During interphase, the centrioles and other components of the centrosomal matrix are duplicated, but remain bound into a single unit on one side of the nucleus, as presented in the fluorescence micrograph. At the onset of mitosis, the centrosome complex divides with each centriole pair becoming the center of an organized network of radial microtubules. When observed through the microscope, the cell nucleus is well defined and surrounded by the nuclear envelope or membrane during interphase.
Within the nuclear membrane are one or more nucleoli, which appear as spherical, dense structures when stained with fluorescent or absorbing dyes. On the outer periphery of the nucleus are two important organelles termed centrosomes , which were formed by duplication of a single copy during the last division cycle. Each centrosome contains a pair of centrioles that extrude visible microtubule networks through the cytoplasm in radial arrays, known as asters derived from the term stars.
Even though the chromosomes have been duplicated during the DNA synthesis S phase, individual chromatids are not visible in late interphase because the chromosomes still exist in the form of loosely packed chromatin fibers.
In higher eukaryotic cells, the non-membranous centrosome also commonly referred to as the microtubule-organizing center functions through the cell cycle to organize the intracellular network of spindle and cytoskeletal microtubules.
The pair of centrioles located at the center of the centrosome is not essential for cell division. In fact, most plants lack centrioles, and the organelles can be eliminated in animal cells through laser microsurgery with no apparent effect on spindle formation during mitosis. Although very little activity is observable in the cell nucleus with fluorescence microscopy during interphase and the period is not considered to be a formal step in mitosis, this stage represents an essential preparation for cell division because the chromosomes are replicated during interphase.
In addition to the synthesis phase, which occurs during the central portion of interphase, the cell cycle also consists of two gap abbreviated G stages that precede and follow the synthesis phase. For most animal cells, the interphase portion accounts for approximately 90 percent of the cell cycle, whereas mitosis is accomplished in the remaining period. The first gap period is referred to as G 1 and starts shortly after mitosis, but prior to the synthesis S phase. The second gap period G 2 follows the synthesis phase and occurs just before mitosis.
These gap periods exhibit intensive biochemical activity resulting in a doubling of the cell size with a similar increase in critical enzymes, ribosomes, mitochondria, carbohydrates, structural proteins, lipids, and other biomolecules and organelles that will be needed as the cell prepares for division. Collectively, the G and S periods are known as interphase. The timeframe for completion of the entire cell cycle the gap, synthesis, mitosis, and cytokinesis phases varies depending upon the organism.
Indeed, in the case of interphase chromatin, mobility has been most commonly inferred indirectly from observed changes in organization and distribution Manuelidis, ; Bartholdi, ; Ferguson and Ward, ; Janevski et al. Second, the mobility of nuclear constituents should reflect their function. In the case of interphase chromatin, a functional significance for mobility is strongly suggested by recent observations of changes in centromere and chromosome distribution in response to cell differentiation, transcription signals, and stage of the cell cycle Manuelidis, ; Bartholdi, ; Ferguson and Ward, ; Funabiki et al.
To date, data bearing on the mobility of chromatin in interphase nuclei are not only limited, they are also somewhat contradictory. For example, some studies suggest that chromatin is relatively immobile during interphase. Notable examples include observations of nonrandom organization of chromosomal substructures and of chromosome confinement to domains in fixed interphase nuclei Moroi et al. Moreover, in living HeLa cells, centromeres are generally motionless during interphase Shelby et al.
Finally, photobleaching studies of chromatin in isolated interphase nuclei indicate that chromatin reorientational mobility is highly restricted Selvin et al. In contrast, some studies have shown that chromatin can reposition during interphase; notable examples include the three-dimensional movement of heterochromatin in nuclei of living interphase neurons De Boni and Mintz, , the coalescing and dispersing of centromeres in cultured cells in late G 2 and early G 1 , respectively Manuelidis, , and the occasional slow movement of a centromere in living HeLa cells Shelby et al.
Data bearing on the mobility of nonchromosomal molecules and other objects in interphase nuclei are also somewhat limited and contradictory. Studies of the trajectories of large, naturally occurring cytoplasmic inclusions have shown that the mobility of inclusion-sized objects in the nuclei of newt pneumocytes is several hundredfold slower than in dilute solution Alexander and Rieder, In contrast, translational FRAP experiments on fluorescently labeled dextrans 3— kD have shown that translation of dextrans in the nuclei of Hepatoma cells is only about sevenfold slower than in dilute solution Lang et al.
These rather disparate results leave considerable uncertainty surrounding the rates and determinants of motion in the nucleus.
In this study, we have directly monitored the translational motion of fluorescently labeled chromatin in the nuclei of living Swiss 3T3 and HeLa cells using FRAP. These cell lines were chosen because microscopy studies of nuclear organization have frequently employed cultured cells, including Swiss 3T3 and HeLa, generating a considerable body of germane literature e.
Our results show that interphase euchromatin and heterochromatin are substantially immobile in these cell lines. This immobility is consistent with chromatin attachment to nuclear substructures and with chromosome confinement to discrete domains during interphase. Moreover, this immobility provides a dynamics-based foundation for the many observations of nonrandom organization of chromosomal substructures in fixed interphase cells.
Swiss 3T3 fibroblasts and HeLa cells were grown on mm-diam round glass No. The longest labeling times and highest dihydroethidium concentrations were used to prepare cells for photography. Dihydroethidium is a membrane permeant, chemically reduced derivative of ethidium bromide that is dehydrogenated to ethidium bromide.
Cell viability during and after photobleaching experiments was assayed in two ways. The latter molecule is nonfluorescent and membrane permeant but is converted into the green fluorescent molecule calcein by intracellular esterases in viable cells.
After conversion, calcein is retained by cells only so long as their plasma membranes are not compromised. Thus, green cells are viable cells, and cell death can be assayed by monitoring for a loss of cytoplasmic green fluorescence. This was done visually and electronically to demonstrate short-term viability during photobleaching experiments.
In addition, to demonstrate that calcein is not retained after cells are compromised, calcein-labeled cells were placed in buffer containing 0. Second, long-term viability and absence of DNA damage were demonstrated by verifying that cells were able to divide and proliferate after they were photobleached. To facilitate identification of cells over long periods of time, cells were grown on coverslips that had been scratched with a diamond pen.
Cells were labeled with dihydroethidium and calcein AM well before they had reached confluence and were then transferred to a sterile Sykes-Moore chamber for photobleaching experiments. Cells were then examined frequently under a dissecting microscope; individual bleached cells could thus be tracked at low cell density even if they moved slowly. As a FRAP control, samples containing a fluorescently labeled macromolecule undergoing Brownian diffusion were prepared by diluting a rhodamine-labeled goat anti—rabbit antibody into Hanks' buffer.
In a FRAP experiment, a brief, intense pulse of laser light is used to bleach render nonfluorescent many of the fluorescent molecules in a small subregion of a fluorescent sample. The return of fluorescence to this subregion is then monitored by shining much attenuated laser light onto the bleached spot and monitoring the temporal dependence of the post-bleach probe fluorescence. The time-scale over which the fluorescence recovers is determined by the rate at which the fluorescently labeled molecules diffuse and can be used in simple systems to determine a diffusion coefficient.
Failure of the fluorescence to recover indicates that the fluorescently labeled molecules are immobile. An aperture diaphragm placed in the image plane of the microscope minimizes detection of background and out-of-focus light.
The nm green line of the argon-ion laser was used to excite fluorescence; excitation of ethidium with visible light, as contrasted with ultraviolet radiation, reduces the probability of radiation-induced damage to the cells. Photographs of cells were taken at defined times before and after photobleaching using Kodak TMX film.
Cells were illuminated for photography by inserting a defocusing lens into the optical path of the photobleaching apparatus, thereby expanding the laser beam to cover the entire field of view. Diffusion coefficients and mobile fractions were obtained by fitting photobleaching data to an equation describing photobleaching recovery in a system containing one immobile fraction and one mobile fraction diffusing in two dimensions Axelrod et al.
Complex generalizations of this equation describing diffusion in three dimensions have recently been derived Blonk et al. These generalized equations show that the recovery time along each dimension is proportional to the square of the characteristic distance along that dimension.
This implies that the fastest recovery time will be dominated by diffusion along the smallest dimension. Thus, for the focused spot used in these experiments, the simpler photobleaching recovery equations describing diffusion in two dimensions can be used. A several hundred millisecond bleaching pulse was sufficient to bleach the ethidium molecules in the illuminated region and create a dark spot in the euchromatin Fig. This spot persisted for at least 1 h Fig.
Moreover, the spot did not move within the nucleus, providing further evidence for chromatin immobilization during interphase. Pre- a and post-bleach b and c photographs of two dihydroethidium-labeled Swiss 3T3 cells in interphase. The ethidium stains nucleic acid in both the nucleus and cytoplasm, and reveals the distribution of euchromatin and heterochromatin within the nucleus. The failure of this spot to fill back up with fluorescently labeled chromatin demonstrates that a large fraction of the chromatin is immobile.
The focus may have shifted slightly or the cell may have moved slightly in c after the sample spent 60 min on the microscope stage. Some nonuniformity in fluorescence in the photographs may arise from interference in the illuminating light. More quantitative translational mobility data were obtained by monitoring the temporal dependence of the post-bleach fluorescence using FRAP curves.
Both euchromatic and heterochromatic regions of the nucleus were studied. The FRAP curves were smoothed using an eleven-point fit to a fourth-order polynomial Savitzky and Golay, to facilitate distinguishing the different recovery curves. The bleach pulse was 10 ms in duration, and data were collected in ms increments. The upper diamonds and lower squares curves in each graph were obtained from relatively smaller focused and larger defocused spots, respectively.
The defocused spot was obtained by translating a lens along the optical path so that the light was not focused exactly on the sample plane. Appropriate neutral density filters were inserted into the path of the bleach beam so that the bleach depths for the two curves in each graph were comparable; results did not depend on bleach depth data not shown , because relatively shallow bleach depths were employed.
Two important features are qualitatively apparent from the FRAP curves. First, a large fraction of the fluorescence fails to recover, indicating that a large fraction of the chromatin is immobile.
Second, the recovery time associated with the fraction of the fluorescence that does recover increases with increasing spot size, indicating that molecular motion is the source of the initial recovery.
FRAP curves were fitted to the theoretical expression for photobleaching recovery in two dimensions, assuming one mobile and one immobile fraction. These results were independent of the depth of bleach. The mobile fractions obtained from the fits are fairly large but will be shown to represent a population of less tightly bound ethidium molecules, and not mobile chromatin.
There are two important differences between the results shown in the photographs and in the photobleaching recovery curves. First, fluorescence from out-of-focus planes inevitably appears in the photographs Scalettar et al. Because laser beam divergence causes the spot radius to increase in out-of-focus planes, out-of-focus pick-up may cause the spots shown in the photographs to appear larger than their in-focus size.
The recovery time associated with this component was studied as a function of the size of the illuminated spot to identify its origin. If molecular motion is the source of the initial partial recovery, the recovery time should increase systematically as the size of the illuminated spot is increased.
For example, for a recovery driven by Brownian diffusion, the recovery half-time is expected to increase as the square of the spot size Axelrod et al. As with the antibody samples, the initial recovery time for the nuclei samples increases as spot size is increased, implying that molecular motion is the source of the initial recovery.
The upper curve represents an average of 1, experiments and the lower an average of 3, experiments, and each was smoothed using an point fit to a fourth-order polynomial. The fluorescence recovers almost completely, indicating that all molecules are mobile.
In addition, the recovery time increases with increasing spot size. A motion-derived origin for the partial recovery is also suggested by results from two additional experiments; see Fig. First, when samples are dried down and immobilized, the recovery largely disappears. Second, when hydrated samples are deoxygenated, the recovery time is essentially unaltered, which indicates that the initial recovery does not arise from reversible photobleaching see Discussion.
It is important to eliminate this latter possibility because the effects of reversible photobleaching have previously been observed in FRAP experiments on DNA and chromatin Scalettar et al. Typical FRAP curves obtained from Swiss 3T3 cells that were labeled with dihydroethidium and then a dried down or b placed in a deoxygenated buffer diamonds or an atmosphere-equilibrated buffer squares.
Bleach and data collection times were 10 ms. The virtual absence of the initial recovery in a sample immobilized by drying a supports the idea that the recovery is motion derived. The failure of the initial relaxation time to vary appreciably with oxygen concentration in the hydrated samples b indicates that the initial relaxation does not represent reversible photobleaching.
Six times more power was required to achieve bleaching in the deoxygenated sample comparable to that achieved in the nondeoxygenated sample. This reflects the greater difficulty of doing irreversible bleaching in the absence of oxygen, and shows that the deoxygenation was successful.
The mobile fraction could represent chromatin, RNA, or a subpopulation of the ethidium bromide that transiently comes off the chromatin. To distinguish these possibilities, FRAP control experiments were performed on cells fixed and labeled with either ethidium bromide or ethidium homodimer-1, which binds essentially irreversibly to DNA and RNA Gaugain et al.
Fixation should largely inhibit motion of protein-containing macromolecules on the FRAP distance scale. This, together with the fact that only the nucleic acid label differs between the two control samples, strongly suggests that the mobile fraction is a subpopulation of the ethidium bromide that transiently comes off the chromatin and diffuses freely see Discussion.
The mobile chromatin fraction is thus small or zero. Typical FRAP curves obtained from fixed ethidium bromide—labeled diamonds and ethidium homodimer-1—labeled squares Swiss 3T3 cells. Note that the initial component is absent when the homodimer is used as a label. A potential artifact in interpretation could arise if the lack of full fluorescence recovery is due to depletion of the finite reservoir of fluorescence in the nucleus, and not to an immobile chromatin fraction.
However, the photographs show that the volume of the bleached region is small, indicating that the bleach is unlikely to significantly deplete the total nuclear fluorescence.
This was not found to be the case. Finally, the long-lived spots shown in the photographs demonstrate that the nucleus does not become uniformly less fluorescent with time after the bleach. Artifacts in interpretation could also arise if the cells were not viable or were damaged during the FRAP experiments.
For this reason, cell viability during and after photobleaching experiments was tested using the two methods described under Materials and Methods. Moreover, bleached cells in an easily identified scratched region of the coverslip were observed to divide and proliferate after being returned to the incubator. Several other results, observations, and aspects of the experimental protocol argue against light-induced artifacts in the FRAP experiments.
First, the mobility results were independent of light intensity and bleaching time. Second, only a very small fraction of the nuclear volume was typically exposed to light. Finally, ethidium was excited through its visible absorption band, using green light; this is likely to result in significantly less nuclear damage than would excitation of ethidium through its UV absorption band.
The experiments described here were directed at a measuring the translational mobility of chromatin, especially euchromatin, in living interphase cells using FRAP; and b relating the results to current ideas about chromatin function and organization during interphase. Even in the absence of photobleaching data, it is evident that there are some constraints on chromatin motion in living interphase Swiss 3T3 cells. Mitchison, T. Mitosis: A history of division.
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Citation: O'Connor, C. Nature Education 1 1 The five phases of mitosis and cell division tightly coordinate the movements of hundreds of proteins. How did early biologists unravel this complex dance of chromosomes? Aa Aa Aa. Mitosis Occupies a Portion of the Cell Cycle. Figure 2. Figure 1. Figure Detail. Figure 3. Ascaris megalocephala bivalens, as drawn by Boveri in The figure shows chromosomes in the middle of the dividing cell, as well as the spindle, two centrosomes, and two centrioles within each centrosome.
Note that the cytoplasm is perceived as being structured. Figure 6. Figure 5. Figure 7. Figure 8. Figure Telophase and Cytokinesis. References and Recommended Reading Cheeseman, I.
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