Lysosome Dispersion in Osteoblasts Accommodates Enhanced Collagen Production during Differentiation

Abstract

Lysosomes are essential organelles for intracellular degradation and are generally sequestered near the cell center to receive vesicles with contents targeted for destruction. During ascorbic acid (AA)-induced differentiation of osteogenic cells (Beck, G. R., Jr., Zerler, B., and Moran, E. (2001) Cell Growth Differ. 12, 61-83), we saw a marked increase in total lysosome organelles in osteoblastic cells, in addition to an enhanced endocytic rate. Interestingly, lysosomes were dispersed toward the cell periphery in differentiating osteoblasts. We determined that lysosome dispersion in differentiated osteoblasts required intact microtubules for long range transport and was dependent on kinesin motors but did not involve cytosolic acidification. Impairment of lysosome dispersion markedly reduced AA-induced osteoblast differentiation. Lysosomes were not secreted in differentiated osteoblasts, implicating them instead in intracellular degradation. We assayed the degradative capacity and saw a significant increase in DQ-ovalbumin fluorescence in differentiated osteogenic cells compared with undifferentiated control cells. Osteogenic cells are specialized for type I collagen production, and we noted enhanced secreted and intracellular collagen in AA-differentiated osteoblasts versus control cells. Importantly, osteoblasts displayed procollagen-containing vesicles that were distributed throughout the cytoplasm, a portion of which colocalized with lysosomes. Treatment of cells with 2,2-dipyridyl to inhibit procollagen trimerization enhanced colocalization of lysosomes with procollagen-containing organelles, implicating dispersed lysosomes in collagen processing in osteogenic cells.Proper organelle positioning is essential in all eukaryotic cells for inter-organelle trafficking, protein targeting, cellular homeostasis, and response to extracellular stimuli (2). The intracellular organellar architecture is dictated by the protein scaffold of the cell, namely the cytoskeleton. The major cytoskeletal component that dictates the final location of organelles is the microtubule (MT) 3 cytoskeleton (3), with intermediate filaments also playing a role for certain organelles (4). MTs are long polarized polymers that interact indirectly with organelles through motor and adaptor proteins (5). The relative abundance and/or activity of motor proteins on the organelles is believed to determine whether the organelle will be positioned in the cell center (toward the MT minus ends) or toward the plasma membrane (at MT plus ends). Organelles, such as mitochondria and melanosomes, are held toward the cell periphery through the actions of the kinesin motor (6, 7), whereas the minus end-directed motor, dynein, is responsible for holding the nucleus, Golgi complex, lysosomes, and recycling endosomes within the cell interior (8).Although most eukaryotic cells have a similar spatial organization of organelles, there are cell-specific differences in organelle quantities and positioning. The relative abundance of organelles can vary from cell to ce...

Role of Ostm1 Cytosolic Complex with Kinesin 5B in Intracellular Dispersion and Trafficking

“…Such functional roles for Ostm1 are supported by our dynamic studies that showed intracellular organelle dispersion and protein corelocalization. Since both Ostm1 and KIF5B are ubiquitously expressed in a wide range of cells, this interaction could underlie a general cellular mechanism where Ostm1 in a complex with KIF5B would transport and deliver key molecules by trafficking to organelles or to specific sites within cells (45)(46)(47).…”

Temporal Profiling and Pulsed SILAC Labeling Identify Novel Secreted Proteins During Ex Vivo Osteoblast Differentiation of Human Stromal Stem Cells

“…The high representation of cathepsins in cluster 1 indicates a role of lysosomal protein degradation during osteoblastic differentiation of MSC. A marked increase in total lysosome organelles and endocytosis in osteoblastic cells as well as implication of lysosomes in procollagen processing has been reported (79).…”

Loss-of-Function Mutations in Rab Escort Protein 1 (REP-1) Affect Intracellular Transport in Fibroblasts and Monocytes of Choroideremia Patients

“…Since an increase in lysosomal pH leads to the inhibition of lysosomal hydrolases, this could dramatically affect the ability of these cells to process and degrade engulfed material [32], [36]. Indeed, using a self-quenched DQ-ovalbumin particle system, we confirmed that the decreased acidification of lysosomes of CHM patients significantly affected proteolytic abilities of the monocytes [25], [26]. Monocytes from different CHM patients demonstrated significant variation in the rates of uptake of material, lysosomal acidification and protein degradation, which could be explained by the effects of different mutations in the CHM gene on structure and function of REP-1.…”

“…Ovalbumin conjugates are internalized via the mannose receptor–mediated endocytosis pathway and are recognized by antigen-presenting cells [25], [26]. The increase in fluorescence corresponding to the rate of degradation of engulfed DQ-ovalbumin was measured using FACS analysis at different time points following the feeding period.…”