The phospholipid flippase ATP8B1 is required for lysosomal fusion in macrophages

Abstract

ATP8B1 is a phospholipid flippase and member of the type 4 subfamily of P-type ATPases (P4-ATPase) subfamily. P4-ATPases catalyze the translocation of phospholipids across biological membranes, ensuring proper membrane asymmetry, which is crucial for membrane protein targeting and activity, vesicle biogenesis, and barrier function. Here we have investigated the role of ATP8B1 in the endolysosomal pathway in macrophages. Depletion of ATP8B1 led to delayed degradation of content in the phagocytic pathway and in overacidification of the endolysosomal system. Furthermore, ATP8B1 knockdown cells exhibited large multivesicular bodies filled with intraluminal vesicles. Similar phenotypes were observed in CRISPR-generated ATP8B1 knockout cells. Importantly, induction of autophagy led to accumulation of autophagosomes in ATP8B1 knockdown cells. Collectively, our results support a novel role for ATP8B1 in lysosomal fusion in macrophages, a process crucial in the terminal phase of endolysosomal degradation.

Keywords: ATP8B1; CDC50A; PFIC1; flippase; late endosomes; lysosomal fusion; multivesicular bodies; phospholipid.

Figure 1

CDC50A is involved in phagocytosis and autophagy. (A) CDC50A expression levels in CDC50A knockdown cells. Data were normalized to shControl and expressed as means ± SD of six different samples from two independent experiments. (B) E. coli‐GFP accumulation in CDC50A knockdown cells after 1 h incubation. Data were normalized to shControl and expressed as mean fluorescent values ± SD of three to four biological replicates from four independent experiments. (C) Fluid‐phase endocytosis in CDC50A knockdown cells. Data are corrected for fluorescence after incubation on ice, normalized to shControl and expressed as means ± SD of seven different samples from two independent experiments. Statistical significance by unpaired t‐test for Figure 1A−C. (D) LC3‐II levels after incubation with 25 nM rapamycin for the indicated time points. ATP1A1 was included as a loading control. Densitometric analysis of four independent experiments is shown. Statistical significance by multiple unpaired t‐testing. SD, standard deviation.

Figure 2

ATP8B1 and CDC50A are important for phagocytic degradation in THP‐1 macrophages. (A) ATP8B1 and CDC50A expression in CDC50A and ATP8B1 knockdown cells. Data were normalized to shControl and expressed as means ± SD of six to nine different samples from two to three independent experiments. Statistical significance by one‐way ANOVA with Kruskal−Wallis correction for multiple testing. (B) E. coli‐GFP uptake. Results are corrected for cytochalasin D treatment and are expressed as MFI ± SD of triplicate wells. A representative experiment of three independent experiments is shown. Statistical significance by one‐way ANOVA with Bonferroni's correction for multiple testing. (C) Bacterial clearance in knockdown macrophages. Data of a representative experiment of two independent experiments are shown. Data are expressed as mean values normalized to the amount of CFUs at t = 0 ±SD of quadruple samples. Statistical significance by one‐way ANOVA with Bonferroni's correction for multiple testing. (D) LC3‐II and p62 protein levels in total lysates. Cells were incubated with indicated compounds for 2 h. β‐Actin is included as loading control. Densitometric analysis of four independent experiments is shown. Statistical significance by multiple unpaired t‐testing. ANOVA, analysis of variance; CFU, colony forming units; MFI, mean fluorescence intensity; SD, standard deviation.

Figure 3

The acidic volume of the endolysosomal system is enhanced in ATP8B1‐ and CDC50A knockdown THP‐1 cells. (A) Immunofluorescence detection of LAMP2. Nuclear DAPI staining in blue. Scale bar: shControl, 7.5 µm; CDC50A knockdown, 5 µm; ATP8B1 knockdown, 10 µm. (B) LAMP2 protein levels. ATP1A1 is included as loading control. LAMP2 levels were quantified by densitometric analysis and were normalized to ATP1A1 and expressed as mean ± SD. Statistical significance was determined with one‐way ANOVA with Bonferroni's correction for multiple testing. (C) Lysotracker accumulation in THP‐1 macrophages. Results are expressed as MFI ± SD of one representative experiment of four independent experiments. Statistical significance by two‐way ANOVA with Bonferroni's correction for multiple testing. ANOVA, analysis of variance; MFI, mean fluorescence intensity; SD, standard deviation.

Figure 4

ATP8B1 and CDC50A knockdown cells show overacidification of the endolysosomal system. (A) and (B) Vehicle‐ and bafilomycin‐treated CDC50A (A) and ATP8B1 (B) knockdown cells were incubated with lysosensor Yellow/Blue at 37°C and fluorescence intensity was measured with a CLARIOstar monochromator microplate reader. pH standard curves (shown as inserts in the graphs) were generated for each experiment by calculating the ratio of light excited at 340‐10 versus 380‐10 nm from cells calibrated in MES buffer containing 15 μM monensin and 15 μM nigericin pH 3.5−7.0. pH values for both cell lines were extrapolated from the corresponding standard curves and are shown as means ± SD of five independent experiments. Statistical significance was tested with two‐way ANOVA with Bonferroni's correction for multiple testing. (C) Immunofluorescence analysis of V₀D₁ by confocal analysis. Nuclear DAPI staining in blue. Scale bar: 10 µm. (D) V₀D₁ protein levels in total lysates. ATP1A1 is included as loading control. Band intensities were quantified by densitometric analysis and are expressed as mean protein levels normalized to ATP1A1 ± SD. Statistical significance by one‐way ANOVA with Bonferroni's correction for multiple testing. ANOVA, analysis of variance; SD, standard deviation.

Figure 5

ATP8B1 knockout cells phenocopy ATP8B1 knockdown cells. (A) ATP8B1 levels in ATP8B1 knockout lines, two control lines (3.1 and 3.2) and a parental line (WT). ATP1A1 is included as loading control. (B) Localization of GFP‐tagged ATP8B1 in ATP8B1 knockout THP‐1 cells. Arrows indicate PM localization. Nuclear DAPI staining in blue. Scale bar: 10 µm. (C) Lysotracker accumulation in ATP8B1^Ko2.3 THP‐1 macrophages. Results are expressed as MFI ± SD of triplicate wells. Statistical significance by one‐way ANOVA with Bonferroni's correction for multiple testing. (D) Endolysosomal pH measurements by lysosensor as described in Figure 4. Data shown are means ± SD of three independent experiments. Statistical significance by two‐way ANOVA with Bonferroni's correction for multiple testing. ANOVA, analysis of variance; MFI, mean fluorescence intensity; PM, plasma membrane; SD, standard deviation.

Figure 6

ATP8B1 depletion leads to accumulation of large multivesicular bodies. (A) Immunofluorescence staining of CD63. Nuclear DAPI staining in blue. Scale bar: 5 µm. Arrows indicate enlarged vesicles. (B) CD63 levels in shControl and ATP8B1 knockdown cells. Both gels are from the same blot from which nonrelevant samples were spliced. (C) Immunogold‐labeling of the MVB marker CD63 on thin sections of shControl, ATP8B1 knockdown, and ATP8B1^KO2.3 THP‐1 cells. Black dashed squares highlight CD63‐positive compartments and are shown at high magnification on the right. ATP8B1‐depleted and ATP8B1^KO2.3 cells exhibit large CD63‐positive compartments containing intraluminal vesicles.

Figure 7

ATP8B1 knockdown impairs autophagosome‐lysosome fusion. (A) shControl and ATP8B1 knockdown THP‐1 cells expressing mRFP‐GFP‐LC3 were incubated in full medium or EBSS (for starvation) during 4 h before fixation. (B) The number of autophagosomes was quantified as described in the materials and methods. Data are relative mean puncta per cell ± SEM of 60−70 cells per condition. Statistical significance by two‐way ANOVA with Bonferroni's correction for multiple testing. Data of a representative experiment of two independent experiments are shown. (C) Immunofluorescence costaining of RAB7 and LAMP2. RAB7 costained with LAMP2‐positive endolysosomal compartments (merge). Nuclear DAPI staining in blue. Scale bar: 7.5 µm. ANOVA, analysis of variance; EBSS, Earle's balanced salt solution.