Nuclear size control in fission yeast

Along-standing biological question is how a eukaryotic cell controls the size of its nucleus. We report here that in fission yeast, nuclear size is proportional to cell size over a 35-fold range, and use mutants to show that a 16-fold change in nuclear DNA content does not influence the relative size of the nucleus. Multi-nucleated cells with unevenly distributed nuclei reveal that nuclei surrounded by a greater volume of cytoplasm grow more rapidly. During interphase of the cell cycle nuclear growth is proportional to cell growth, and during mitosis there is a rapid expansion of the nuclear envelope. When the nuclear/cell (N/C) volume ratio is increased by centrifugation or genetic manipulation, nuclear growth is arrested while the cell continues to grow; in contrast, low N/C ratios are rapidly corrected by nuclear growth. We propose that there is a general cellular control linking nuclear growth to cell size.     

The size-wise nucleus: nuclear volume control in eukaryotes

Eukaryotic cells have an “awareness” of their volume and organellar volumes, and maintain a nuclear size that is proportional to the total cell size. New studies in budding and fission yeast have examined the relationship between cell and nuclear volumes. It was found that the size of the nucleus remains proportional to cell size in a wide range of genetic backgrounds and growth conditions that alter cell volume and DNA content. Moreover, in multinucleated fission yeast cells, Neumann and Nurse (see p. 593 of this issue) found that the sizes of individual nuclei are controlled by the relative amount of cytoplasm surrounding each nucleus. These results highlight a role of the cytoplasm in nuclear size control.     

Regulation of cell size in the yeast Saccharomyces cerevisiae.

For cells of the yeast Saccharomyces cerevisiae, the size at initiation of budding is proportional to growth rate for rates from 0.33 to 0.23 h-1. At growth rates lower than 0.23 h-1, cells displayed a minimum cell size at bud initiation independent of growth rate. Regardless of growth rate, cells displayed an increase in volume each time budding was initiated. When abnormally small cells, produced by starvation for nitrogen, were placed in fresh medium containing nitrogen but with different carbon sources, they did not initiate budding until they had grown to the critical size characteristic of that medium. Moreover, when cells were shifted from a medium supporting a low growth rate and small size at bud initiation to a medium supporting a higher growth rate and larger size at bud initiation, there was a transient accumulation of cells within G1. These results suggest that yeast cells are able to initiate cell division at different cell sizes and that regulation of cell size occurs within G1.     

A comparison of volume growth during bud and mycelium formation in Candida albicans: a single cell analysis

When stationary phase cells of the dimorphic yeast Candida albicans are diluted into fresh medium at pH 4.5 (low pH), they synchronously form ellipsoidal buds, but when diluted into the same medium at pH 6.7 (high pH), they synchronously form elongate mycelia. Using a perfusion chamber to monitor single cells, we have compared the rates of volume growth between budding and mycelium-forming cells. Results are presented which demonstrate that: (1) after release from stationary phase into medium of low or high pH, each original sphere grows in volume to the time of initial evagination, but does not grow subsequently; (2) successive budding on the original mother cell occurs without interruption resulting in continuous volume growth; however, an interruption in volume growth of the initial bud (B1) occurs before it in turn evaginates; and (3) the rate of volume growth of the first bud at low pH is identical to the rate of volume growth of the mycelium at high pH even though the surface to volume ratios are quite different. The last result is unexpected and is therefore considered in relation to cell wall deposition.     

cAMP-mediated increase in the critical cell size required for the G1 to S transition in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, cyclic AMP is required for cellular growth. In this study we show that cAMP also specifically inhibits the G1-S transition of the S. cerevisiae cell cycle by increasing the critical cell size required at start, the major yeast cell cycle control step. In fact: (a) addition of cAMP delays the time of entering into the S budded phase of small G1 cells, while it is ineffective on large fast-growing cells. (b) If cell growth is strongly depressed, cAMP permanently inhibits cell cycle commitment of cells arrested at the alpha-factor-sensitive step. The cell fraction inhibited by cAMP is inversely correlated with the average cell size of treated populations. (c) The critical protein content (Ps) and the critical cell volume (VB) required for budding in unperturbed exponentially growing yeast populations are largely increased by cAMP. On these bases, we propose a new cAMP role at start.     

A cytological study of Nematospora coryli pegl

A study is made of nuclear division in Nematospora coryli, a pathogenic yeast. The DNA of cells (grown on a V-8-medium) was stained with leuco-basic fuchsin (Feulgen test) at pH 3.5. After budding has started the rounded nucleus elongates and some differentiation into chromosomes is perceptible. A few slides suggest the number of chromosomes being 4. After some time the nucleus appears to have duplicated. This nucleus migrates towards the isthmus between mother cell and bud. In the isthmus, or just in front of it, the two daughter nuclei proceed to disjoin and move along each other to opposite directions. One daughter nucleus moves into the bud; the other one migrates back into the mother cell.Samples from synchronously growing cultures show that a fraction of the young yeast cells are destined to grow out to asci, in which after about 6 hours the presence of bivalents seems highly probable. The succeeding nuclear divisions take place in the same way as described for the vegetative cells and stop when the majority of the enlarged asci contain 8 nuclei.Problems of haploidy and diploidy are discussed.Small, densely stained bodies are observed in certain vegetative and some meiotic stages. As these bodies contain DNA, their function and possible homology with centrioles is discussed.     

Bud-Localization of CLB2 mRNA Can Constitute a Growth Rate Dependent Daughter Sizer

Maintenance of cellular size is a fundamental systems level process that requires balancing of cell growth with proliferation. This is achieved via the cell division cycle, which is driven by the sequential accumulation and destruction of cyclins. The regulatory network around these cyclins, particularly in G₁, has been interpreted as a size control network in budding yeast, and cell size as being decisive for the START transition. However, it is not clear why disruptions in the G₁ network may lead to altered size rather than loss of size control, or why the S-G₂-M duration also depends on nutrients. With a mathematical population model comprised of individually growing cells, we show that cyclin translation would suffice to explain the observed growth rate dependence of cell volume at START. Moreover, we assess the impact of the observed bud-localisation of the G₂ cyclin CLB2 mRNA, and find that localised cyclin translation could provide an efficient mechanism for measuring the biosynthetic capacity in specific compartments: The mother in G₁, and the growing bud in G₂. Hence, iteration of the same principle can ensure that the mother cell is strong enough to grow a bud, and that the bud is strong enough for independent life. Cell sizes emerge in the model, which predicts that a single CDK-cyclin pair per growth phase suffices for size control in budding yeast, despite the necessity of the cell cycle network around the cyclins to integrate other cues. Size control seems to be exerted twice, where the G₂/M control affects bud size through bud-localized translation of CLB2 mRNA, explaining the dependence of the S-G₂-M duration on nutrients. Taken together, our findings suggest that cell size is an emergent rather than a regulatory property of the network linking growth and proliferation.     

Roles of the CDC24 gene product in cellular morphogenesis during the Saccharomyces cerevisiae cell cycle

Temperature-sensitive yeast mutants defective in gene CDC24 continued to grow (i.e., increase in cell mass and cell volume) at restrictive temperature (36 degrees C) but were unable to form buds. Staining with the fluorescent dye Calcofluor showed that the mutants were also unable to form normal bud scars (the discrete chitin rings formed in the cell wall at budding sites) at 36 degrees C; instead, large amounts of chitin were deposited randomly over the surfaces of the growing unbudded cells. Labeling of cell-wall mannan with fluorescein isothiocyanate-conjugated concanavalin A suggested that mannan incorporation was also delocalized in mutant cells grown at 36 degrees C. Although the mutants have well-defined execution points just before bud emergence, inactivation of the CDC24 gene product in budded cells led both to selective growth of mother cells rather than of buds and to delocalized chitin deposition, indicating that the CDC24 gene product functions in the normal localization of growth in budded as well as in unbudded cells. Growth of the mutant strains at temperatures less than 36 degrees C revealed allele-specific differences in behavior. Two strains produced buds of abnormal shape during growth at 33 degrees C. Moreover, these same strains displayed abnormal localization of budding sites when growth at 24 degrees C (the normal permissive temperature for the mutants); in each case, the abnormal pattern of budding sites segregated with the temperature sensitivity in crosses. Thus, the CDC24 gene product seems to be involved in selection of the budding site, formation of the chitin ring at that site, the subsequent localization of new cell wall growth to the budding site and the growing bud, and the balance between tip growth and uniform growth of the bud that leads to the normal cell shape.     

Astral Microtubule Dynamics in Yeast: A Microtubule-based Searching Mechanism for Spindle Orientation and Nuclear Migration into the Bud

Localization of dynein–green fluorescent protein (GFP) to cytoplasmic microtubules allowed us to obtain one of the first views of the dynamic properties of astral microtubules in live budding yeast. Several novel aspects of microtubule function were revealed by time-lapse, three-dimensional fluorescence microscopy. Astral microtubules, about four to six in number for each pole, exhibited asynchronous dynamic instability throughout the cell cycle, growing at 0.3–1.5 μm/min toward the cell surface then switching to shortening at similar velocities back to the spindle pole body (SPB). During interphase, a conical array of microtubules trailed the SPB as the nucleus traversed the cytoplasm. Microtubule disassembly by nocodozole inhibited these movements, indicating that the nucleus was pushed around the interior of the cell via dynamic astral microtubules. These forays were evident in unbudded G1 cells, as well as in late telophase cells after spindle disassembly. Nuclear movement and orientation to the bud neck in S/G2 or G2/M was dependent on dynamic astral microtubules growing into the bud. The SPB and nucleus were then pulled toward the bud neck, and further microtubule growth from that SPB was mainly oriented toward the bud. After SPB separation and central spindle formation, a temporal delay in the acquisition of cytoplasmic dynein at one of the spindle poles was evident. Stable microtubule interactions with the cell cortex were rarely observed during anaphase, and did not appear to contribute significantly to spindle alignment or elongation into the bud. Alterations of microtubule dynamics, as observed in cells overexpressing dynein-GFP, resulted in eventual spindle misalignment. These studies provide the first mechanistic basis for understanding how spindle orientation and nuclear positioning are established and are indicative of a microtubule-based searching mechanism that requires dynamic microtubules for nuclear migration into the bud.     

Polarized growth in fungi: symmetry breaking and hyphal formation

Cell shape is a critical determinant for function. The baker's yeast Saccharomyces cerevisiae changes shape in response to its environment, growing by budding in rich nutrients, forming invasive pseudohyphal filaments in nutrient poor conditions and pear shaped shmoos for growth towards a partner during mating. The human opportunistic pathogen Candida albicans can switch from budding to hyphal growth, in response to numerous environmental stimuli to colonize and invade its host. Hyphal growth, typical of filamentous fungi, is not observed in S. cerevisiae. A number of internal cues regulate when and where yeast cells break symmetry leading to polarized growth and ultimately distinct cell shapes. This review discusses how cells break symmetry using the yeast S. cerevisiae paradigm and how polarized growth is initiated and maintained to result in dramatic morphological changes during C. albicans hyphal growth.     

Functions of microtubules in the Saccharomyces cerevisiae cell cycle

We used the inhibitor nocodazole in conjunction with immunofluorescence and electron microscopy to investigate microtubule function in the yeast cell cycle. Under appropriate conditions, this drug produced a rapid and essentially complete disassembly of cytoplasmic and intranuclear microtubules, accompanied by a rapid and essentially complete block of cellular and nuclear division. These effects were similar to, but more profound than, the effects of the related drug methyl benzimidazole carbamate (MBC). In the nocodazole-treated cells, the selection of nonrandom budding sites, the formation of chitin rings and rings of 10-nm filaments at those sites, bud emergence, differential bud enlargement, and apical bud growth appeared to proceed normally, and the intracellular distribution of actin was not detectably perturbed. Thus, the cytoplasmic microtubules are apparently not essential for the establishment of cell polarity and the localization of cell-surface growth. In contrast, nocodazole profoundly affected the behavior of the nucleus. Although spindle-pole bodies (SPBs) could duplicate in the absence of microtubules, SPB separation was blocked. Moreover, complete spindles present at the beginning of drug treatment appeared to collapse, drawing the opposed SPBs and associated nuclear envelope close together. Nuclei did not migrate to the mother-bud necks in nocodazole-treated cells, although nuclei that had reached the necks before drug treatment remained there. Moreover, the double SPBs in arrested cells were often not oriented toward the budding sites, in contrast to the situation in normal cells. Thus, microtubules (cytoplasmic, intranuclear, or both) appear to be necessary for the migration and proper orientation of the nucleus, as well as for SPB separation, spindle function, and nuclear division.     

cAMP-mediated increase in the critical cell size required for the G1 to S transition in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, cyclic AMP is required for cellular growth. In this study we show that cAMP also specifically inhibits the G1-S transition of the S. cerevisiae cell cycle by increasing the critical cell size required at start, the major yeast cell cycle control step. In fact: (a) addition of cAMP delays the time of entering into the S budded phase of small G1 cells, while it is ineffective on large fast-growing cells. (b) If cell growth is strongly depressed, cAMP permanently inhibits cell cycle commitment of cells arrested at the α-factor-sensitive step. The cell fraction inhibited by cAMP is inversely correlated with the average cell size of treated populations. (c) The critical protein content (P_s) and the critical cell volume (V_B) required for budding in unperturbed exponentially growing yeast populations are largely increased by cAMP. On these bases, we propose a new cAMP role at start.     

Method for immobilizing microbial cells on gel surface for dynamic AFM studies.

The processes of cell growth and budding of the yeast cells Saccharomyces cerevisiae, which were gently immobilized on 3% agar and submerged in culture medium, were successfully imaged with an atomic force microscope for 6-7 h. Similar experiments on chemically fixed cells did not detect any appreciable change in their appearance except in a few scannings at the very beginning, indicating that the dissolution of agar and/or scraping of its surface by the scanning tip, if any, did not significantly interfere with the images taken thereafter. The increment in the height of many of the untreated cells, accompanied by their lateral enlargement, was taken as an indication of successful imaging of the growth process of yeast cells, together with an image of a growing daughter cell attached to its mother cell.     

Differential distribution of factors involved in pre-mRNA processing in the yeast cell nucleus.

The yeast cell nucleus has previously been shown to be divided into two regions by a variety of microscopic approaches. We used antibodies specific for the 2,2,7-trimethylguanosine cap structure of small nuclear ribonucleic acids (snRNAs) and for a protein component of small nuclear ribonucleoprotein particles to identify the distribution of small nuclear ribonucleoprotein particles within the yeast cell nucleus. These studies were performed with the fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae. By using immunofluorescence microscopy and immunoelectron microscopy, most of the abundant snRNAs were localized to the portion of the nucleus which has heretofore been referred to as the nucleolus. This distribution of snRNAs is different from that found in mammalian cells and suggests that the nucleolar portion of the yeast nucleus contains functional domains in addition to those associated with RNA polymerase I activity.     

Herpesvirus Envelopment

The growth and envelopment processes of three representative herpesviruses, equine abortion, pseudorabies, and herpes simplex, were examined in baby hamster kidney (BHK 21/13) cells by bioassay (plaque-forming units) and electron microscopy. The envelopment process was identical for all three viruses. After assembly in the nucleus, the nucleocapsid acquired an envelope by budding from the inner nuclear membrane. This membrane was reduplicated as the enveloped particle was released so that the budding process did not result in disruption of the continuity of the nuclear membrane. That portion of the nuclear membrane which comprised the viral envelope was appreciably thicker than the remainder of the membrane and exhibited numerous projections on its surface. Once enveloped, the viral particles were seen in vesicles and vacuoles in the cell cytoplasm. These appeared to open at the cytoplasmic membrane, releasing the virus from the cell. There was no detectable difference in the size or appearance of enveloped particles in intra- or extracellular locations.     

Cellular dimensions affecting the nucleocytoplasmic volume ratio

Although it has long been appreciated that larger eukaryotic cells have larger nuclei, little is known about how this size relationship is maintained. Here we describe a method for measuring the aqueous volume ratio of nucleus to cytoplasm, two compartments which are interconnected via the pores in the nuclear envelope. We then use that method to identify proportional cellular dimensions in variously treated cells and in different cell types. Cells were scrape loaded with a mixture of fluorescent dextrans: Texas red dextran, average mol wt = 10,000 (TRDx10), and fluorescein isothiocyanate dextran, average mol wt = 70,000 (FDx70). After introduction into the cytoplasmic space, the TRDx10 distributed into both the nucleus and cytoplasm, whereas the FDx70 was restricted to cytoplasm, due to size exclusion by the nuclear pores. The aqueous nucleocytoplasmic volume ratio (RN/C) was determined by measuring, from fluorescence images of spread cells, total cellular fluorescence of each of the two probes and the fluorescence ratio of those probes in the cytoplasm. RN/C was unaffected by the measurement procedure or by varying temperatures between 23 degrees and 37 degrees C. Loading excess unlabeled dextrans had little effect on RN/C, with the single exception that high concentrations of large dextrans could lower RN/C in endothelial cells. Expanding intracellular membranous compartments of macrophages by phagocytosis of latex beads decreased RN/C. Expanding the same compartment by pinocytosis of sucrose, which nearly doubled total cell volume, had little effect on RN/C, indicating that nuclear volume was more closely linked to the cytoplasmic volume, exclusive of vesicular organelles, than to total cell volume. RN/C was the same in mononucleate and binucleate endothelial cells. Finally, measurements of RN/C in murine bone marrow-derived macrophages, bovine aortic endothelial cells, Swiss 3T3 fibroblasts, PtK2 cells, and CV-1 cells revealed that nuclear volume scaled allometrically with cell volume. The allometric relationship indicated that cell volume was proportional to nuclear surface area.     

Morphologically-structured models of growing budding yeast populations

It has been well recognized that many key aspects of cell cycle regulation are encoded into the size distributions of growing budding yeast populations due to the tight coupling between cell growth and cell division present in this organism. Several attempts have been made to model the cell size distribution of growing yeast populations in order to obtain insight on the underlying control mechanisms, but most were based on the age structure of asymmetrically dividing populations. Here we propose a new framework that couples a morphologically-structured representation of the population with population balance theory to formulate a dynamic model for the size distribution of growing yeast populations. An advantage of the presented framework is that it allows derivation of simpler models that are directly identifiable from experiments. We show how such models can be derived from the general framework and demonstrate their utility in analyzing yeast population data. Finally, by employing a recently proposed numerical scheme, we proceed to integrate numerically the full distributed model to provide predictions of dynamics of the cell size structure of growing yeast populations.