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Cytokinesis involves the concerted efforts of the microtubule and actin cytoskeletons as well as vesicle trafficking and membrane remodeling to form the cleavage furrow and complete daughter cell separation. The exact mechanisms that support membrane remodeling during cytokinesis remain largely undefined. In this study, we report that the large GTPase dynamin, a protein involved in membrane tubulation and vesiculation, is essential for successful cytokinesis. Using biochemical and morphological methods, we demonstrate that dynamin localizes to the spindle midzone and the subsequent intercellular bridge in mammalian cells and is also enriched in spindle midbody extracts. In Caenorhabditis elegans, dynamin localized to newly formed cleavage furrow membranes and accumulated at the midbody of dividing embryos in a manner similar to dynamin localization in mammalian cells. Further, dynamin function appears necessary for cytokinesis, as C. elegans embryos from a dyn-1 ts strain, as well as dynamin RNAi-treated embryos, showed a marked defect in the late stages of cytokinesis. These findings indicate that, during mitosis, conventional dynamin is recruited to the spindle midzone and the subsequent intercellular bridge, where it plays an essential role in the final separation of dividing cells.

The substitution of iron for cobalt in the monomeric insect hemoglobin CTT (Chironomus thummi thummi) III does not alter the Bohr effect for O2-binding. The cobalt substitution in this hemoglobin allows us to identify not only the O-O and Co-O2 stretching mode but also the Co-O-O bending mode by resonance Raman spectroscopy. The assignments were made via 16O2/18O2 isotope exchange. The modes associated with the Co-O-O moiety are pH-dependent. These pH-induced changes of the resonance Raman spectra are correlated with the t = r conformation transition. At high pH (high-affinity state) two unperturbed O-O stretching modes are observed at 1,068 cm-1 (major component) and 1,093 cm-1 (minor component) for the 18O2 complex. These frequencies correspond to split modes at 1,107 cm-1 and 1,136 cm-1 and an unperturbed mode at approximately 1,153 cm-1 for the 16O2 complex. At low pH (low-affinity state) the minor component becomes the major component and vice versa. The Co-O2 stretching frequency varies for approximately 520 cm-1 (pH 5.5) to 537 cm-1 (pH 9.5) indicating a stronger (hence shorter) Co-O2 bond in the high-affinity state. On the other hand, the O-O bond is weakened upon the conversion of the low- to the high-affinity state. The Co-O-O bending mode changes from 390 cm-1 (pH 9.5) to 374 cm-1 (pH 5.5). In the deoxy form the resonance Raman spectra are essentially pH-insensitive except for a vinyl mode at 414 cm-1 (pH 5.5), which is shifted to 416 cm-1 (pH 5.5).

Resonance Raman spectroscopy has been employed to probe the effects of proximal base strain on the bonding of O2 and CO in three synthetic hemins with covalently linked imidazole ligands. The strain is introduced by varying the length of the imidazole-containing side chain and by restricting the side chain flexibility with a phenyl ring. These hemins are abbreviated as "long," "short," and "stiff" hemins, respectively. In the deoxy state, the iron-imidazole stretching frequencies [nu(Fe--N epsilon)] for long, short, and stiff hemins are detected at 200, 207, and 204 cm-1, respectively. The strain induced in the iron-imidazole bond by the short hemin results in a higher nu(Fe--N epsilon) frequency, in contrast to the strain induced by sterically hindered 2-methylimidazole or 1,2-dimethylimidazole complexes in which the Fe--N epsilon bond is tilted and lengthened, but the imidazole ring remains perpendicular to the heme plane. However, in the short hemin, the plane of the imidazole ring may not be perpendicular to the plane of the porphyrin, altering the amount of pi-interaction (hence the strength of Fe--N epsilon bond) and the nature of normal mode containing Fe--N epsilon bond stretching. Upon CO binding, we have observed the nu(Fe--CO) stretching frequencies at 497 (long), 499 (short), and 496 cm-1 (stiff), somewhat lower than those reported by Mitchell et al. (Inorg. Chem., 1985, 24:967) for the chelated-heme X CO complexes (i.e., 501-506 cm-1). This is the first report of an iron-oxygen-associated vibration observed in solution for an unprotected heme. The oxy complexes were formed by introducing dioxygen to the deoxy complexes at -700C. The isotope-sensitive line was detected at 576 cm- (1602) in oxy stiff hemin, which was shifted to 545 cm-1 upon 1802 substitution. This is perhaps the largest isotope shift (31 cm-') observed to date, compared with the usual 22-24 cm-'.For the long and short hemins, the iron-oxygen-associated vibration was detected at 574 and 573 cm-', respectively.These values are very similar to those observed(N-methylimidazole) and myoglobin/hemoglobin.