The cytoskeleton is composed mainly of microtubules (MT), microfilaments, and intermediate filaments (IF) that form a structural network which connects cellular membranes, cytoplasmic organelles, and the nucleus. Since the cytoskeleton may be involved in modulating signal transduction and in the morphological and structural changes that occur during cellular proliferation and differentiation, cytoskeletal changes were measured by immunofluorescence microscopy and fluorescence-activated cell sorter analysis during the differentiation of HL-60 leukemia cells induced by retinoic acid (RA). Differentiated HL-60 cells exhibited increased staining intensity and altered organization of MT and IF, as visualized by immunofluorescence microscopy with anti-tubulin monoclonal antibody and anti-vimentin antibody, respectively. A new procedure was developed and used to measure the content of the cytoskeletal components of HL-60 cells during the process of maturation. HL-60 cells were fixed with formaldehyde in an MT-stabilizing buffer, permeabilized using l-lysophosphatidylcholine, stained for immunofluorescent measurement with antibodies specific for particular cytoskeletal components, and analyzed by flow cytometry. Terminally differentiated cells produced by exposure to RA contained larger amounts of MT and the IF vimentin. During the course of the maturation process, a transient increase in the amounts of the microtubule-associated proteins, (MAPs) MAP2 and tau, occurred. An RA-supersensitive clone, designated HL-60/S4, and an RA-resistant clone, designated HL-60/R3, were developed by mutagenization and selection. Use of these clones supported the concept that the observed changes in MT, MAPs, and vimentin were associated with the differentiation process rather than being due to other effects produced by the retinoid. Thus, the findings suggest that changes in MT, MAPs, and IF are important to the terminal maturation of leukemia cells.

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