Conservation of Mechanisms Controlling Entry into Mitosis Budding Yeast Wee1 Delays Entry into Mitosis and Is Required for Cell Size Control

AbstractBackground: In fission yeast, the Wee1 kinase delays entry into mitosis until a critical cell size has been reached; however, a similar role for Wee1-related kinases has not been reported in other organisms. SWE1, the budding yeast homolog of wee1, is thought to function in a morphogenesis checkpoint that delays entry into mitosis in response to defects in bud morphogenesis.Results: In contrast to previous studies, we found that budding yeast swe1Δ cells undergo premature entry into mitosis, leading to birth of abnormally small cells. Additional experiments suggest that conditions that activate the morphogenesis checkpoint may actually be activating a G2/M cell size checkpoint. For example, actin depolymerization is thought to activate the morphogenesis checkpoint by inhibiting bud morphogenesis. However, actin depolymerization also inhibits bud growth, suggesting that it could activate a cell size checkpoint. Consistent with this possibility, we found that actin depolymerization fails to induce a G2/M delay once daughter buds pass a critical size. Other conditions that activate the morphogenesis checkpoint block bud formation, which could also activate a size checkpoint if cell size at G2/M is monitored in the daughter bud. Previous work reported that Swe1 is degraded during G2, which was proposed to account for failure of large-budded cells to arrest in response to actin depolymerization. However, we found that Swe1 is present throughout G2 and undergoes hyperphosphorylation as cells enter mitosis, as found in other organisms.Conclusions: Our results suggest that the mechanisms known to coordinate entry into mitosis in other organisms have been conserved in budding yeast

Grapes(Chk1) prevents nuclear CDK1 activation by delaying cyclin B nuclear accumulation

Entry into mitosis is characterized by a dramatic remodeling of nuclear and cytoplasmic compartments. These changes are driven by cyclin-dependent kinase 1 (CDK1) activity, yet how cytoplasmic and nuclear CDK1 activities are coordinated is unclear. We injected cyclin B (CycB) into Drosophila melanogaster embryos during interphase of syncytial cycles and monitored effects on cytoplasmic and nuclear mitotic events. In untreated embryos or embryos arrested in interphase with a protein synthesis inhibitor, injection of CycB accelerates nuclear envelope breakdown and mitotic remodeling of the cytoskeleton. Upon activation of the Grapes(checkpoint kinase 1) (Grp(Chk1))-dependent S-phase checkpoint, increased levels of CycB drives cytoplasmic but not nuclear mitotic events. Grp(Chk1) prevents nuclear CDK1 activation by delaying CycB nuclear accumulation through Wee1-dependent and independent mechanisms

Regulation of Mih1/Cdc25 by protein phosphatase 2A and casein kinase 1

The Cdc25 phosphatase promotes entry into mitosis by removing cyclin-dependent kinase 1 (Cdk1) inhibitory phosphorylation. Previous work suggested that Cdc25 is activated by Cdk1 in a positive feedback loop promoting entry into mitosis; however, it has remained unclear how the feedback loop is initiated. To learn more about the mechanisms that regulate entry into mitosis, we have characterized the function and regulation of Mih1, the budding yeast homologue of Cdc25. We found that Mih1 is hyperphosphorylated early in the cell cycle and is dephosphorylated as cells enter mitosis. Casein kinase 1 is responsible for most of the hyperphosphorylation of Mih1, whereas protein phosphatase 2A associated with Cdc55 dephosphorylates Mih1. Cdk1 appears to directly phosphorylate Mih1 and is required for initiation of Mih1 dephosphorylation as cells enter mitosis. Collectively, these observations suggest that Mih1 regulation is achieved by a balance of opposing kinase and phosphatase activities. Because casein kinase 1 is associated with sites of polar growth, it may regulate Mih1 as part of a signaling mechanism that links successful completion of growth-related events to cell cycle progression

The Septins Function in G1 Pathways that Influence the Pattern of Cell Growth in Budding Yeast

The septins are a conserved family of proteins that have been proposed to carry out diverse functions. In budding yeast, the septins become localized to the site of bud emergence in G1 but have not been thought to carry out important functions at this stage of the cell cycle. We show here that the septins function in redundant mechanisms that are required for formation of the bud neck and for the normal pattern of cell growth early in the cell cycle. The Shs1 septin shows strong genetic interactions with G1 cyclins and is directly phosphorylated by G1 cyclin-dependent kinases, consistent with a role in early cell cycle events. However, Shs1 phosphorylation site mutants do not show genetic interactions with the G1 cyclins or obvious defects early in the cell cycle. Rather, they cause an increased cell size and aberrant cell morphology that are dependent upon inhibitory phosphorylation of Cdk1 at the G2/M transition. Shs1 phosphorylation mutants also show defects in interaction with the Gin4 kinase, which associates with the septins during G2/M and plays a role in regulating inhibitory phosphorylation of Cdk1. Phosphorylation of Shs1 by G1 cyclin-dependent kinases plays a role in events that influence Cdk1 inhibitory phosphorylation

PP2ARts1 is a master regulator of pathways that control cell size

Cell size checkpoints ensure that passage through G1 and mitosis occurs only when sufficient growth has occurred. The mechanisms by which these checkpoints work are largely unknown. PP2A associated with the Rts1 regulatory subunit (PP2ARts1) is required for cell size control in budding yeast, but the relevant targets are unknown. In this paper, we used quantitative proteome-wide mass spectrometry to identify proteins controlled by PP2ARts1. This revealed that PP2ARts1 controls the two key checkpoint pathways thought to regulate the cell cycle in response to cell growth. To investigate the role of PP2ARts1 in these pathways, we focused on the Ace2 transcription factor, which is thought to delay cell cycle entry by repressing transcription of the G1 cyclin CLN3. Diverse experiments suggest that PP2ARts1 promotes cell cycle entry by inhibiting the repressor functions of Ace2. We hypothesize that control of Ace2 by PP2ARts1 plays a role in mechanisms that link G1 cyclin accumulation to cell growth

Electrical characterization of gel collected from shark electrosensors

To investigate the physical mechanism of the electric sense, we present an initial electrical characterization of the glycoprotein gel that fills the electrosensitive organs of marine elasmobranchs ͑sharks, skates, and rays͒. We have collected samples of this gel, postmortem, from three shark species, and removed the majority of dissolved salts in one sample via dialysis. Here we present the results of dc conductivity measurements, low-frequency impedance spectroscopy, and electrophoresis. Electrophoresis shows a range of large proteinbased molecules fitting the expectations of glycoproteins, but the gels of different species exhibit little similarity. The electrophoresis signature is unaffected by thermal cycling and measurement currents. The dc data were collected at various temperatures, and at various electric and magnetic fields, showing consistency with the properties of seawater. The impedance data collected from a dialyzed sample, however, show large values of static permittivity and a loss peak corresponding to an unusually long relaxation time, about 1 ms. The exact role of the gel is still unknown, but our results suggest its bulk properties are well matched to the sensing mechanism, as the minimum response time of an entire electric organ is on the order of 5 ms

Electrical characterization of gel collected from shark electrosensors

To investigate the physical mechanism of the electric sense, we present an initial electrical characterization of the glycoprotein gel that fills the electrosensitive organs of marine elasmobranchs (sharks, skates, and rays). We have collected samples of this gel, postmortem, from three shark species, and removed the majority of dissolved salts in one sample via dialysis. Here we present the results of dc conductivity measurements, low-frequency impedance spectroscopy, and electrophoresis. Electrophoresis shows a range of large protein-based molecules fitting the expectations of glycoproteins, but the gels of different species exhibit little similarity. The electrophoresis signature is unaffected by thermal cycling and measurement currents. The dc data were collected at various temperatures, and at various electric and magnetic fields, showing consistency with the properties of seawater. The impedance data collected from a dialyzed sample, however, show large values of static permittivity and a loss peak corresponding to an unusually long relaxation time, about 1 ms. The exact role of the gel is still unknown, but our results suggest its bulk properties are well matched to the sensing mechanism, as the minimum response time of an entire electric organ is on the order of 5 ms

The dark haloes of early-type galaxies in low-density environments: XMM-Newton and Chandra observations of NGC 57, NGC 7796 and IC 1531

We present analysis of Chandra and XMM-Newton observations of three early-type galaxies, NGC 57, NGC 7796 and IC 1531. All three are found in very low density environments, and appear to have no neighbours of comparable size. NGC 57 has a halo of kT~0.9 keV, solar metallicity gas, while NGC 7796 and IC 1531 both have ~0.55 keV, 0.5-0.6 Zsol haloes. IC 1531 has a relatively compact halo, and we consider it likely that gas has been removed from the system by the effects of AGN heating. For NGC 57 and NGC 7796 we estimate mass, entropy and cooling time profiles and find that NGC 57 has a fairly massive dark halo with a mass-to-light ratio of 44.7 (4.0,-8.5) Msol/Lsol (1 sigma uncertainties) at 4.75 Re. This is very similar to the mass-to-light ratio found for NGC 4555 and confirms that isolated ellipticals can possess sizable dark matter haloes. We find a significantly lower mass-to-light ratio for NGC 7796, 10.6 (+2.5,-2.3) Msol/Lsol at 5 Re, and discuss the possibility that NGC 7796 hosts a galactic wind, causing us to underestimate its mass.Comment: 14 pages, 9 figures, accepted for publication in MNRA