Stimulus-dependent relocation of the microtubule organizing center in human polymorphonuclear leukocytes

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.