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Stimulus-dependent relocation of the microtubule organizing center in human polymorphonuclear leukocytes
J M Chiplonkar1, D D Vandré, J M Robinson
1Department of Cell Biology, Neurobiology and Anatomy, Ohio State University, Columbus 43210.
Abstract:
Polymorphonuclear leukocytes (PMNs) exhibit extensive directional migration (chemotaxis) and phagocytic activities. We have developed an in vitro model to evaluate the organization of the microtubule organizing center (MTOC) in PMNs as the latter interact with various substrata, including immobilized antigen-antibody complexes. PMNs were layered on poly-L-lysine substrata containing ferritin (PL+F) or ferritin-antiferritin complex (PL+F+AF) and the location of MTOCs was determined by indirect immunofluorescence of tubulin using conventional epifluorescence microscopy and confocal laser scanning microscopy. The MTOCs in the majority of the PMNs attached to PL+F occupied an apical location (81.29% +/- 3.34%), while in the majority of PMNs layered onto PL+F+AF, a basal location (79.37% +/- 5.26%) was observed. Following disruption of microtubules (MTs) by nocodazole before layering the cells on the substrata, the proportions of PMNs with apical MTOCs were 65.2% +/- 6.27% for PL+F and 47.2% +/- 4.1% for PL+F+AF substrata, while the proportions of PMNs with basal MTOCs were 26.11% +/- 8.89% for PL+F and 39.6% +/- 4.4 for PL+F+AF substrata. The results indicate that MTOCs in human PMNs in vitro (i) occupied a 'pre-defined' apical location; (ii) translocated to a 'newly defined' basal location upon stimulation with immobilized antigen-antibody complex; (iii) and depended on intact MTs for placement of MTOCs in both situations.
Insights
Polymorphonuclear leukocytes (PMNs) relocate their microtubule organizing centers (MTOCs) to a basal position upon immune complex stimulation. This repositioning relies on intact microtubules for proper cell function.
Area of Science:
- Cell Biology
- Immunology
- Cytoskeleton Dynamics
Background:
- Polymorphonuclear leukocytes (PMNs) are crucial for immune responses, exhibiting chemotaxis and phagocytosis.
- The microtubule organizing center (MTOC) plays a key role in cell polarization and migration.
- Understanding MTOC organization is vital for comprehending PMN function.
Purpose of the Study:
- To investigate the in vitro organization of the MTOC in human PMNs.
- To determine how PMN interaction with different substrata affects MTOC localization.
- To elucidate the role of microtubules in MTOC positioning during PMN activation.
Main Methods:
- Development of an in vitro model using poly-L-lysine substrata with or without ferritin-antiferritin complexes.
- Layering of human PMNs onto prepared substrata.
- Determination of MTOC location using indirect immunofluorescence of tubulin and confocal microscopy.
- Assessment of MTOC positioning after microtubule disruption with nocodazole.
Main Results:
- PMNs on non-specific substrata (PL+F) predominantly showed apical MTOCs (81.29%).
- PMNs stimulated with immobilized antigen-antibody complexes (PL+F+AF) exhibited a significant shift to basal MTOCs (79.37%).
- Microtubule disruption affected MTOC localization, indicating dependence on intact microtubules for positioning.
Conclusions:
- Human PMN MTOCs adopt a pre-defined apical location in vitro.
- Immune complex stimulation induces translocation of MTOCs to a basal position.
- Intact microtubules are essential for correct MTOC placement in both resting and stimulated PMNs.