Figure 1.
Plekhm1 is a novel LIS1-interacting protein in osteoclasts.
(A and B) Endogenous LIS1 binds to RUN and PH1 domains of Plekhm1. GST and GST fusion proteins of individual domain of mouse Plekhm1 were expressed in E. coli and purified. Proteins bound to glutathione-Sepharose beads were incubated with mature osteoclast lysate, and co-purified proteins were visualized by Coomassie blue staining and identified by mass spectrometry (A) or immunoblotting (B). (C) HA tagged Plekhm1 is co-immunoprecipitated with endogenous LIS1 from mature osteoclast lysate. Bone marrow macrophages were retrovirally transduced with empty vector (V), full-length (FL), N-terminal half (N), and C-terminal half (C) of mouse Plekhm1. Mature osteoclasts were lysed and subjected to immunoprecipitation experiments with anti-HA antibody. Immunoprecipitated proteins were then analyzed by western blotting with anti-LIS1, anti-dynein intermediate chain (DIC) and anti-HA antibodies, respectively. (D) RUN and PH1 domains of Plekhm1 mediate its interaction with LIS1. Bone marrow macrophages were retrovirally transduced with N-terminal half of Plekhm1 without RUN domain (N-ΔRUN), C-terminal half (C) and C-terminal half depleted in PH1 domain (C-ΔPH1). Endogenous LIS1 was Immunoprecipitated by a rabbit polyclonal antibody from mature osteoclasts and the binding of Plekhm1 was detected by monoclonal anti-HA antibody. The multiple low molecular-weight bands shown in the TCL HA blot for C-terminal Plekhm1 are probably due to the degradation of the protein. MW, molecular mass; WB, Western blot; IP, immunoprecipitation; TCL, total cell lysate.
Figure 2.
LIS1 is essential for osteoclast formation and function.
(A) Knockdown of LIS1 expression by lentivirus mediated shRNAs. Bone marrow macrophages were transduced with lentiviral vectors expressing a control shRNA (LUC-sh) or LIS1 specific shRNAs (LIS1a-sh and LIS1b-sh), respectively. Bone marrow macrophages (m), pre-osteoclasts (p) and mature osteoclasts (o) were lysed and analyzed by western blotting. (B) LIS1 down-regulation attenuates multinucleated TRAP+ osteoclast formation. Scale bar = 10 µm. (C) The number of TRAP+, multinucleated osteoclasts per well of 48-well plate were counted, n = 6. (D) Decreased LIS1 expression blocks osteoclastic bone resorption. Osteoclasts were cultured on cortical bovine bone slices and resorption pits were labeled with peroxidase-conjugated wheat germ agglutinin. Scale bar = 10 µm. (E) The percentage of resorbed area/bone slice as measured by Ossteomeasure software. n = 6. (F) CTx-I level in culture medium was measured by ELISA (E), n = 6. WB, Western blot. = . ** p<0.01 vs LUC-sh by Student's t-test.
Figure 3.
LIS1 controls macrophage proliferation, differentiation and survival via modulating M-CSF and RANKL signaling pathways and NFATc1.
(A) Low level of LIS1 inhibits M-CSF induced ERK and AKT activation. LUC-sh and LIS1-sh transduced BMMs were serum and cytokine starved for 12 hours before stimulated with 50 ng/ml M-CSF for indicated time. ERK and AKT activation were detected by western blots with anti-phospho-ERK (p-ERK) and anti-phospho-AKT (p-AKT) antibodies. The knockdown efficiency was examined by LIS1 western blot. Western blots for total ERK (t-ERK), AKT (t-AKT) and DIC serve as loading controls. (B) Depletion of LIS1 results in sustained RANKL induced JNK activation without changing in NF-κB activation as determined by western blots with anti-phospho-JNK (p-JNK) and anti-phospho-IκB (p- IκB) antibodies. Total JNK (t-JNK) and IκB (t- IκB) serve as controls. (C) LIS1 knockdown BMMs are less proliferative, as measured by Brdu incorporation ELISA kit. (D) Loss of LIS1 accelerates osteoclast apoptosis. Pre-osteoclasts were either untreated or were serum and cytokine starved for 3 hours. Apoptosis was assessed by a fluorometric caspase-3 activity assay. (E) LIS1-depletiojn leads to decreased NFATc1 expression in osteoclasts. Bone marrow macrophages (m), pre-osteoclasts (p), and mature osteoclasts (o) were lysed and the level of NFATc1 and acetylated tubulin (ac-tubulin) was detected by Western blots. β-tubulin serves as a loading control. * p<0.05, ** p<0.01 vs LUC-sh by Student's t-test.
Figure 4.
LIS1 regulates macrophage and pre-osteoclast motility.
BMMs (A) and pre-osteoclasts (B) were cultured in Bioptechs dishes and the cells were imaged by time-lapse video microscopy. Images were obtained using Scion imaging software at 10 minute intervals for 8 hours. The 8-hour movements of ten motile osteoclast precursors in each cell group were tracked and analyzed with ImageJ MTrackJ plug-in (A and B). (C and D) Cumulative length of each track was depicted as microns displaced over 48 images. ** p<0.01 vs LUC-sh by Student's t-test. Scale bar = 100 µm.
Figure 5.
LIS1 is essential for cathepsin K secretion in osteoclasts.
(A and B) Decreased LIS1 expression inhibits cathepsin K secretion in osteoclasts. F-actin and Cathepsin K in osteoclasts cultured on glass coverslips (A) and cortical bovine bone slices (B) were labeled by phalloidin and monoclonal anti-Cathepsin K antibody, respectively. Arrows in (A) demonstrate the aggregation of cathepsin K around the nucleus in LIS-1 knockdown osteoclasts. Arrow heads in (B) indicate the secretion of cathepsin K into the resorption lacuna inside the actin-rings in control osteoclasts. Scale bars = 10 µm.
Figure 6.
LIS1 interacts with dynein/dynactin complex in osteoclasts.
(A and C) V5 tagged LIS1 (LIS1-V5) binds to and co-localizes with endogenous p150glued subunit of dynactin complex in mature osteoclasts. Bone marrow macrophages were retrovirally transduced with empty vector (Vec) and LIS1-V5. Mature osteoclasts were lysed and subjected to immunoprecipitation experiments with anti-LIS1 antibody. Immunoprecipitated proteins were analyzed by Western blotting with anti- p150glued and anti-V5 antibodies, respectively (A). Osteoclasts cultured on glass coverslips were fixed and processed for immunofluorescence with anti-V5 and p150 antibodies (C). (B and D) LIS1 binds to DIC and co-localizes with dynein heavy chain (DHC) in mature osteoclasts. WB, Western blot; IP, immunoprecipitation; TCL, total cell lysate. Scale bar = 10 µm.
Figure 7.
LIS1 regulates microtubule organization and EB1 distribution in osteoclasts.
(A and B) LIS1 reduction induces condensed microtubules focused around nucleus. Filamentous actin (F-actin) and microtubules in osteoclasts cultured on glass coverslips (A) and cortical bovine bone slices (B) were labeled by Alexa-488 conjugated phalloidin and anti-tubulin antibody and were examined by conventional (A) and laser confocal microscopy (B), respectively. (C) EB1 is clustered around the nucleus and is less transported by dynein to the cell periphery in LIS1 knockdown osteoclasts (right panel) than in control cells (left panel). Cells were labeled by Hoechst 33258, phalloidin and anti-EB1 monoclonal antibody, respectively. Scale bars = 10 µm.
Figure 8.
LIS1 modulates cytoplasmic dynein activity in osteoclasts.
(A) Loss of LIS1 in osteoclasts does not affect dynein/dynactin complex integrity as measured by a sucrose gradient sedimentation assay. (B) Decreased LIS1 expression in osteoclasts causes dispersed Golgi apparatus (arrows in upper panels and inset 1, 3 and 5) and reduced transportation of CLIP170 to the periphery of osteoclasts. Arrows in the middle panels indicate an increased accumulation of CLIP170 around the nucleus.
Figure 9.
A schematic model for LIS1 in osteoclast formation and function.