Fig 1.
Generation of osteoclasts, odontoclasts and clasts in cell culture.
Presence of tartrate-resistant acid phosphatase (TRAP) positive cells at the end of the cell culture period on (A) osteoclasts on bone slice; (B) odontoclasts on dentin slice; (C) clasts on plastic tissue culture plate. All photos are at the same magnification. Note that on plastic some giant cells with more than 10 nuclei form whereas on bone or dentin rarely are cells with more than 5 nuclei detected. To confirm that osteoclasts (D) and odontoclasts (E) were resorbing, actin rings, which are surrogate markers for resorbing osteoclasts and odontoclasts, were stained with rhodamine-phalloidin. The scale bar is 66 μm in A-C and 12 μm in D and E.
Fig 2.
Overview of protein composition of EVs from clastic cells.
(A) Venn diagram of proteins detected from osteoclasts, odontoclasts and clasts. (B) Comparison of the gene ontology biological pathways represented by 10% or more of the total proteins in EVs. (C) Selected primary cellular location of proteins detected in clastic EVs. These locations were abundant and indicative of the general feature that there was little difference between the groups of clastic EVs.
Fig 3.
Categorization of proteins by cellular location.
The 200 most abundant proteins in extracellular vesicles (EVs) were categorized based on their expected cellular location. Numbers at the top indicate the percentage of the proteins in the category reported to be extracellular vesicle (EV) proteins in ExoCarta. EVs from all types of clastic cells had mostly cytosolic proteins and very few transmembrane proteins.
Table 1.
Selected transmembrane proteins in EVs with number of peptides and Z-scores.
Fig 4.
Immunoblot confirms that reduced levels of RANK are present in EVs from odontoclasts.
Left panel; 3 X 107 EVs (by nanoparticle tracking) isolated from clasts, odontoclasts and osteoclast were separated by SDS-PAGE, blotted to Immobilon P, and probed with anti-RANK antibody (Biorbyt, Cat # orb8560). Note the reduced level of RANK in the EVs from odontoclasts. Middle panel; 100 μg of protein from whole cell extracts of clasts, odontoclasts and osteoclasts was probed with the anti-RANK antibody. Levels of RANK were similar in all cell types. Right panel; The same cell extracts from clasts, odontoclasts and osteoclasts were probed with anti-actin (Sigma, Cat # A2066) to show that the loading of proteins was similar.
Table 2.
Protein complexes found in clastic cells and other cell types.
Fig 5.
Evidence for protein complexes in clastic EVs.
(A) Comparison of the relative abundance by 2D LC/MS/MS of hexaminidase alpha and beta, which are known to exist as a tight dimer across 6 experiments. (B-H) Various complexes found in clastic EVs, with relative abundance plotted against the expected amount of protein (molecular weight X expected stoichiometry). Note that the relative abundance estimate stays similar within all elements of a complex in a group; in most cases all elements of a complex in one group are similarly enriched or reduced compared to the other groups.
Fig 6.
Evidence for a PRR-containing V-ATPase sub-complex in clastic EVs.
(A) Relative abundance of the V-ATPase subunits and the PRR found in clastic EVs. (B) Left, Western blot of whole EVs stained with anti-PRR; right anti-E-subunit and IgG control immunoprecipitates (IP) from detergent-extracted EVs from clasts that were then blotted and probed with the anti-PRR antibody. HC and LC refer to the heavy and light chains of the antibodies used in the IPs. (C) Left is a schematic representation of the intact V-ATPase. The PRR has been positioned hypothetically in the intact enzyme. Right is a schematic of the subunits detected in osteoclast EVs assuming they are part of a single complex.