25. Although the kinetic analysis of the effect of li- poproteins on the number of CFUs of M. tubercu- losis does not differentiate between microbicidal and microbiostatic mechanisms [Web fig. 6 (12)], antimicrobial proteins can mediate activity by ei- ther mechanism. Although it is well known that defensins clearly lyse microbial pathogens in vitro, in vivo, inside a cell, it appears that the principal action is growth inhibition (29). Over time, the cumulative effect would be to cause death and elimination of the organism. As the infection is contained, T cell responses, including the release of granulysin (27), could contrib- ute to killing of the organism. 26. S. Stenger et al., Science 276, 1684 (1997). 27. S. Stenger et al., Science 282, 121 (1998). 28. D. Jullien et al., J. Immunol. 158, 800 (1997). 29. M. A. Couto, L. Liu, R. I. Lehrer, T. Ganz, Infect. Immun. 62, 2375 (1994). 30. This work was supported in part by grants from the National Institutes of Health (AI 22553, AR 40312, AI 47868, AI 07118), the Deutsche Forschungsge- meinschaft (SFB263), and the AIDS Stipendienpro- gramm of the Ministry of Science and Technology (BMFT), Germany. 8 December 2000; accepted 16 January 2001 Requirement of a Centrosomal Activity for Cell Cycle Progression Through G 1 into S Phase Edward H. Hinchcliffe, 1 Frederick J. Miller, 1 Matthew Cham, 1 Alexey Khodjakov, 2 Greenfield Sluder 1 * Centrosomes were microsurgically removed from BSC-1 African green monkey kidney cells before the completion of S phase. Karyoplasts (acentrosomal cells) entered and completed mitosis. However, postmitotic karyoplasts arrested before S phase, whereas adjacent control cells divided repeatedly. Postmitotic karyoplasts assembled a microtubule-organizing center containing -tubulin and pericentrin, but did not regenerate centrioles. These observations reveal the existence of an activity associated with core centrosomal structures— distinct from elements of the microtubule-organizing center—that is required for the somatic cell cycle to progress through G 1 into S phase. Once the cell is in S phase, these core structures are not needed for the G 2 -M phase transition. The centrosome in mammalian cells consists of a pair of centrioles associated with a cloud of pericentriolar material containing the -tu- bulin ring complexes that nucleate microtu- bules during interphase and mitosis (1). The centrioles, along with their associated struc- tures, represent “core centrosomal structures” that determine the precise one-to-two dupli- cation of the centrosome in preparation for mitosis (2). After removal of the centrosome, both somatic and embryonic cells can regen- erate a microtubule-organizing center (MTOC) (3–5) but do not regenerate centrioles (2, 4 ), even though the cytoplasm (in the case of zy- gotes) contains enough subunits to assemble many complete centrosomes (6 ). It has been generally understood that both the duplication of the centrosome and varia- tions in its microtubule-nucleating capacity are driven by cell cycle– dependent changes in the cytoplasmic environment (7 ). The no- tion that the centrosome is a necessary par- ticipant in cell cycle progression through in- terphase was raised by a report that BSC-1 African green monkey karyoplasts (acentro- somal cells) do not enter mitosis even though they grow to larger than normal size (4 ). This finding, coupled with the observation that cyclin-dependent kinase 1–cyclin B (Cdk1- B) is concentrated at the centrosome (8), led to the proposals that the presence or duplica- tion (or both) of an intact centrosome is required for the activation of Cdk1-B and entry into mitosis (4, 9). However, these pro- posals lacked direct experimental support be- cause the karyoplasts were not continuously followed in vivo. To investigate the role of the centrosome in cell cycle progression, we physically cut BSC-1 cells during interphase between the nucleus and the centrosome to form karyo- plasts (4, 10) and continuously followed the karyoplasts for several days by time-lapse videomicroscopy (11). The fact that the cen- trosome is slightly separated from the nucleus and lies at the center of a mass of granules makes this cell type favorable for this micro- surgery (12). We brought the microneedle down at the edge of the nucleus, which dis- placed the centrosome from the nucleus and segregated it into the anucleate cytoplast as the needle approached the cover slip (Fig. 1A). In no case did we cut or fragment the nucleus. Although we cannot know at what point in the cell cycle the cells were cut, 5-bromo-2'-deoxyuridine (BrdU) incorpora- tion experiments (13) revealed that they were cut before the completion of S phase (14 ), consistent with previous findings (4 ). None were cut in early G 1 or in prophase. During the first 1 to 3 hours after the operation, the cytoplasmic granules became organized into a spherical mass at the center of the cytoplast, indicative of the presence of the centrosome, while the granules in the karyoplast remained randomly distributed in the vicinity of the nucleus (Fig. 1B). Normal- ly we removed the cytoplast with the mi- croneedle so that it would not interfere with observations of karyoplast behavior. Within an hour of the microsurgery, karyoplasts ex- tended lamellipodia and resumed movement across the cover slip (Fig. 2A). Later, they grew in area and regenerated their Golgi ap- paratus to control levels, as judged by in vivo labeling with Bodipy FL C 5 -ceramide (4, 12, 15). In 37 experiments, 32 karyoplasts entered mitosis (Fig. 2A), four remained in interphase until the recordings were terminated 24 hours after the microsurgery, and one died within 12 hours. The interval from the microsurgical operation to the onset of mitosis was on average 12.5 hours (range 4 to 24 hours), which is within the normal interphase dura- tion for control cells in our preparations (av- erage 15.5 hours, range 11 to 26 hours, N = 25). In mitosis, karyoplasts aligned chromo- somes into a metaphase plate, separated two groups of chromosomes in anaphase, and formed a cleavage furrow (14 ). This indicates that karyoplasts organized a functional, albeit acentrosomal, bipolar spindle [see also (5, 16 )]. Karyoplasts spent a longer and a more variable amount of time in mitosis (average 197 min, range 68 to 557 min) than did control cells (average 56 min, range 24 to 99 min; N = 40), presumably because of the need for extra time to organize an acentroso- mal spindle. In telophase all karyoplasts ini- tiated bipolar cleavage. However, in 13 of 32 cases (41%), the cleavage furrow regressed and the karyoplasts exited mitosis as a single cell with one or more nuclei (12, 17 ). We unexpectedly found that in 28 of 32 experiments, the postmitotic karyoplasts— whether they divided or not—arrested in inter- phase for the duration of the observations, up to 60 hours after mitosis (Fig. 2A) (12). This was not attributable to loss of cell viability in our preparations, because the karyoplasts showed continuous lamellipod extension, cell motility, and movement of phase-dense granules toward 1 Department of Cell Biology, University of Massachu- setts Medical School, Worcester, MA 01605, USA. 2 Laboratory of Cell Regulation, Wadsworth Center, Albany, NY 12201, USA. *To whom correspondence should be addressed. E- mail: greenfield.sluder@umassmed.edu R EPORTS www.sciencemag.org SCIENCE VOL 291 23 FEBRUARY 2001 1547