Simultaneous production of buds on mother and daughter cells of Saccharomyces cerevisiae in the presence of hydroxyurea

Individual budding yeast cells, Saccharomyces cerevisiae, enclosedin small culture chambers were observed through two buddingcycles to examine their behavior during growth and division.In the nutrient medium (YHG medium), the duration of the buddingcycles was 77 min for mother cells and 90 min for daughter cells;a 13-min time lag between the two durations. Continuous exposureof cells to 16 or 32 mM hydroxyurea extended the duration ofthe cycles and increased the volume of the cells, resultingin the formation of abnormally large and equal-sized mother-daughterpairs. Each cell of these pairs subsequently produced buds simultaneously.Stained cell nuclei showed simultaneous nuclear division. Thissynchronous budding on mother-daughter pairs was repeated inthe next budding cycle. The coordination of growth with divisionis discussed in relation to these results. (Received August 11, 1979; )     

Effect of Reversible Inhibition of Deoxyribonucleic Acid Synthesis on the Yeast Cell Cycle

Hydroxyurea (HU) preferentially inhibited deoxyribonucleic acid (DNA) replication and division in Saccharomyces cerevisiae. Growth, ribonucleic acid synthesis, and protein synthesis were less sensitive to this drug. Upon addition of HU, cells underwent one cycle of budding and the nuclei migrated into the necks between the mother cells and buds. Neither the nucleus nor the cells divided. Removal of HU allowed immediate resumption of DNA synthesis. Nuclear division, budding, and cell division occurred 1.5, 2, and 4 hr, respectively, after HU was removed. If protein synthesis was blocked at the time HU was removed, budding and cell division did not occur. These results were interpreted to indicate that HU prevents accumulation of the potential to initiate a new cell cycle.     

Artificial tethering to nuclear pores promotes partitioning of extrachromosomal DNA during yeast asymmetric cell division

SUMMARY: Asymmetric cell division in unicellular organisms enables sequestration of senescence factors to specific subpopulations. Accumulation of autonomously replicating sequence (ARS) plasmids, which frequently emerge from recombination within the highly repetitive ribosomal DNA locus, is linked to limited replicative life span of Saccharomyces cerevisiae cells [1]. During budding yeast cell division, ARS plasmids are retained in the ageing mother cell, such that only 1 out of 10 plasmids enters the rejuvenated bud [2]. Binding of ARS plasmids to nuclear structures retained in the mother cell was speculated to explain asymmetric plasmid segregation [2]. Association with nuclear pore complexes (NPCs) was proposed to underlie retention of ARS plasmids in the mother cell [3]. However, the role of NPCs in segregation of ARS plasmids is unclear, as NPCs are partitioned between mother and bud nuclei during mitosis [4,5]. Here we analyzed how segregation of ARS plasmids is influenced by their interaction with NPCs. We found that artificial tethering to NPCs promotes transport of ARS plasmids into the bud. Moreover, our experiments provide support for the notion that interaction with ARS plasmids does not affect movement of NPCs into the bud. We conclude that binding to NPCs cannot by itself contribute to asymmetric segregation of ARS plasmids.     

F-actin redistributions at the division site in livingTradescantia stomatal complexes as revealed by microinjection of rhodamine-phalloidin

Summary Microinjection of rhodamine-phalloidin into living cells of isolatedTradescantia leaf epidermis and visualisation by confocal microscopy has extended previous results on the distribution of actin in mitotic cells of higher plants and revealed new aspects of actin arrays in stomatal cells and their initials. Divisions in the stomatal guard mother cells and unspecialised epidermal cells are symmetrical. Asymmetrical divisions occur in guard mother precursor cells and subsidiary mother cells. Each asymmetrical division is preceded by migration of the nucleus and the subsequent accumulation of thick bundles of anticlinally oriented actin filaments localised to the area of the anticlinal wall closest to the polarised nucleus. During prophase, in all cell types, a subset of cortical actin filaments coaligns to form a band, which, like the preprophase band of microtubules, accurately delineates the site of insertion of the future cell wall. Following the breakdown of the nuclear envelope, F-actin in these bands disassembles but persists elsewhere in the cell cortex. Thus, cortical F-actin marks the division site throughout mitosis, firstly as an appropriately positioned band and then by its localised depletion from the same region of the cell cortex. This sequence has been detected in all classes of division inTradescantia leaf epidermis, irrespective of whether the division is asymmetrical or symmetrical, or whether the cell is vacuolate or densely cytoplasmic. Taken together with earlier observations on stamen hair cells and root tip cells it may therefore be a general cytoskeletal feature of division in cells of higher plants.Abbreviations GMC guard mother cell - MT microtubule - PPB preprophase band - Rh rhodamine - SMC subsidiary mother cell     

Mitotic Exit and Separation of Mother and Daughter Cells

Productive cell proliferation involves efficient and accurate splitting of the dividing cell into two separate entities. This orderly process reflects coordination of diverse cytological events by regulatory systems that drive the cell from mitosis into G1. In the budding yeast Saccharomyces cerevisiae, separation of mother and daughter cells involves coordinated actomyosin ring contraction and septum synthesis, followed by septum destruction. These events occur in precise and rapid sequence once chromosomes are segregated and are linked with spindle organization and mitotic progress by intricate cell cycle control machinery. Additionally, critical parts of the mother/daughter separation process are asymmetric, reflecting a form of fate specification that occurs in every cell division. This chapter describes central events of budding yeast cell separation, as well as the control pathways that integrate them and link them with the cell cycle.     

Lysis of growing cells ofSaccharomyces cerevisiae induced by papulacandin B

Light and electron microscopy was used to study the effect of papulacandin B onSaccharomyces cerevisiae in the exponential growth phase. At 1–2 μg/mL cell division in the culture continued almost in parallel with the control, at 4 μg/mL cell proliferation was reduced and the culture contained some cells with 2–9 buds which were not separated from the mother cell by a septum, and at higher concentrations (8, 16 and 32 μg/mL) the proliferation stopped within 2 h. Cessation of proliferation was due to lysis of budding cells in the bud region including perforation of thinned cell wall (most often at the bud basis and sometimes at its apex), extrusion of cytoplasm and death of cell. Lysis was also observed in cells without visible buds. Dividing cells died without visible lysis.     

Patterns of asexual reproduction in <Emphasis Type="Italic">Nannochloris bacillaris</Emphasis> and <Emphasis Type="Italic">Marvania geminata</Emphasis> (Chlorophyta,Trebouxiophyceae)

Non-flagellated vegetative green algae of the Trebouxiophyceae propagate mainly by autosporulation. In this manner, the mother cell wall is shed following division of the protoplast in each round of cell division. Binary fission type Nannochloris and budding type Marvania are also included in the Trebouxiophyceae. Phylogenetic trees based on the actin sequences of Trebouxiophyceae members revealed that the binary fission type Nannochloris bacillaris and the budding type Marvania geminata are closely related in a distal monophyletic group. Our results suggest that autosporulation is the ancestral mode of cell division in Trebouxiophyceae. To elucidate how non-autosporulative mechanisms such as binary fission and budding evolved, we focused on the cleavage of the mother cell wall. Cell wall development was analyzed using a cell wall-specific fluorescent dye, Fluostain I. Exfoliation of the mother cell wall was not observed in either N. bacillaris or M. geminata. We then compared the two algae by transmission electron microscopy with rapid freeze fixation and freeze substitution; in both algae, the mother cell wall was cleaved at the site of cell division, but remained adhered to the daughter cell wall. In N. bacillaris, the cleaved mother cell wall gradually degenerated and was not observed in the next cell cycle. In contrast, M. geminata daughter cells entered the growth phase of the next cell cycle bearing the mother and grandmother cell walls, causing the uncovered portion of the plane of division to bulge outward. Such a delay in the degeneration and shedding of the mother cell wall probably led to the development of binary fission and budding.     

Microtubules and actin cytoskeleton in Cryptococcus neoformans compared with ascomycetous budding and fission yeasts

Actin cytoskeleton and microtubules were studied in a human fungal pathogen, the basidiomycetous yeast Cryptococcus neoformans (haploid phase of Filobasidiella neoformans), during its asexual reproduction by budding using fluorescence and electron microscopy. Staining with rhodamine-conjugated phalloidin revealed an F-actin cytoskeleton consisting of cortical patches, cables and cytokinetic ring. F-actin patches accumulated at the regions of cell wall growth, i. e. in sterigma, bud and septum. In mother cells evenly distributed F-actin patches were joined to F-actin cables, which were directed to the growing sterigma and bud. Some F-actin cables were associated with the cell nucleus. The F-actin cytokinetic ring was located in the bud neck, where the septum originated. Antitubulin TAT1 antibody revealed a microtubular cytoskeleton consisting of cytoplasmic and spindle microtubules. In interphase cells cytoplasmic microtubules pointed to the growing sterigma and bud. As the nucleus was translocated to the bud for mitosis, the cytoplasmic microtubules disassembled and were replaced by a short intranuclear spindle. Astral microtubules then emanated from the spindle poles. Elongation of the mitotic spindle from bud to mother cell preceded nuclear division, followed by cytokinesis (septum formation in the bud neck). Electron microscopy of ultrathin sections of chemically fixed and freeze-substituted cells revealed filamentous bundles directed to the cell cortex. The bundles corresponded in width to the actin microfilament cables. At the bud neck numerous ribosomes accumulated before septum synthesis. We conclude: (i) the topology of F-actin patches, cables and rings in C. neoformans resembles ascomycetous budding yeast Saccharomyces, while the arrangement of interphase and mitotic microtubules resembles ascomycetous fission yeast Schizosaccharomyces. The organization of the cytoskeleton of the mitotic nucleus, however, is characteristic of basidiomycetous yeasts. (ii) A specific feature of C. neoformans was the formation of a cylindrical sterigma, characterized by invasion of F-actin cables and microtubules, followed by accumulation of F-actin patches around its terminal region resulting in development of an isodiametrical bud.     

Interactions among Rax1p, Rax2p, Bud8p, and Bud9p in marking cortical sites for bipolar bud-site selection in yeast

In the budding yeast Saccharomyces cerevisiae, selection of the bud site determines the axis of polarized cell growth and eventual oriented cell division. Bud sites are selected in specific patterns depending on cell type. These patterns appear to depend on distinct types of marker proteins in the cell cortex; in particular, the bipolar budding of diploid cells depends on persistent landmarks at the birth-scar-distal and -proximal poles that involve the proteins Bud8p and Bud9p, respectively. Rax1p and Rax2p also appear to function specifically in bipolar budding, and we report here a further characterization of these proteins and of their interactions with Bud8p and Bud9p. Rax1p and Rax2p both appear to be integral membrane proteins. Although commonly used programs predict different topologies for Rax2p, glycosylation studies indicate that it has a type I orientation, with its long N-terminal domain in the extracytoplasmic space. Analysis of rax1 and rax2 mutant budding patterns indicates that both proteins are involved in selecting bud sites at both the distal and proximal poles of daughter cells as well as near previously used division sites on mother cells. Consistent with this, GFP-tagged Rax1p and Rax2p were both observed at the distal pole as well as at the division site on both mother and daughter cells; localization to the division sites was persistent through multiple cell cycles. Localization of Rax1p and Rax2p was interdependent, and biochemical studies showed that these proteins could be copurified from yeast. Bud8p and Bud9p could also be copurified with Rax1p, and localization studies provided further evidence of interactions. Localization of Rax1p and Rax2p to the bud tip and distal pole depended on Bud8p, and normal localization of Bud8p was partially dependent on Rax1p and Rax2p. Although localization of Rax1p and Rax2p to the division site did not appear to depend on Bud9p, normal localization of Bud9p appeared largely or entirely dependent on Rax1p and Rax2p. Taken together, the results indicate that Rax1p and Rax2p interact closely with each other and with Bud8p and Bud9p in the establishment and/or maintenance of the cortical landmarks for bipolar budding.     

A cell cycle checkpoint monitors cell morphogenesis in budding yeast

Checkpoint controls are regulatory pathways that inhibit cell cycle progression in cells that have not faithfully completed a prior step in the cell cycle. In the budding yeast Saccharomyces cerevisiae, DNA replication and spindle assembly are monitored by checkpoint controls that prevent nuclear division in cells that have failed to complete these processes. During the normal cell cycle, bud formation is temporally coincident with DNA replication and spindle assembly, and the nucleus divides along the mother-bud axis in mitosis. In this report, we show that inhibition of bud formation also causes a dramatic delay in nuclear division. This allows cells to recover from a transient disruption of cell polarity without becoming binucleate. The delay occurs after DNA replication and spindle assembly, and results from delayed activation of the master cell cycle regulatory kinase, Cdc28. Cdc28 activation is inhibited by phosphorylation of Cdc28 on tyrosine 19, and by delayed accumulation of the B-type cyclins Clb1 and Clb2. These results suggest the existence of a novel checkpoint that monitors cell morphogenesis in budding yeast.     

Lack of invaginations of the plasma membrane during budding and cell division of Saccharomyces cerevisiae and Schizosaccharomyces pombe

Abstract The plasma membrane of Saccharomyces cerevisiae and Schizosaccharomyces pombe in stationary phase had abundant invaginations. A round uninvaginated area emerged before budding when S. cerevisiae cells were given fresh medium. Middle-sized buds had some invaginations, whereas the neck between the bud and mother had very few. S. pombe which has neither the neck nor the predetermined position to divide had no uninvaginated ring area even in long cells during elongation in fresh medium. However, an uninvaginated ring area emerged as the earliest noticeable stage of cytokinesis. The uninvaginated state of the plasma membrane appeared to be correlated with budding and cell division.     

A retention mechanism for distribution of mitochondria during cell division in budding yeast.

Mitochondria are indispensable for normal eukaryotic cell function. As they cannot be synthesized de novo and are self-replicating, mitochondria must be transferred from mother to daughter cells. Studies in the budding yeast Saccharomyces cerevisiae indicate that mitochondria enter the bud immediately after bud emergence, interact with the actin cytoskeleton for linear, polarized movement of mitochondria from mother to bud, but are equally distributed among mother and daughter cells [1] [2] [3]. It is not clear how the mother cell maintains its own supply of mitochondria. Here, we found that mother cells retain mitochondria by immobilization of some mitochondria in the 'retention zone', the base of the mother cell distal to the bud. Retention requires the actin cytoskeleton as mitochondria colocalized with actin cables in the retention zone, and mutations that perturb actin dynamics or actin-mitochondrial interactions produced retention defects. Our results support the model that equal distribution of mitochondria during cell division is a consequence of two actin-dependent processes: movement of some mitochondria into the daughter bud and immobilization of others in the mother cell.     

A novel actin-related protein is associated with daughter cell formation in Toxoplasma gondii

Cell division in Toxoplasma gondii occurs by an unusual budding mechanism termed endodyogeny, during which twin daughters are formed within the body of the mother cell. Cytokinesis begins with the coordinated assembly of the inner membrane complex (IMC), which surrounds the growing daughter cells. The IMC is compiled of both flattened membrane cisternae and subpellicular filaments composed of articulin-like proteins attached to underlying singlet microtubules. While proteins that comprise the elongating IMC have been described, little is known about its initial formation. Using Toxoplasma as a model system, we demonstrate that actin-like protein 1 (ALP1) is partially redistributed to the IMC at early stages in its formation. Immunoelectron microscopy localized ALP1 to a discrete region of the nuclear envelope, on transport vesicles, and on the nascent IMC of the daughter cells prior to the arrival of proteins such as IMC-1. The overexpression of ALP1 under the control of a strong constitutive promoter disrupted the formation of the daughter cell IMC, leading to delayed growth and defects in nuclear and apicoplast segregation. Collectively, these data suggest that ALP1 participates in the formation of daughter cell membranes during cell division in apicomplexan parasites.     

Sharing the wealth: peroxisome inheritance in budding yeast

Eukaryotic cells have evolved molecular mechanisms to ensure the faithful partitioning of cellular components during cell division. The budding yeast Saccharomyces cerevisiae has to actively deliver about half of its organelles to the growing bud, while retaining the remaining organelles in the mother cell. Until lately, little was known about the inheritance of peroxisomes. Recent studies have identified the peroxisomal proteins Inp1p and Inp2p as two key regulators of peroxisome inheritance that perform antagonistic functions. Inp1p is required for the retention of peroxisomes in mother cells, whereas Inp2p promotes the bud-directed movement of these organelles. Inp1p anchors peroxisomes to the cell cortex by interacting with specific structures lining the cell periphery. On the other hand, Inp2p functions as the peroxisome-specific receptor for the class V myosin, Myo2p, thereby linking peroxisomes to the translocation machinery that propels peroxisome movement. Tight coordination between Inp1p and Inp2p ensures a fair and harmonious spatial segregation of peroxisomes upon cell division.     

Control of spindle polarity and orientation in Saccharomyces cerevisiae

Control of mitotic spindle orientation represents a major strategy for the generation of cell diversity during development of metazoans. Studies in the budding yeast Saccharomyces cerevisiae have contributed towards our present understanding of the general principles underlying the regulation of spindle positioning in an asymmetrically dividing cell. In S. cerevisiae, the mitotic spindle must orient along the cell polarity axis, defined by the site of bud emergence, to ensure correct nuclear division between the mother and daughter cells. Establishment of spindle polarity dictates this process and relies on the concerted control of spindle pole function and a precise program of cues originating from the cell cortex that directs cytoplasmic microtubule attachments during spindle morphogenesis. These cues cross talk with the machinery responsible for bud-site selection, indicating that orientation of the spindle in yeast cells is mechanistically coupled to the definition of a polarity axis and the division plane. Here, we propose a model integrating the inherently asymmetric properties of the spindle pathway with the program of positional information contributing towards orienting the spindle in budding yeast. Because the basic machinery orienting the spindle in higher-eukaryotic cells appears to be conserved, it might be expected that similar principles govern centrosome asymmetry in the course of metazoan development.     

Nuclear envelope fission is linked to cytokinesis in budding yeast

We have investigated the relationship between nuclear envelope fission and cytokinesis during mitotic cell division in budding yeast. By carrying out time-lapse and optical sectioning video microscopy analysis of cells that express green fluorescent protein (GFP)-tagged nuclear envelope and actomyosin ring components, we found that nuclear division is temporally coupled to cytokinesis. Light and electron microscopy analysis also showed that nuclear envelope fission and the division of the nucleoplasm are severely delayed in cytokinesis mutants, resulting in discoupling between the nuclear division cycle and the budding cycle. These results suggest that homotypic membrane fusion may be activated by components or the mechanical action of cytokinetic structures and presents a mechanism for the equal partitioning of the nucleus and the temporal coordination of this event with chromosome segregation during mitosis.     

Severing all ties between mother and daughter: cell separation in budding yeast

At the end of nuclear division in the budding yeast, acto-myosin ring contraction and cytokinesis occur between mother and daughter cells. This is followed by cell separation, after which mother and daughter cells go their separate ways. While cell separation may be the last event that takes place between the two cells, it is nonetheless under tight regulation which ensures that both cells are viable upon separation. It is becoming increasingly clear that the components of the cell separation machinery are controlled at various levels, including the temporal and spatial regulation of the genes encoding for the components and the specific localization of the components to the neck. In addition, these regulatory controls are co-ordinated with exit from mitosis, thereby placing a mechanistic link between the end of mitosis and cell separation. More importantly, the success of the cell separation event is contingent upon the presence of a trilaminar septum, whose assembly is dependent on a host of proteins which localize to the neck over the span of one cell division cycle.     

Induction of respiration-deficient mutants by ultraviolet radiation in a synchronous yeast cell culture

Haploid cells ofSaccharomyces cerevisiae in a synchronous culture were exposed in different phases of division to UV radiation lethal for 98% of the cells and the occurrence of respiration-deficient mutants among the survivors was determined. Comparison of the various phases showed significantly higher occurrence of these mutants at the onset of budding. It was concluded that the mitochondrial DNA also replicates synchronously in a synchronous yeast cell culture, but in a different phase from the nuclear DNA.