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Changes in microtubules, microtubule-associated proteins, and intermediate filaments during the differentiation of
M F Leung1, J A Sokoloski, A C Sartorelli
1Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510.
Abstract:
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.
Insights
Retinoic acid (RA) induces changes in the cytoskeleton, including microtubules (MT) and intermediate filaments (IF), during HL-60 leukemia cell differentiation. These cytoskeletal alterations, particularly increased MT and vimentin, are crucial for leukemia cell maturation.
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- The cytoskeleton, comprising microtubules (MT), microfilaments, and intermediate filaments (IF), provides structural support and is implicated in cell signaling and differentiation.
- Changes in cytoskeletal organization are observed during cellular proliferation and differentiation processes.
Purpose of the Study:
- To investigate cytoskeletal alterations during retinoic acid (RA)-induced differentiation of HL-60 leukemia cells.
- To quantify changes in MT, intermediate filaments (IF), and microtubule-associated proteins (MAPs) during HL-60 cell maturation.
Main Methods:
- Immunofluorescence microscopy and fluorescence-activated cell sorter (FACS) analysis were used to visualize and quantify cytoskeletal components.
- A novel flow cytometry-based procedure was developed to measure cytoskeletal protein content in fixed and permeabilized HL-60 cells.
- RA-supersensitive (HL-60/S4) and RA-resistant (HL-60/R3) clones were utilized to confirm RA-specific effects.
Main Results:
- Differentiated HL-60 cells showed increased staining intensity and altered organization of MT and IF (vimentin).
- Terminally differentiated cells contained significantly higher amounts of MT and vimentin.
- A transient increase in MAP2 and tau was observed during the maturation process.
- Changes in MT, MAPs, and vimentin were confirmed to be associated with RA-induced differentiation.
Conclusions:
- Cytoskeletal remodeling, involving microtubules, MAPs, and intermediate filaments like vimentin, plays a critical role in the terminal maturation of HL-60 leukemia cells.
- The findings highlight the importance of cytoskeletal dynamics in the differentiation of cancer cells.