Structural analysis of human neutrophil migration: Centriole, microtubule, and microfilament orientation and function during chemotaxis

Abstract

Orientation of nucleus, centriole, microtubules, and microfilaments within human neutrophils in a gradient of chemoattractant (5 percent Escherichia coli endotoxin-activated serum) was evaluated by electron microscopy. Purified neutropils (hypaque-Ficoll) were placed in the upper compartment of chemotactic chambers. Use of small pore (0.45 μm) micropore filters permitted pseudopod penetration, but impeded migration. Under conditions of chemotaxis with activated serum beneath the filter, the neutrophil population oriented at the filter surface with nuclei located away from the stimulus, centrioles and associated radial array of microtubules beneath the nuclei, and microfilament-rich pseudopods penetrating the filter pores. Reversal of the direction of the gradient of the stimulus (activated serum above cells) resulted in a reorientation of internal structure which preceded pseudopod formation toward the activated serum and migration off the filter. Coordinated orientation of the entire neutrophil population did not occur in buffer (random migration) or in a uniform concentration of activated serum (activated random migration). Conditions of activated random migration resulted in increased numbers of cells with locomotory morphology, i.e. cellular asymmetry with linear alignment of nucleus, centriole, microtubule array, and pseudopods. Thus, activated serum increased the number of neutrophils exhibiting locomotory morphology, and a gradient of activated serum induced the alignment of neutrophils such that this locomotory morphology was uniform in the observed neutrophil populayion. In related studies, cytochalasin B and colchicines were used to explore the role of microfilaments and microtubules in the neutrophil orientation and migration response to activated serum. Cytochalasin B (3.0 μg/ml) prevented migration and decreased the microfilaments seen, but allowed normal orientation of neutrophil structures. In an activated serum gradient, colchicines, but not lumicolchicine, decreased the orientation of nuclei and centrioles, and caused a decrease in centriole-associated microtubules in concentrations as low as 10(-8) to 10(-7) M. These colchicines effects were associated with the rounding of cells and impairment of pseudopod formation. The impaired pseudopod formation was characterized by an inability to form pseudopods in the absence of a solid substrate, a formation of narrow pseudopods within a substrate, and a defect in pseudopod orientation in an activated serum gradient. Functional studies of migration showed that colchicines, but not lumicolchicine, minimally decreased activated random migration and markedly inhibited directed migration, but had not effect on random migration. These studies show that, although functioning microfilaments are probably necessary for neutrophil migration, intact microtubules are essential for normal pseudopod formation and orientation, and maximal unidirectional migration during chemotaxis.

C-reactive Protein Inhibits Chemotactic Peptide-induced p38 Mitogen-activated Protein Kinase Activity and Human Neutrophil Movement

“…By inhibiting phosphorylation of hsp27, CRP may be preventing the formation of membrane ruffles necessary for movement. Finally, microtubules are necessary for neutrophil polarization and microtubule inhibitors decrease neutrophil motility (47), revealing the importance of polarization to the movement process. Activation of p38 MAP kinase may play an important role in proper arrangement of microtubules and CRP may be inhibiting movement at this step.…”

The position of the microtubule-organizing center in directionally migrating fibroblasts depends on the nature of the substratum.

“…An involvement in the expression of a locomotory phenotype is revealed in the experimental observation that microtubule depolymerization impairs cell locomotion (e.g., see refs. 2 and 3), and the morphological finding that, in certain migrating cells, the MTOC is located ahead of the nucleus and behind the advancing lamellipodium (4)(5)(6). This intriguing correlation was interpreted to mean that MTOC reorientation to the front of the cell not only accompanies the onset of cell migration but actually "may play a role in determining the direction of cell movement" (ref.…”

The position of the microtubule‐organizing center relative to the nucleus is independent of the direction of cell migration in dictyostelium discoideum

“…It is clear that the polarity depends on microtubules [Malech et al, 1977;Gotlieb et al, 19831 and forms after encountering the stimulating gradient of chemoattractant (Fig. 3b,e) [see also Malech et al, 1977;Anderson et al, 1982;Schliwa et al, 19821. In addition, it may be controlled by cell-cell adhesion as suggested here.…”

“…In aggregating Dictyostelium [Yumura and Fukui, 1983;Rubino et al, 19841 and migrating cells [Malech et al, 1977;Albrecht-Buehler and Bushnell, 1979;Gotlieb et al, 1983;Koonce et al, 19841 cultured under submerged conditions, the microtubule-organizing center (MTOC) is usually located in advance of the nucleus, which suggests that it may play a role in determining the direction of cell locomotion. However, in submerged cultures, Dictyostelium are denied the air-water interface necessary to complete differentiation [Bonner, 1947;Bozzaro and Roseman, 19831.…”