Predictive-focus illumination for reducing photodamage in live-cell microscopy.

Due to photobleaching and phototoxicity induced by high-intensity excitation light, the number of fluorescence images that can be obtained in live cells is always limited. This limitation becomes particularly prominent in multidimensional recordings when multiple Z-planes are captured at every time point. Here we present a simple technique, termed predictive-focus illumination (PFI), which helps to minimize cells' exposure to light by decreasing the number of Z-planes that need to be captured in live-cell 3D time-lapse recordings. PFI utilizes computer tracking to predict positions of objects of interest (OOIs) and restricts image acquisition to small dynamic Z-regions centred on each OOI. Importantly, PFI does not require hardware modifications and it can be easily implemented on standard wide-field and spinning-disc confocal microscopes.

The centrosome in vertebrates: more than a microtubule-organizing center.

The somatic cells of all higher animals contain a single minute organelle called the centrosome. For years, the functions of the centrosome were thought to revolve around its ability to nucleate and organize the various microtubule arrays seen in interphase and mitosis. But the centrosome is more than just a microtubule-organizing center. Recent work reveals that this organelle is essential for cell-cycle progression and that this requirement is independent of its ability to organize microtubules. Here, we review the various functions attributed to the centrosome and ask which are essential for the survival and reproduction of the cell, the organism, or both.

Centrosomes enhance the fidelity of cytokinesis in vertebrates and are required for cell cycle progression.

When centrosomes are destroyed during prophase by laser microsurgery, vertebrate somatic cells form bipolar acentrosomal mitotic spindles (Khodjakov, A., R.W. Cole, B.R. Oakley, and C.L. Rieder. 2000. Curr. Biol. 10:59-67), but the fate of these cells is unknown. Here, we show that, although these cells lack the radial arrays of astral microtubules normally associated with each spindle pole, they undergo a normal anaphase and usually produce two acentrosomal daughter cells. Relative to controls, however, these cells exhibit a significantly higher (30-50%) failure rate in cytokinesis. This failure correlates with the inability of the spindle to properly reposition itself as the cell changes shape. Also, we destroyed just one centrosome during metaphase and followed the fate of the resultant acentrosomal and centrosomal daughter cells. Within 72 h, 100% of the centrosome-containing cells had either entered DNA synthesis or divided. By contrast, during this period, none of the acentrosomal cells had entered S phase. These data reveal that the primary role of the centrosome in somatic cells is not to form the spindle but instead to ensure cytokinesis and subsequent cell cycle progression.

Kinetochores moving away from their associated pole do not exert a significant pushing force on the chromosome.

We used video-light microscopy and laser microsurgery to test the hypothesis that as a bioriented prometaphase chromosome changes position in PtK1 cells, the kinetochore moving away from its associated pole (AP) exerts a pushing force on the centromere. When we rapidly severed congressing chromosomes near the spindle equator between the sister kinetochores, the kinetochore that was originally "leading" the motion towards a pole (P) always (17/17 cells) continued moving P whereas the "trailing" kinetochore moving AP always stopped moving as soon as the operation was completed. This trailing kinetochore then initiated motion towards the pole it was originally moving away from up to 50 s later. The same result was observed (15/15 cells) when we selectively destroyed the leading (P moving) kinetochore on a congressing chromosome positioned > or = 3 microns from the pole it was moving away from. When we conducted this experiment on congressing chromosomes positioned within 3 microns of the pole, the centromere region either stopped moving, before switching into motion towards the near pole (2/4 cells), or it continued to move AP for 30-44 s (2/4 cells) before switching into P motion. Finally, kinetochore-free chromosome fragments, generated in the polar regions of PtK1 spindles, were ejected AP and often towards the spindle equator at approximately 2 microns/min. From these data we conclude that the kinetochore moving AP on a moving chromosome does not exert a significant pushing force on the chromosome. Instead, our results reveal that, when not generating a P force, kinetochores are in a "neutral" state that allows them to remain stationary or to coast AP in response to external forces sufficient to allow their K-fiber to elongate.

The sudden recruitment of gamma-tubulin to the centrosome at the onset of mitosis and its dynamic exchange throughout the cell cycle, do not require microtubules.

gamma-Tubulin is a centrosomal component involved in microtubule nucleation. To determine how this molecule behaves during the cell cycle, we have established several vertebrate somatic cell lines that constitutively express a gamma-tubulin/green fluorescent protein fusion protein. Near simultaneous fluorescence and DIC light microscopy reveals that the amount of gamma-tubulin associated with the centrosome remains relatively constant throughout interphase, suddenly increases during prophase, and then decreases to interphase levels as the cell exits mitosis. This mitosis-specific recruitment of gamma-tubulin does not require microtubules. Fluorescence recovery after photobleaching (FRAP) studies reveal that the centrosome possesses two populations of gamma-tubulin: one that turns over rapidly and another that is more tightly bound. The dynamic exchange of centrosome-associated gamma-tubulin occurs throughout the cell cycle, including mitosis, and it does not require microtubules. These data are the first to characterize the dynamics of centrosome-associated gamma-tubulin in vertebrate cells in vivo and to demonstrate the microtubule-independent nature of these dynamics. They reveal that the additional gamma-tubulin required for spindle formation does not accumulate progressively at the centrosome during interphase. Rather, at the onset of mitosis, the centrosome suddenly gains the ability to bind greater than three times the amount of gamma-tubulin than during interphase.

The checkpoint delaying anaphase in response to chromosome monoorientation is mediated by an inhibitory signal produced by unattached kinetochores.

During mitosis in Ptk1 cells anaphase is not initiated until, on average, 23 +/- 1 min after the last monooriented chromosome acquires a bipolar attachment to the spindle--an event that may require 3 h (Rieder, C. L., A. Schultz, R. W. Cole, and G. Sluder. 1994. J. Cell Biol. 127:1301-1310). To determine the nature of this cell-cycle checkpoint signal, and its site of production, we followed PtK1 cells by video microscopy prior to and after destroying specific chromosomal regions by laser irradiation. The checkpoint was relieved, and cells entered anaphase, 17 +/- 1 min after the centromere (and both of its associated sister kinetochores) was destroyed on the last monooriented chromosome. Thus, the checkpoint mechanism monitors an inhibitor of anaphase produced in the centromere of monooriented chromosomes. Next, in the presence of one monooriented chromosome, we destroyed one kinetochore on a bioriented chromosome to create a second monooriented chromosome lacking an unattached kinetochore. Under this condition anaphase began in the presence of the experimentally created monooriented chromosome 24 +/- 1.5 min after the nonirradiated monooriented chromosome bioriented. This result reveals that the checkpoint signal is not generated by the attached kinetochore of a monooriented chromosome or throughout the centromere volume. Finally, we selectively destroyed the unattached kinetochore on the last monooriented chromosome. Under this condition cells entered anaphase 20 +/- 2.5 min after the operation, without congressing the irradiated chromosome. Correlative light microscopy/elctron microscopy of these cells in anaphase confirmed the absence of a kinetochore on the unattached chromatid. Together, our data reveal that molecules in or near the unattached kinetochore of a monooriented PtK1 chromosome inhibit the metaphase-anaphase transition.

Merotelic kinetochore orientation is a major mechanism of aneuploidy in mitotic mammalian tissue cells.

In mitotic cells, an error in chromosome segregation occurs when a chromosome is left near the spindle equator after anaphase onset (lagging chromosome). In PtK1 cells, we found 1.16% of untreated anaphase cells exhibiting lagging chromosomes at the spindle equator, and this percentage was enhanced to 17.55% after a mitotic block with 2 microM nocodazole. A lagging chromosome seen during anaphase in control or nocodazole-treated cells was found by confocal immunofluorescence microscopy to be a single chromatid with its kinetochore attached to kinetochore microtubule bundles extending toward opposite poles. This merotelic orientation was verified by electron microscopy. The single kinetochores of lagging chromosomes in anaphase were stretched laterally (1.2--5.6-fold) in the directions of their kinetochore microtubules, indicating that they were not able to achieve anaphase poleward movement because of pulling forces toward opposite poles. They also had inactivated mitotic spindle checkpoint activities since they did not label with either Mad2 or 3F3/2 antibodies. Thus, for mammalian cultured cells, kinetochore merotelic orientation is a major mechanism of aneuploidy not detected by the mitotic spindle checkpoint. The expanded and curved crescent morphology exhibited by kinetochores during nocodazole treatment may promote the high incidence of kinetochore merotelic orientation that occurs after nocodazole washout.

Chromosome fragments possessing only one kinetochore can congress to the spindle equator.

We used laser microsurgery to cut between the two sister kinetochores on bioriented prometaphase chromosomes to produce two chromosome fragments containing one kinetochore (CF1K). Each of these CF1Ks then always moved toward the spindle pole to which their kinetochores were attached before initiating the poleward and away-from-the-pole oscillatory motions characteristic of monooriented chromosomes. CF1Ks then either: (a) remained closely associated with this pole until anaphase (50%), (b) moved (i.e., congressed) to the spindle equator (38%), where they usually (13/19 cells) remained stably positioned throughout the ensuing anaphase, or (c) reoriented and moved to the other pole (12%). Behavior of congressing CF1Ks was indistinguishable from that of congressing chromosomes containing two sister kinetochores. Three-dimensional electron microscopic tomographic reconstructions of CF1Ks stably positioned on the spindle equator during anaphase revealed that the single kinetochore was highly stretched and/or fragmented and that numerous microtubules derived from the opposing spindle poles terminated in its structure. These observations reveal that a single kinetochore is capable of simultaneously supporting the function of two sister kinetochores during chromosome congression and imply that vertebrate kinetochores consist of multiple domains whose motility states can be regulated independently.

The respective contributions of the mother and daughter centrioles to centrosome activity and behavior in vertebrate cells.

We have generated several stable cell lines expressing GFP-labeled centrin. This fusion protein becomes concentrated in the lumen of both centrioles, making them clearly visible in the living cell. Time-lapse fluorescence microscopy reveals that the centriole pair inherited after mitosis splits during or just after telophase. At this time the mother centriole remains near the cell center while the daughter migrates extensively throughout the cytoplasm. This differential behavior is not related to the presence of a nucleus because it is also observed in enucleated cells. The characteristic motions of the daughter centriole persist in the absence of microtubules (Mts). or actin, but are arrested when both Mts and actin filaments are disrupted. As the centrioles replicate at the G1/S transition the movements exhibited by the original daughter become progressively attenuated, and by the onset of mitosis its behavior is indistinguishable from that of the mother centriole. While both centrioles possess associated gamma-tubulin, and nucleate similar number of Mts in Mt repolymerization experiments. during G1 and S only the mother centriole is located at the focus of the Mt array. A model, based on differences in Mt anchoring and release by the mother and daughter centrioles, is proposed to explain these results.

The force for poleward chromosome motion in Haemanthus cells acts along the length of the chromosome during metaphase but only at the kinetochore during anaphase.

The force for poleward chromosome motion during mitosis is thought to act, in all higher organisms, exclusively through the kinetochore. We have used time-lapse. video-enhanced, differential interference contrast light microscopy to determine the behavior of kinetochore-free "acentric" chromosome fragments and "monocentric" chromosomes containing one kinetochore, created at various stages of mitosis in living higher plant (Haemanthus) cells by laser microsurgery. Acentric fragments and monocentric chromosomes generated during spindle formation and metaphase both moved towards the closest spindle pole at a rate (approximately 1.0 microm/min) similar to the poleward motion of anaphase chromosomes. This poleward transport of chromosome fragments ceased near the onset of anaphase and was replaced. near midanaphase, by another force that now transported the fragments to the spindle equator at 1.5-2.0 microm/min. These fragments then remained near the spindle midzone until phragmoplast development, at which time they were again transported randomly poleward but now at approximately 3 microm/min. This behavior of acentric chromosome fragments on anastral plant spindles differs from that reported for the astral spindles of vertebrate cells, and demonstrates that in forming plant spindles, a force for poleward chromosome motion is generated independent of the kinetochore. The data further suggest that the three stages of non-kinetochore chromosome transport we observed are all mediated by the spindle microtubules. Finally, our findings reveal that there are fundamental differences between the transport properties of forming mitotic spindles in plants and vertebrates.

Centriole disassembly in vivo and its effect on centrosome structure and function in vertebrate cells.

Glutamylation is the major posttranslational modification of neuronal and axonemal tubulin and is restricted predominantly to centrioles in nonneuronal cells (Bobinnec, Y., M. Moudjou, J.P. Fouquet, E. Desbruyères, B. Eddé, and M. Bornens. 1998. Cell Motil. Cytoskel. 39:223-232). To investigate a possible relationship between the exceptional stability of centriole microtubules and the compartmentalization of glutamylated isoforms, we loaded HeLa cells with the monoclonal antibody GT335, which specifically reacts with polyglutamylated tubulin. The total disappearance of the centriole pair was observed after 12 h, as judged both by immunofluorescence labeling with specific antibodies and electron microscopic observation of cells after complete thick serial sectioning. Strikingly, we also observed a scattering of the pericentriolar material (PCM) within the cytoplasm and a parallel disappearance of the centrosome as a defined organelle. However, centriole disappearance was transient, as centrioles and discrete centrosomes ultimately reappeared in the cell population. During the acentriolar period, a large proportion of monopolar half-spindles or of bipolar spindles with abnormal distribution of PCM and NuMA were observed. However, as judged by a quasinormal increase in cell number, these cells likely were not blocked in mitosis. Our results suggest that a posttranslational modification of tubulin is critical for long-term stability of centriolar microtubules. They further demonstrate that in animal cells, centrioles are instrumental in organizing centrosomal components into a structurally stable organelle.

Characterization of a minus end-directed kinesin-like motor protein from cultured mammalian cells.

Using the CHO2 monoclonal antibody raised against CHO spindles (Sellitto, C., M. Kimble, and R. Kuriyama. 1992. Cell Motil. Cytoskeleton. 22:7-24) we identified a 66-kD protein located at the interphase centrosome and mitotic spindle. Isolated cDNAs for the antigen encode a 622-amino acid polypeptide. Sequence analysis revealed the presence of 340-amino acid residues in the COOH terminus, which is homologous to the motor domain conserved among other members of the kinesin superfamily. The protein is composed of a central alpha-helical portion with globular domains at both NH2 and COOH termini, and the epitope to the monoclonal antibody resides in the central alpha-helical stalk. A series of deletion constructs were created for in vitro analysis of microtubule interactions. While the microtubule binding and bundling activities require both the presence of the COOH terminus and the alpha-helical domain, the NH2-terminal half of the antigen lacked the ability to interact with microtubules. The full-length as well as deleted proteins consisting of the COOH-terminal motor and the central alpha-helical stalk supported microtubule gliding, with velocity ranging from 1.0 to 8.4 microns/minute. The speed of microtubule movement decreased with decreasing lengths of the central stalk attached to the COOH-terminal motor. The microtubules moved with their plus end leading, indicating that the antigen is a minus end-directed motor. The CHO2 sequence shows 86% identify to HSET, a gene located at the centromeric end of the human MHC region in chromosome 6 (Ando, A., Y. Y. Kikuti, H. Kawata, N. Okamoto, T. Imai, T. Eki, K. Yokoyama, E. Soeda, T. Ikemura, K. Abe, and H. Inoko. 1994. Immunogenetics. 39:194-200), indicating that HSET might represent a human homologue of the CHO2 antigen.

Requirement of a centrosomal activity for cell cycle progression through G1 into S phase.

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 gamma-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 G1 into S phase. Once the cell is in S phase, these core structures are not needed for the G2-M phase transition.

Centrosome-independent mitotic spindle formation in vertebrates.

BACKGROUND: In cells lacking centrosomes, the microtubule-organizing activity of the centrosome is substituted for by the combined action of chromatin and molecular motors. The question of whether a centrosome-independent pathway for spindle formation exists in vertebrate somatic cells, which always contain centrosomes, remains unanswered, however. By a combination of labeling with green fluorescent protein (GFP) and laser microsurgery we have been able to selectively destroy centrosomes in living mammalian cells as they enter mitosis. RESULTS: We have established a mammalian cell line in which the boundaries of the centrosome are defined by the constitutive expression of gamma-tubulin-GFP. This feature allows us to use laser microsurgery to selectively destroy the centrosomes in living cells. Here we show that this method can be used to reproducibly ablate the centrosome as a functional entity, and that after destruction the microtubules associated with the ablated centrosome disassemble. Depolymerization-repolymerization experiments reveal that microtubules form in acentrosomal cells randomly within the cytoplasm. When both centrosomes are destroyed during prophase these cells form a functional bipolar spindle. Surprisingly, when just one centrosome is destroyed, bipolar spindles are also formed that contain one centrosomal and one acentrosomal pole. Both the polar regions in these spindles are well focused and contain the nuclear structural protein NuMA. The acentrosomal pole lacks pericentrin, gamma-tubulin, and centrioles, however. CONCLUSIONS: These results reveal, for the first time, that somatic cells can use a centrosome-independent pathway for spindle formation that is normally masked by the presence of the centrosome. Furthermore, this mechanism is strong enough to drive bipolar spindle assembly even in the presence of a single functional centrosome.

Cleavage furrows formed between centrosomes lacking an intervening spindle and chromosomes contain microtubule bundles, INCENP, and CHO1 but not CENP-E.

PtK1 cells containing two independent mitotic spindles can cleave between neighboring centrosomes, in the absence of an intervening spindle, as well as at the spindle equators. We used same-cell video, immunofluorescence, and electron microscopy to compare the structure and composition of normal equatorial furrows with that of ectopic furrows formed between spindles. As in controls, ectopic furrows contained midbodies composed of microtubule bundles and an electron-opaque matrix. Despite the absence of an intervening spindle and chromosomes, the midbodies associated with ectopic furrows also contained the microtubule-bundling protein CHO1 and the chromosomal passenger protein INCENP. However, CENP-E, another passenger protein, was not found in ectopic furrows but was always present in controls. We also examined cells in which the ectopic furrow initiated but relaxed. Although relaxing furrows contained overlapping microtubules from opposing centrosomes, they lacked microtubule bundles as well as INCENP and CHO1. Together these data suggest that the mechanism defining the site of furrow formation during mitosis in vertebrates does not depend on the presence of underlying microtubule bundles and chromosomes or on the stable association of INCENP or CHO1. The data also suggest that the completion of cytokinesis requires the presence of microtubule bundles and specific proteins (e.g., INCENP, CHO1, etc.) that do not include CENP-E.

A specific light chain of kinesin associates with mitochondria in cultured cells.

The motor protein kinesin is implicated in the intracellular transport of organelles along microtubules. Kinesin light chains (KLCs) have been suggested to mediate the selective binding of kinesin to its cargo. To test this hypothesis, we isolated KLC cDNA clones from a CHO-K1 expression library. Using sequence analysis, they were found to encode five distinct isoforms of KLCs. The primary region of variability lies at the carboxyl termini, which were identical or highly homologous to carboxyl-terminal regions of rat KLC B and C, human KLCs, sea urchin KLC isoforms 1-3, and squid KLCs. To examine whether the KLC isoforms associate with different cytoplasmic organelles, we made an antibody specific for a 10-amino acid sequence unique to B and C isoforms. In an indirect immunofluorescence assay, this antibody specifically labeled mitochondria in cultured CV-1 cells and human skin fibroblasts. On Western blots of total cell homogenates, it recognized a single KLC isoform, which copurified with mitochondria. Taken together, these data indicate a specific association of a particular KLC (B type) with mitochondria, revealing that different KLC isoforms can target kinesin to different cargoes.

Anticancer drugs induce increased mitochondrial cytochrome c expression that precedes cell death.

Recent studies have demonstrated that cytochrome c plays an important role in cell death. In the present study, we report that teniposide and various other chemotherapeutic agents induced a dose-dependent increase in the expression of the mitochondrial respiratory chain proteins cytochrome c, subunits I and IV of cytochrome c oxidase, and the free radical scavenging enzyme manganous superoxide dismutase. The teniposide-induced increase of cytochrome c was inhibited by cycloheximide, indicating new protein synthesis. Elevated cytochrome c levels were associated with enhanced cytochrome c oxidase-dependent oxygen uptake using TMPD/ascorbate as the electron donor, suggesting that the newly synthesized proteins were functional. Cytochrome c was released into the cytoplasm only after maximal levels had been reached in the mitochondria, but there was no concomitant decrease in mitochondrial membrane potential or caspase activation. Our results suggest that the increase in mitochondrial protein expression may play a role in the early cellular defense against anticancer drugs.

Increased mitochondrial cytochrome c levels and mitochondrial hyperpolarization precede camptothecin-induced apoptosis in Jurkat cells.

Mitochondria play a central role in apoptosis through release of cytochrome c and activation of caspases. In the present study, we showed that, in Jurkat human T cells, camptothecin-induced apoptosis is preceded by (i) an increase in cytochrome c and subunit IV of cytochrome c oxidase (COX IV) levels in mitochondria; and (ii) an elevation of the mitochondrial membrane potential (Delta(Psi)m). These events are followed by cytochrome c release into the cytosol, cytochrome c and COX IV depletion from mitochondria, externalization of phosphatidylserine (PS), disruption of Delta(Psi)m, caspase activation, poly(ADP-ribose)polymerase cleavage and DNA fragmentation. The pan-caspase inhibitor z-VAD.fmk blocked camptothecin-induced PS externalization, disruption of Delta(Psi)m and DNA fragmentation, suggesting that these events are mediated by caspase activation. In contrast, z-VAD did not prevent cytochrome c release, despite preventing cytochrome c and COX IV depletion from mitochondria. Together, these data suggest that mitochondrial cytochrome c and COX IV enrichment are early events preceding the onset of apoptosis and that cytochrome c release is upstream of caspase activation and loss of Delta(Psi)m. Furthermore, prevention by z-VAD of cytochrome c and COX IV depletion in mitochondria suggests the possibility that a caspase-like activity in mitochondria is involved in the proteolytic depletion of respiratory chain proteins. Activation of this activity may play an important role in drug-induced apoptosis.

Mitosis and checkpoints that control progression through mitosis in vertebrate somatic cells.

During mitosis in vertebrates the sister kinetochores on each replicated chromosome interact with two separating arrays of astral microtubules to form a bipolar spindle that produces and/or directs the forces for chromosome motion. In order to ensure faithful chromosome segregation cells have evolved mechanisms that delay progress into and out of mitosis until certain events are completed. At least two of these mitotic "checkpoint controls" can be identified in vertebrates. The first prevents nuclear envelope breakdown, and thus spindle formation, when the integrity of some nuclear component(s) is compromised. The second prevents chromosome disjunction and exit from mitosis until all of the kinetochores are attached to the spindle.

Identification of ubiquitous high-molecular-mass, heat-stable microtubule-associated proteins (MAPs) that are related to the Drosophila 205-kDa MAP but are not related to the mammalian MAP-4.

AX3, a monoclonal antibody raised against isolated microtubule-organizing centers of Dictyostelium discoideum, stains microtubule-containing structures in species ranging from Dictyostelium to human. On immunoblots, the AX3 antibody recognizes heat-stable proteins in the 260- to 280-kDa molecular-mass range in a number of different species. The AX3 antigens from HeLa and embryonic mouse fibroblast cells coprecipitate with microtubules in vitro, indicating that these antigens are, indeed, MAPs. The AX3 antigens are not immunologically related to the mammalian MAP-2 or MAP-4 but are related to the 205-kDa MAP of Drosophila. This report describes a structural-type MAP in Dictyostelium and a MAP that is detected in a wide variety of species. The Drosophila 205-kDa MAP had previously been proposed to represent a member of the MAP-4 class of proteins. From the results reported here, however, it is suggested that proteins recognized by AX3 monoclonal antibody, including the Drosophila 205-kDa MAP, represent a distinct class of MAPs that has been widely conserved through evolution.