Recombinant VSV G proteins reveal a novel raft-dependent endocytic pathway in resorbing osteoclasts.
Transcytotic membrane flow delivers degraded bone fragments from the ruffled border to the functional secretory domain, FSD, in bone resorbing osteoclasts. Here we show that there is also a FSD-to-ruffled border trafficking pathway that compensates for the membrane loss during the matrix uptake process and that rafts are essential for this ruffled border-targeted endosomal pathway. Replacing the cytoplasmic tail of the vesicular stomatitis virus G protein with that of CD4 resulted in partial insolubility in Triton X-100 and retargeting from the peripheral non-bone facing plasma membrane to the FSD. Recombinant G proteins were subsequently endosytosed and delivered from the FSD to the peripheral fusion zone of the ruffled border, which were both rich in lipid rafts as suggested by viral protein transport analysis and visualizing the rafts with fluorescent recombinant cholera toxin. Cholesterol depletion by methyl-beta-cyclodextrin impaired the ruffled border-targeted vesicle trafficking pathway and inhibited bone resorption dose-dependently as quantified by measuring the CTX and TRACP 5b secreted to the culture medium and by measuring the resorbed area visualized with a bi-phasic labeling method using sulpho-NHS-biotin and WGA-lectin. Thus, rafts are vital for membrane recycling from the FSD to the late endosomal/lysosomal ruffled border and bone resorption.
Mulari
Mika T K
MT
Department of Anatomy, Institute of Biomedicine, University of Turku, Kiinamyllynkatu 10, Fin-20520, Turku, Finland. mika.mulari@utu.fi <mika.mulari@utu.fi>
Nars
Martin
M
Laitala-Leinonen
Tiina
T
Kaisto
Tuula
T
Metsikkö
Kalervo
K
Sun
Yi
Y
Väänänen
H Kalervo
HK
D000818:Animals; D001862:Bone Resorption; D015704:CD4 Antigens / Q000737:chemistry; D016764:Cell Polarity; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism; D003902:Detergents; D004705:Endocytosis; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000032:analysis; D008562:Membrane Glycoproteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism*; D017830:Octoxynol; D010010:Osteoclasts / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D021381:Protein Transport; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D012995:Solubility; D014759:Viral Envelope Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics
D015704:CD4 Antigens; D003902:Detergents; C028391:G protein, vesicular stomatitis virus; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D017830:Octoxynol; D002784:Cholesterol