PLEKHM1/DEF8/RAB7 complex regulates lysosome positioning and bone homeostasis
Toshifumi Fujiwara, … , Stavros C. Manolagas, Haibo Zhao
Toshifumi Fujiwara, … , Stavros C. Manolagas, Haibo Zhao
Published October 20, 2016
Citation Information: JCI Insight. 2016;1(17):e86330. https://doi.org/10.1172/jci.insight.86330.
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Research Article Bone biology

PLEKHM1/DEF8/RAB7 complex regulates lysosome positioning and bone homeostasis

Abstract

Mutations of the Plekhm1 gene in humans and rats cause osteopetrosis, an inherited bone disease characterized by diminished bone resorption by osteoclasts. PLEKHM1 binds to RAB7 and is critical for lysosome trafficking. However, the molecular mechanisms by which PLEKHM1 regulates lysosomal pathways remain unknown. Here, we generated germline and conditional Plekhm1-deficient mice. These mice displayed no overt abnormalities in major organs, except for an increase in trabecular bone mass. Furthermore, loss of PLEKHM1 abrogated the peripheral distribution of lysosomes and bone resorption in osteoclasts. Mechanistically, we indicated that DEF8 interacts with PLEKHM1 and promotes its binding to RAB7, whereas the binding of FAM98A and NDEL1 with PLEKHM1 connects lysosomes to microtubules. Importantly, suppression of these proteins results in lysosome positioning and bone resorption defects similar to those of Plekhm1-null osteoclasts. Thus, PLHKEM1, DEF8, FAM98A, and NDEL1 constitute a molecular complex that regulates lysosome positioning and secretion through RAB7.

Authors

Toshifumi Fujiwara, Shiqiao Ye, Thiago Castro-Gomes, Caylin G. Winchell, Norma W. Andrews, Daniel E. Voth, Kottayil I. Varughese, Samuel G. Mackintosh, Yunfeng Feng, Nathan Pavlos, Takashi Nakamura, Stavros C. Manolagas, Haibo Zhao

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Figure 5

PLEKHM1 regulates lysosome peripheral distribution and ruffled border formation in osteoclasts.

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PLEKHM1 regulates lysosome peripheral distribution and ruffled border fo...
(A) An illustration of the polarized structures of an active osteoclast cultured on bone. The levels (upper level and bone surface level) of two representative sections of confocal microscopic images shown in B and C are indicated. (B) Immunofluorescent staining of LAMP-2 and (C) immunofluorescent staining of cathepsin K (CTSK) in wild-type and Plekhm1–/– (KO) osteoclasts cultured on glass coverslips and bone slices. White arrows in top rows point out the distribution of LAMP-2–positive lysosomes and CTSK at the periphery of WT osteoclasts. White arrowheads in the bottom rows point out the localization of LAMP-2 at the ruffled border and secretion of CTSK in the resorption lacuna inside actin rings of WT osteoclasts cultured on bone. Each image is a representative of 6 coverslips or bone slices/group/culture from at least 3 independent cultures from different mice. Scale bar: 20 μm. (D) Electron microscopic images of WT and KO osteoclasts cultured on bone slices in vitro. Images are representatives of 6 osteoclasts/group. SZ, sealing zone; RB, ruffled border. Black arrows in bottom panel point out enlarged vesicles accumulated in KO osteoclasts. Scale bar: 5 μm.