Human liver cell trafficking mutants: characterization and whole exome sequencing

Abstract

The HuH7 liver cell mutant Trf1 is defective in membrane trafficking and is complemented by the casein kinase 2α subunit CK2α''. Here we identify characteristic morphologies, trafficking and mutational changes in six additional HuH7 mutants Trf2-Trf7. Trf1 cells were previously shown to be severely defective in gap junction functions. Using a Lucifer yellow transfer assay, remarkable attenuation of gap junction communication was revealed in each of the mutants Trf2-Trf7. Electron microscopy and light microscopy of thiamine pyrophosphatase showed that several mutants exhibited fragmented Golgi apparatus cisternae compared to parental HuH7 cells. Intracellular trafficking was investigated using assays of transferrin endocytosis and recycling and VSV G secretion. Surface binding of transferrin was reduced in all six Trf2-Trf7 mutants, which generally correlated with the degree of reduced expression of the transferrin receptor at the cell surface. The mutants displayed the same transferrin influx rates as HuH7, and for efflux rate, only Trf6 differed, having a slower transferrin efflux rate than HuH7. The kinetics of VSV G transport along the exocytic pathway were altered in Trf2 and Trf5 mutants. Genetic changes unique to particular Trf mutants were identified by exome sequencing, and one was investigated in depth. The novel mutation Ile34Phe in the GTPase RAB22A was identified in Trf4. RNA interference knockdown of RAB22A or overexpression of RAB22AI34F in HuH7 cells caused phenotypic changes characteristic of the Trf4 mutant. In addition, the Ile34Phe mutation reduced both guanine nucleotide binding and hydrolysis activities of RAB22A. Thus, the RAB22A Ile34Phe mutation appears to contribute to the Trf4 mutant phenotype.

Figure 1. Dye transfer assay.

(A) Lucifer yellow was microinjected into single cells in a confluent monolayer and dye spread to neighboring cells after 3 min (shown as green) was visualized using fluorescence microscopy. Scale bar 50 µm. (B) The extent of dye transfer was quantified by counting the number of fluorescent cells around each injected cell. Data are expressed as mean ± SEM. Numbers above each bar indicate the number of cells injected. Significance was determined by Student’s t-test. **p<0.01, ***p<0.001. Data were obtained in two experiments with HuH7 cells included in each experiment giving, respectively, 17±2.8 (n = 8) and 11±1.5 (n = 3) cells with dye. These numbers are not significantly different (p = 0.24).

Figure 2. The Golgi apparatus in Trf mutants.

(A) Representative EM images showing perinuclear Golgi apparatus membrane stacks (arrowhead) in HuH7 and Trf mutant cells. Scale bar: 0.5 µm. (B) Representative histochemical staining of the Golgi apparatus lumen with TPPase substrate. Scale bar: 10 µm.

Figure 3. Surface Tfn binding and TfnR distribution.

(A) Cells were washed and incubated with Alexa Fluor 488-labeled Tfn on ice for 30 min prior to fixation. Fixed cells were imaged using a widefield microscope. Scale bar: 10 µm. (B) The mean fluorescence of surface Tfn was measured by NIH ImageJ software and is presented as mean ± SEM (n = 11). The data are representative of two independent experiments. (C) Total and cell surface TfnR expression were analyzed by flow cytometry of fixed cells incubated with either FITC-conjugated anti-TfnR antibody or isotype control, with or without cell permeabilization. Total and surface TfnR profiles for Trf3 and Trf4 are shown with a solid line and HuH7 TfnR profiles are shown with a dashed line. The data are representative of three experiments. (D) The histogram represents cell surface or total TfnR normalized to HuH7 cells and are expressed as mean ± SEM, n = 3. Statistical analysis was performed using a Student’s t-test. *p<0.05, **p<0.01, ***p<0.001.

Figure 4. Transferrin internalization and efflux.

(A) After serum starvation, cells were treated with or without 10 µg/ml Alexa Fluor 488-labeled Tfn at 37°C for up to 60 min. Following acid washing to remove surface-bound Tfn, internalized Tfn was determined by flow cytometry. Data are expressed as mean ± SEM of three independent experiments. (B) The internalization rate was calculated using MFI at each time point normalized to MFI at 60 min for each mutant and presented as mean ± SEM. (C) Alexa Fluor 488-labeled Tfn was allowed to internalize at 37°C for 20 min prior to chase for the indicated times at 37°C in the presence of 0.1 mM desferroxamine and excess unlabeled Tfn. The amount of fluorescent Tfn remaining in cells was determined by flow cytometry. The efflux rate was normalized to 0 min efflux (100%) in each cell line, and is represented as mean ± SEM of three independent experiments. Significance was determined using a Student’s t-test. *p<0.05, **p<0.01.

Figure 5. VSV G transport from ER to the cell surface.

(A–C) Cells transfected with plasmids encoding VSV G-EGFP were incubated at 40°C overnight. The cells were then shifted to 32°C in medium containing cycloheximide for various times, fixed and imaged by fluorescence microscopy to detect VSV G-EGFP. Scale bar: 10 µm. (D) Surface VSV G-EGFP was determined using I1-hybridoma supernatant containing an antibody recognizing the extracellular domain of VSV G , and normalized against total VSV G-EGFP expression quantitated by flow cytometry analysis. Results are shown as mean ± SEM of three experiments. Significance was determined by Student’s t-test. *p<0.05.

Figure 6. Depletion of RAB22A in HuH7 cells.

(A) Western blot showing endogenous RAB22A in HuH7 and Trf4, as well as in HuH7 expressing empty vector (Ctrl) or RAB22A shRNAs (sh1 and sh2), as detected with anti-RAB22A antibody. Actin was a loading control. The data are representative of blots from three independent lysates of stable HuH7 sh1 and sh2 populations. The ratio of RAB22A to actin bands determined by NIH ImageJ was not significantly different for HuH7 versus HuH7 vector or Trf4 but RAB22A was significantly reduced in sh1 (p<0.04) and sh2 (p<0.0001). (B) The independent lysates from (A) were examined with anti-RAB31 antibody. There was no significant difference RAB31 expression between HuH7, Trf4 and HuH7 cells expressing sh1 or sh2. (C) Surface Tfn binding was analyzed after incubation of Alexa Fluor 488-labeled Tfn with cells on ice for 30 min followed by flow cytometry. Results were normalized to surface Tfn in HuH7 cells and are shown as mean ± SEM, n = 4. (D) MTT assay of cells incubated with different concentrations of PEx at 37°C for 4 days. Cells were treated with MTT solution at 37°C for 3 hours. Cell viability was reflected by the color of the formazan crystals solubilized in DMSO. The data are representative of two independent experiments. Statistical significance was determined by Student’s t-test. *p<0.05, ***p<0.001.

Figure 7. GTP binding and GTPase activity of sfGFP-RAB22AI34F.

sfGFP-tagged RAB22A and mutant proteins expressed by HEK-293T cells were captured from lysate on anti-GFP agarose, eluted and resolved by SDS-PAGE, and the blot transferred to NC membranes. GTP overlay was performed to determine GTP binding and hydrolysis as described in Materials and Methods. (A) Western blot analysis was performed using anti-GFP antibody. The two replicate blots were incubated with [γ ³²P]GTP for one hour at RT then incubated in hydrolysis buffer containing Mg²⁺ either at 4°C or at 37°C for 2 hours. (B) GTP binding was normalized to the binding of sfGFP-RAB22A WT. (C) GTP hydrolysis was quantified as the percent reduction in radioactivity after incubation at 37°C relative to GTP bound at 4°C (100%). Results are shown as mean ± range of two independent experiments. Significance was determined by Student’s t-test. *p<0.05, **p<0.01, ***p<0.001.

Figure 8. Enlarged vesicular structures in sfGFP-RAB22AI34F expressed in HEK-293 cells.

Live HEK-293 cells were imaged using spinning-disk confocal microscope 48 hours after transfection with various sfGFP-RAB22A constructs as shown (A). Scale bar: 10 µm. From 61-372 cells were examined for enlarged spherical structures and the percentage is expressed as mean ± SEM of three independent experiments (B). Significance was determined by Student’s t-test. *p<0.05, **p<0.01.