Characterization of Rab32- and Rab38-positive lysosome-related organelles in osteoclasts and macrophages

Abstract

Both the biogenesis and functions of osteoclasts and macrophages involves dynamic membrane traffic. We screened transcript levels for Rab family small GTPases related to osteoclasts and identified Rab38. Rab38 expression is upregulated during osteoclast differentiation and maturation. In osteoclasts, both Rab38 and its paralog, Rab32, colocalize to lysosome-related organelles (LROs). In macrophages, Rab32 is also found in LROs. LROs are part of the endocytic pathway but are distinct from lysosomes. After receptor activator of NF-κB ligand stimulation, LROs contain cathepsin K and tartrate-resistant acid phosphatase inside and help both proteins to accumulate around bone resorption pits. After osteoclast maturation, these enzymes are hardly found within LROs. In macrophages derived from Rab32 and Rab38 double knockout mice, both acidification and V-ATPase a3 localization were severely compromised. Both the double knockout macrophage and bafilomycin-treated wildtype macrophage show an increase in Lamp1-positive organelles, implying that biogenesis of lysosomes and LROs are related. These results indicate that Rab32 and Rab38 both play a crucial role in LRO biogenesis in macrophages and in osteoclasts.

Keywords: Rab32; Rab38; lysosome-related organelles; macrophage; osteoclast.

Figure 1

Rab32 and Rab38 expression patterns in macrophages and osteoclasts. A, macrophages cultured on cover glasses were induced to differentiate into osteoclasts by RANKL treatment. Cells were fixed and stained with endogenous Rab32 (Santa Cruz, B-4, sc-3901784) or Rab38 (Santa Cruz, A-8, sc-3901768) and actin. The localization of Rab32-or Rab38-positive ring structures within the actin ring was determined by confocal fluorescence microscopy. Scale bar: 25 μm. B, gene expression was determined by microarray using mRNA derived from macrophages and cells on day 6 of RANKL stimulation. Expression levels and changes in expression rates (arbitrary units) of each Rab mRNA in osteoclasts are shown. C, macrophages (RANKL-stimulation day 0, day 3, and day 5) were collected. Cell lysates were subjected to Western blotting with specific antibodies to detect the expression of Rab32, Rab38, cathepsin K, and actin protein. The band intensity of the Rab32 signal was measured using the ImageJ software. The average and SD of four independent experiments are shown. RANKL, receptor activator of NF-κB ligand.

Figure 2

Rab32 and Rab38 colocalize in osteoclasts. A, cells (macrophages) on day 0 and day 5 of RANKL stimulation were labeled with Rab32 and Rab38 antibodies and observed by confocal fluorescence microscopy. Arrows indicate multinucleated osteoclasts, and arrowheads indicate mononucleated osteoclasts. B, macrophages were transduced with lentivirus for either GFP-Rab32 or GFP-Rab38 expression and induced to differentiate into osteoclasts by RANKL stimulation. After fixation, cells were labeled with antibodies against either Rab38 or Rab32 and observed by confocal fluorescence microscopy. Arrows indicate colocalization. Scale bar: 25 μm. RANKL, receptor activator of NF-κB ligand.

Figure 3

Rab32-positive organelles are positive for Lamp1 and Lamp2 but deficient in Rab7 in macrophages.A, macrophages were transduced with lentivirus for either GFP-Rab32 or GFP-Rab38 expression. Transduced cells were fixed and labeled with antibodies against a lysosomal marker, either Lamp1 or Lamp2, and observed by confocal fluorescence microscopy. Arrows indicate colocalization. B, GFP-Rab7 expressing macrophages were labeled with anti-Rab32 antibody and observed by confocal fluorescence microscopy. Arrows indicate colocalization. Scale bar: 25 μm. Lamp1, lysosomal associated membrane protein 1; RANKL, receptor activator of NF-κB ligand.

Figure 4

Phagocytosed fluorescent beads are inside Rab32/38-positive organelles.A, fluorescent beads were added to the macrophage culture medium expressing each fluorescent protein, fixed after 90 min, and observed by confocal fluorescence microscopy. The scale bar: 10 μm. B, in macrophages expressing GFP-Rab7, -Rab32, -Rab38, or Lamp1-RFP, fluorescent beads were added to the medium and fixed at each time point, and the cells were observed by confocal fluorescence microscopy. The percentage of cells with fluorescent beads within each marker-positive organelle was counted for at least 30 cells at each time point in a blinded manner. C, in macrophages expressing GFP-Rab7, -Rab32, -Rab38, or Lamp1-RFP, fluorescent beads were added to the medium, fixed at each time point, and cells were observed by confocal fluorescence microscopy. The percentage of fluorescent beads localized inside the organelle was counted for at least 10 cells per time point in a blinded manner. In figures B and C, the graphs show the SD of four independent experiments. Lamp1, lysosomal associated membrane protein 1.

Figure 5

Endogenous Rab32/38 partially colocalizes with GFP-Rab7 in osteoclasts. A, macrophages were transduced with lentivirus for GFP-Rab7 expression and induced to differentiate into osteoclasts by RANKL stimulation. After fixation, the cells were labeled with Rab32 and Rab38 antibodies and observed by confocal fluorescence microscopy. Arrows indicate colocalization. B, macrophages were transduced with lentivirus for Lamp1-RFP expression and induced to differentiate into osteoclasts by RANKL stimulation. After fixation, cells were labeled with Rab32 and Rab38 antibodies and observed by confocal fluorescence microscopy. Arrows indicate colocalization. Scale bar: 25 μm. Lamp1, lysosomal associated membrane protein 1; RANKL, receptor activator of NF-κB ligand.

Figure 6

Localization of cathepsin K inside Rab32- and Rab38-positive organelles after osteoclast induction. A and B, macrophages were genetically transfected with GFP-Rab32/38 and induced to differentiate into osteoclasts by RANKL stimulation. Magic Red Cathepsin K was added 30 min before fixation and observed by confocal fluorescence microscopy. C, macrophages were transduced with either GFP-Rab32 or GFP-Rab38 expression and RANKL stimulation was performed. After fixation, the cells were labeled with TRAP antibody and observed by confocal fluorescence microscopy. Scale bar: 25 μm. RANKL, receptor activator of NF-κB ligand; TRAP, tartrate-resistant acid phosphatase.

Figure 7

Rab32 and Rab38 affect the establishment of lysosomes, lysosome-like organelles, or both. A, GFP-Rab7 or GFP-Rab32 expressing macrophages were treated with 50 nM LysoTracker for 1 h. Cells were fixed and observed by confocal fluorescence microscopy. The LysoTracker fluorescence intensity of GFP-Rab7- and GFP-Rab32-positive vesicles was detected using the ImageJ program over 100 vesicles in more than four images at each condition. Scale bar: 10 μm. B, bone marrow-derived macrophages isolated from either WT or Rab32 KO or DKO mice were treated with 50 nM LysoTracker for 1 h and fixed with 4% PFA. Cells were labeled with rat anti-Lamp1 antibodies and observed by confocal microscopy. Scale bar: 10 μm. The graph shows the numbers of large vacuoles per cell from over 100 cells in three images in each condition. Large vacuoles were defined by a diameter exceeding 1.5 μm. The fluorescence intensity of LysoTracker in each Lamp1-positive vesicle was collected for over 300 vesicles in three images under each condition tested. These are representative patterns among multiple experiments. Statistical significance was determined by unpaired two-tailed t test (∗∗∗∗p < 0.0001). DKO, double knockout; Lamp1, lysosomal associated membrane protein 1.

Figure 8

Lysosomes positive for V-ATPase a3 subunit were fewer in number and a3 accumulation in plasma membrane were absent in Rab32 and Rab38 DKO osteoclasts. A, BMDMs isolated from either WT or DKO mice were cultured for 3 days and fixed with 4% PFA. Fixed cells were labeled with rat anti-Lamp1, chicken anti-a3 antibodies, and DAPI. In the merged image, green indicates the a3 subunits (Alexa-488), red indicates Lamp1 (Alexa-568), and blue indicates DAPI. White arrows indicate a3 signal on the plasma membrane. Red arrows indicate intracellular a3 positive structure. Scale bar: 7.5 μm. The number of a3 dots per cell was counted over 50 cells from three images in the graph. ∗∗∗ indicates p < 0.001 by unpaired two-tailed t test. B, macrophages were genetically transfected with GFP-Rab32 and incubated with either 50 nM bafilomycin A1 or 20 nM concanamycin A for 24 h. After fixation, the cells were labeled with actin and observed by confocal fluorescence microscopy. Scale bar: 25 μm. BMDM, bone marrow-derived macrophage; DKO, double knockout; Lamp1, lysosomal associated membrane protein 1.