX (ThermoFisher). Media was replaced at 12 hr post transfection. Conditioned media containing virus were harvested at 48 hr post-transfection, centrifuged at 1000g for 10 mins, and the resulting supernatant stored at -80 C for future use. To generate knockout cells, HuH7 cells were transduced with lentivirus carrying the corresponding sgRNA, selected for transduced cells with puromycin, and passaged for two weeks to allow time for target site mutagenesis and turnover of wild type protein. RAB10 knockout clonal cell lines were derived by diluting cell suspensions into 96 well plates. Wells containing a single colony of growth were then expanded. Selected clonal cell lines were analyzed by immunofluorescence and immunoblotting. RAB10 expression constructs were generated by cloning CRISPR resistant cDNA sequences and a blasticidin resistance cassette into the lentiviral expression vector LeGO-iC2(Addgene, 27345) using GIBSON assembly mix purchased from NEB (NEBuilder HiFi DNA Assembly). HEK293T and HuH7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, and 100 U/ml penicillin, 100 mg/ml streptomycin (Thermo Scientific) at 37˚C in a 5% CO2-conditioned, humidified incubator.LDL and transferrin uptake assayCells were seeded in 6 well plates to achieve ∼70%-80% confluence on the day of analysis. For uptake assays, cells were washed with serum free DMEM and then incubated in DMEM containing either 4 μg/ml DyLight550-conjugated LDL (Cayman Chemical) or 5 μg/ml Alexa Fluor 555 conjugated transferrin (ThermoFisher Scientific) at 37˚C for 1 hour or 30 mins, respectively. Cells were harvested with TrypLE express (ThermoFisher Scientific), washed with ice cold PBS, resuspended in 150ul of ice-cold PBS, and analyzed with a Bio-Rad Ze5 flow cytometer. Data analysis was performed with FlowJo (FlowJo).
Western blotCells were cultured at 37˚C in 10 cm dish until 70-80% confluent. Cells collected with trypLE express were washed in PBS, and then lysed in RIPA lysis and extraction buffer (Thermo Scientific) containing complete protease inhibitor cocktail (Roche). After brief sonication, lysed cell suspensions were rotated at 4˚C for 1 hr for protein extraction followed by centrifugation at 15000g. Protein concentration was determined with the Bio-Rad DC assay kit (Bio-Rad, # 500-0111) and SDS-PAGE was performed using NuPAGE™ 4 to 12%, Bis-Tris, mini protein gels (ThermoFisher Scientific # NP0321BOX) according to manufacturer’s instruction. Western blot transfer was done into nitrocellulose membrane (Thermo scientific #IB23002) using the iBlot 2 Dry Blotting System (Thermo Scientific).
Flow cytometryHuH7 cells cultured in 6 well plates were prepared for analysis at 70-80% confluence. For surface staining, collected cells were washed three times with ice cold blocking buffer (PBS, 2% FBS), resuspended at approximately 106 cells in 1 mL blocking buffer and incubated for 30 mins with end-over-end rotation at 4˚C. After centrifugation at 400g for 5 mins, cells were resuspended in fluorescently labelled LDLR antibody or TFR antibody diluted in 100 μl blocking buffer and incubated for 1 hr in the dark at 4oC. Cells were then washed 3 times with ice cold PBS, resuspended in 150 μl cold PBS for final analysis by flow cytometry (Bio-Rad ZE5). For quantification of total cellular LDLR or TFR, harvested cells were fixed with 2% PFA for 10 minutes followed by PBS wash and permeabilization with 500 μl of 0.5% saponin in PBS before proceeding with staining for LDLR and TFR.
Immunofluorescence and confocal microscopyCells cultured on poly-D-lysine coated glass coverslips (Electron Microscopy Sciences, #72294-11) were fixed in 2% paraformaldehyde for 15 mins in the dark at room temperature. After washing three times with PBS, cells were then permeabilized with 0.1% saponin in PBS for 5 mins, incubated for 1 hr in blocking buffer (PBS with 4% FBS and 40mM glycine), stained with primary antibody at indicated dilutions in PBS with 4% FBS for 1 hr, washed with PBS three times, stained with secondary antibody at indicated dilutions in PBS with 4% FBS for 1 hr, and washed with PBS three times. Coverslips were mounted on glass slides with Prolong Diamond antifade mounting reagent (Invitrogen). Images were acquired with a NIKON A1 standard sensitivity (SS) confocal microscope with 60X (NA51.4) oil objective. Colocalization quantification was done using the open-source Fiji (Image J) software. Mander's coefficient and Pearson's coefficient were calculated using JACop in Image J. A total of 10-30 cells from two to three biological replicates were analyzed. For all quantitative analysis, the observer was blinded to cell genotype.
Endocytosis assayAn assay for transferrin receptor endocytosis was adapted from previous reports [32The Connecdenn DENN domain: a GEF for Rab35 mediating cargo-specific exit from early endosomes.]. Briefly, cells grown in 10 cm dishes were serum starved in DMEM for 30 mins, harvested in tryPLE Express (ThermoFisher Scientific), washed in ice cold DMEM, incubated with Alexa Fluor 555-conjugated transferrin in DMEM at 4˚C, and rotated for 1 hr. Unbound excess transferrin was removed by washing cells with PBS and surface bound transferrin internalization was induced by incubating cells in prewarmed complete culture medium at 37˚C for various time points. At each time point, an excess of ice-cold PBS was added to a sample to stop internalization, cells were collected by centrifugation, and surface bound transferrin was removed with ice-cold acid wash buffer (0.1 M glycine and 150 mM NaCl, pH 3) followed by three PBS washes. Cells were resuspended in ice-cold PBS and analyzed by flow cytometry on a BioRad ZE5. 10000-15000 cells were analyzed for each time point.An assay for LDLR endocytosis was adapted from previous reports [34Monoclonal antibodies to the low density lipoprotein receptor as probes for study of receptor-mediated endocytosis and the genetics of familial hypercholesterolemia., 35The adaptor protein Dab2 sorts LDL receptors into coated pits independently of AP-2 and ARH.]. Briefly, after PBS wash, cells were incubated in blocking buffer (2% FBS in PBS) for 30 mins at 4˚C. Surface LDLR was then stained with LDLR antibody for 1 hr at 4˚C and cells were washed with PBS to remove excess antibody. Cells were then incubated with prewarmed media at 37˚C for the indicated duration of time. At each time point, ice-cold blocking buffer was added to the sample, cells were collected by centrifugation, and the remaining surface-exposed LDLR antibody was labeled by incubation with fluorescent secondary antibody for 1 hour at 4˚C followed by three PBS washes. Cells were resuspended in ice-cold PBS. Analysis was performed by flow cytometry on a BioRad ZE5, with 10000-15000 cells analyzed for each time point.Recycling assayAn assay for transferrin recycling was adapted from a previous report [32The Connecdenn DENN domain: a GEF for Rab35 mediating cargo-specific exit from early endosomes.]. Briefly, cells were serum starved for 30 min in DMEM, incubated with Alexa Fluor 555 transferrin for 30 minutes at 37˚C, and washed with ice-cold PBS. Surface-bound transferrin was then removed by cold acid wash (0.1 M glycine and 150 mM NaCl, pH 3) followed by a PBS wash. Cell samples were resuspended in pre-warmed media at 37˚C for the indicated times. A second acid wash followed by PBS wash was done after which samples were analyzed by flow cytometry.DISCUSSIONRecycling of endocytosed membrane proteins to the cell surface plays an important role in maintaining the composition of the plasma membrane and the physiologic functions of the recycled proteins[39Boucrot E. Kirchhausen T. Endosomal recycling controls plasma membrane area during mitosis., 40Grant B.D. Donaldson J.G. Pathways and mechanisms of endocytic recycling., 41O'Sullivan M.J. Lindsay A.J. The Endosomal Recycling Pathway-At the Crossroads of the Cell.]. Together with gene expression, protein secretion, and protein turnover, recycling regulates the steady state level of a given receptor protein on the cell surface. The initial endocytosis of integral membrane proteins shares similar features, with receptors often releasing their ligands in the acidic lumen of early endosomes. After complex dissociation, receptors may then recycle back to the plasma membrane, either directly or via the endocytic recycling compartment (ERC) and late recycling vesicles.Rab GTPases have previously been reported to play broad roles in the regulation of vesicular trafficking. We recently identified the small GTPase RAB10 as a putative modifier of cellular LDL and transferrin uptake [13Genome-scale CRISPR screening for modifiers of cellular LDL uptake.]. In the current report, we confirmed the discordant effects of RAB10 on LDL and transferrin cellular accumulation, with the former decreased and the latter increased upon RAB10 depletion (Fig. 1B-C). Unexpectedly, in contrast to the opposing effects of RAB10 depletion on LDL and transferrin uptake, we observed similar effects on their corresponding receptors, LDLR and TFR. This discrepancy was likely due to the different fates of the two ligands following uptake, with LDL undergoing dissociation from LDLR while transferrin remains in complex with TFR during recycling until its release extracellularly. This process is summarized schematically in Figure 7.Figure 7Differential effects of RAB10 deletion on LDLR and TFR recycling. (A) LDL bound LDLR and holo transferrin bound TFR undergo clathrin mediated endocytosis, upon which LDLR releases LDL and transferrin releases iron molecules within sorting endosome. LDLR is then transported via an endocytic recycling compartment (ERC) and recycled to the cell surface. The majority (80-90%) of apo transferrin bound TFR is recycled through a fast-recycling route to the cell surface. (B) Deletion of RAB10 results in a defect in trafficking of both fast and slow recycling vesicles, leading to accumulation of LDLR in RAB11 positive ERC and TFR in RAB4 positive fast recycling vesicles, resulting in reduced cell surface LDLR and TFR. Despite both LDLR and TFR accumulating within recycling compartments, only transferrin, which remains in complex with TFR, also accumulates in RAB10-deleted cells, as LDL dissociates from LDLR and undergoes degradation within lysosomes.
Several lines of evidence support a model in which RAB10 promotes the recycling of both LDLR and TFR. First, RAB10 depletion caused a decrease in the amount of both receptors on the cell surface without a corresponding decrease in gene expression (Fig. 2). Second, RAB10 depletion also caused an intracellular accumulation of both receptors in recycling organelles consistent with a delay in their plasma membrane recycling (Fig. 4H-I, Fig. 5G-H). Third, a subpopulation of RAB10 was found to colocalize with both receptors and with recycling endosomes. Fourth, the association of GTP locked RAB10 mutant with recycling endosomes was decreased, consistent with this active form accelerating the anterograde transport of cargo vesicles out of this compartment. Finally, kinetic experiments confirmed a delay in TFR recycling to the plasma membrane (Fig. 6D).Previous studies have revealed heterogeneity in the recycling of different receptors, with some, including LDLR, transported along a RAB11-mediated slow recycling pathway involving the ERC, while others, including TFR, utilize both RAB11 and RAB4-mediated rapid recycling pathways [20Mayle K.M. Le A.M. Kamei D.T. The intracellular trafficking pathway of transferrin., 42Mayor S. Presley J.F. Maxfield F.R. Sorting of membrane components from endosomes and subsequent recycling to the cell surface occurs by a bulk flow process., 43The receptor recycling pathway contains two distinct populations of early endosomes with different sorting functions.]. Our results implicate RAB10 in both pathways, as we observed LDLR accumulation in RAB11-positive punctae and TFR accumulation in RAB4-positive punctae upon RAB10 depletion.Small GTPases function as molecular switches, cycling between a GDP bound inactive state and GTP-bound active state that mediates recruitment of effector proteins to membranes. Intriguingly, GTP-bound RAB10 has previously been demonstrated to mediate the insulin-stimulated transport of GLUT4-containing vesicles to the plasma membrane via its recruitment of the exocyst membrane tethering complex [44A potential link between insulin signaling and GLUT4 translocation: Association of Rab10-GTP with the exocyst subunit Exoc6/6b.]. Our prior screen of LDL uptake modifiers likewise identified several exocyst components including EXOC1, EXOC2, EXOC3, EXOC4, EXOC7, EXOC8 that phenocopied RAB10, with depletion of either protein resulting in decreased LDL uptake and increased transferrin accumulation. Association of RAB10 with the exocyst complex has also been reported in renal epithelial cells [45Babbey C.M. Bacallao R.L. Dunn K.W. Rab10 associates with primary cilia and the exocyst complex in renal epithelial cells.]. Taken together, these findings suggest that RAB10 may promote the recycling of LDLR and TFR through the recruitment of the exocyst to recycling vesicles. The previously reported CRISPR screen for modifiers of LDL uptake [13Genome-scale CRISPR screening for modifiers of cellular LDL uptake.] also identified RABIF (Rab interacting factor), a guanine nucleotide exchange factor that stimulates GDP release from various Rab GTPases including RAB10, and which has also been shown to stabilize RAB10 [46RABIF/MSS4 is a Rab-stabilizing holdase chaperone required for GLUT4 exocytosis.]. This screen also identified STX4, a SNARE protein that facilitates docking and fusion of transport vesicle with the cell membrane and has been similarly implicated in the fusion of GLUT4 vesicles with the plasma membrane[47Regulation of glucose transport by insulin: traffic control of GLUT4.]. A recent study based on published proteomic data and CRISPR/Cas9 screens also identified a correlation between RAB10 and STX4[48Data mining for traffic information.]. Taken together, these findings suggest that RAB10, the exocyst, and STX4 may work together to coordinate the trafficking, tethering, and fusion of LDLR and TFR-containing recycling vesicles, similar to their role in GLUT4 vesicular trafficking.RAB10 has also been implicated in diverse areas of membrane trafficking in different cell types, including formation of noncanonical macropinosome tubules in macrophages [49Rab10-Positive Tubular Structures Represent a Novel Endocytic Pathway That Diverges From Canonical Macropinocytosis in RAW264 Macrophages.], vesicle transportation from early endosome to recycling endosome in C. elegans[28RAB-10 is required for endocytic recycling in the Caenorhabditis elegans intestine.], and Golgi to plasma membrane transport in macrophages [29Ras-related protein Rab10 facilitates TLR4 signaling by promoting replenishment of TLR4 onto the plasma membrane.]. Consistent with this wide range of functions, RAB10 has been localized to multiple subcellular compartments in different cell types including the endoplasmic reticulum, trans Golgi network, early endosomes, recycling endosomes, phagosomes, and primary cilia [22Rab10 GTPase regulates ER dynamics and morphology., 28RAB-10 is required for endocytic recycling in the Caenorhabditis elegans intestine., 29Ras-related protein Rab10 facilitates TLR4 signaling by promoting replenishment of TLR4 onto the plasma membrane., 50A Rab10-dependent mechanism for polarized basement membrane secretion during organ morphogenesis., 51Rab10 regulates tubular endosome formation through KIF13A and KIF13B motors., 52Analysis of rab10 localization in sea urchin embryonic cells by three-dimensional reconstruction.]. We also observed significant subpopulations of RAB10 in several subcellular compartments. In further support of the breadth of cellular functions for RAB10, germline deletion of RAB10 in mice results in embryonic lethality[53Targeted disruption of Rab10 causes early embryonic lethality.]. This latter observation limits the direct confirmation of our current findings in an in vivo mouse model and, together with our demonstration that multiple receptors depend on RAB10 for recycling, suggests that the potential for RAB10-mediated LDLR recycling as a therapeutic target is likely to be limited by substantial off-target effects.Funding and additional informationThis research was supported by National Institutes of Health grants R35-HL135793T (DG) and K08-HL148552 (BTE). DG is a Howard Hughes Medical Institute investigator.
The authors declare that they have no conflicts of interest with the contents of this article.
DATA AVAILABILITY: All data are contained within the manuscript.
Author Contribution StatementTaslima Gani Khan: Conceptualization, Methodology, Investigation, Writing-original draft, visualization; David Ginsburg: Conceptualization, Methodology, Writing- review & editing, Supervision, Project administration, Funding acquisition; Brian T. Emmer: Conceptualization, Methodology, Writing- review & editing, Supervision, Project administration, Funding acquisition.
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