STABILITY OF NUCLEAR SEGMENTS IN HUMAN NEUTROPHILS AND EVIDENCE AGAINST A ROLE FOR MICROFILAMENTS OR MICROTUBULES IN THEIR GENESIS DURING DIFFERENTIATION OF HL-60 MYELOCYTES

The nucleus of the mature human neutrophil is segmented into three to five interconnected lobes. The physiological purpose of this segmentat ion is unknown, as is the mechanism by which the lobes are formed duri ng differentiation. Using video observation of migrating human neutrop hils simultaneously illuminated for fluorescence and phase-contrast mi croscopy, we analyzed nuclear movements with respect to cell shape cha nges. The number of nuclear lobes and their relative size remained con stant during observation (up to 1 h). The thin connecting segments bet ween the lobes elongated and attenuated extensively but never separate d. Electron microscopic analysis of neutrophil nuclei revealed no spec ialized nuclear or cytoplasmic structures in the vicinity of connectin g segments. With fluorescence in situ hybridization of whole chromosom e probes, we determined that chromosomes are randomly distributed amon g neutrophil nuclear lobes. HL60 cells are a human myelocytic line tha t, with retinoic acid treatment, segment their nuclei and differentiat e into neutrophil-like cells over several days. Using a rapidly respon ding variant line termed HL60/S4 (Cancer Res, 52, 949-954), we found t hat segmentation could be induced within 24 h. We tested the role of c ytoskeletal elements in the process of nuclear segmentation. Neither t he microtubule inhibitor nocodazole nor the microfilament inhibitor cy tochalasin D prevented nuclear segmentation. Together, our studies sug gest that nuclear lobes in neutrophils are relatively stable structure s that are not generated by microtubule- or microfilament-dependent fo rces.