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...