The anaphase promoting complex (APC) controls the degradation of proteins during exit from mitosis and entry into S-phase. The activity of the APC is regulated by phosphorylation during mitosis. Because the phosphorylation pattern provides insights into the complexity of regulation of the APC, we studied in detail the phosphorylation patterns at a single mitotic state of arrest generated by various antimitotic drugs. We examined the phosphorylation patterns of the APC in HeLa S3 cells after they were arrested in prometaphase with taxol, nocodazole, vincristine, or monastrol. There were 71 phosphorylation sites on nine of the APC subunits. Despite the common state of arrest, the various antimitotic drug treatments resulted in differences in the phosphorylation patterns and phosphorylation stoichiometries. The relative phosphorylation stoichiometries were determined by using a method adapted from the isotope-free quantitation of the extent of modification (iQEM). We could show that during drug arrest the phosphorylation state of the APC changes, indicating that the mitotic arrest is not a static condition. We discuss these findings in terms of the variable efficacy of antimitotic drugs in cancer chemotherapy. mass spectrometry ͉ stable isotope free quantitation ͉ prometaphase ͉ cell cycle arrest

Our results suggest that during early mitosis (from prophase to metaphase) the timing of biochemical events (such as phosphorylation) and morphological events (such as structural changes in the nucleus) is at least partly controlled by the responses of the substrates themselves to a common set of signals.

Reovirus serotype 1 (Lang) can be conjugated with rhodamine B or fluorescein isothiocyanate in a way that preserves viral infectivity. We have used epifluorescence microscopy to detect individual vnirions bound to the surface ofcells and to follow in real time the early stages of reovirus infection in living cells. Following uptake of the virus into endocytic vesicles, the movement of these vesicles can be observed readily. The vesicle movement is inhibited by nocodazole or colchicine, consistent with previous fldings that the movement of intracellular vesicles is often microtubule-based.Epifluorescence microscopy has become one of the essential tools in biology. A major attribute of this technique is its sensitivity: as few as 50 fluorescent molecules in a cubic micrometer can be detected (1). In contrast to electron microscopy, epifluorescence microscopy can be readily adapted to experiments with living cells (1). With the advent of highly sensitive detection systems including improved photographic film and video cameras that can image aggregates offewer than 20 fluorescent molecules, the time seemed ripe for an attempt to detect a single animal virus particle by light microscopy.Fluorescently labeled viruses have been used in other ways to study virus-host interactions. Epstein-Barr virus conjugated with fluorescein isothiocyanate (FITC) has been used to examine the specificity of binding to cellular receptors (2), and FITC diphosphate-conjugated influenza virus has been used to study viral uncoating in endosomes (3). However, it has not been established whether each individual fluorescent particle represents a single virion or an aggregate of several virions; nor have fluorescently labeled viruses been used for studies in a living cell system.Based on the size of most animal viruses and the detection systems available, we estimated that -500 molecules of rhodamine B per virion might be sufficient for detection. Reovirus serotype 1 (Lang) was chosen for these studies because it grows to high titer in cell culture, is relatively stable at high pH, and is therefore more likely t6 survive the conditions of dye conjugation (2). We report here that individual virions of reovirus conjugated with Rither rhodamine or fluorescein can be observed on-the surface of infected L cells at early times after inoculation and that endocytic vesicles containing small cluste~is of virions can be seen moving inside cells in a manner consistent with the translocation of organelles along microtubules. MATERIALS AND METHODSCells and Virus. Mouse L cells (fibroblasts) were grown on glass coverslips or in spinner cultures in "completed MEM" containing Joklik's modified Eagle's medium (Irvine Scientific) supplemented with 2.5% fetal bovine serum (HyClone), 2.5% neonatal bovine serum (Biocell Laboratories), 0.3% glutamine, and 1% penicillin/streptomycin solution (Irvine Scientific).Plaque-purified reovirus serotype 1 (Lang) was used for conjugation. The virus was purified by sonication and Freon extraction (4, 5). The virus was ...