Mitogen activated protein kinases of Aspergillus fumigatus.

In microbial eukaryotes, mitogen activated protein kinase (MAPK) pathways play a pivotal role in regulating cellular physiology. In fungi MAPK pathways have established functions in mating-pheromone responses, maintaining cell wall integrity, responding to changes in osmolarity and nutrient sensing. We have been studying MAPK functions in the human pathogenic fungus Aspergillus fumigatus. The genome of A. fumigatus has four MAPK genes, sakA/hogA, mpkA, mpkB and mpkC. Deletion of the sakA gene produces a strain that does not correctly regulate conidial germination, sense environmental nitrogen or responds to hypertonic stress. The function of the remaining MAPK genes is still under investigation, but by analogy to work in other filamentous fungi, we speculate as to their possible functions in A. fumigatus.

Effect of convection on osteoblastic cell growth and function in biodegradable polymer foam scaffolds.

Culture of seeded osteoblastic cells in three-dimensional osteoconductive scaffolds in vitro is a promising approach to produce an osteoinductive material for repair of bone defects. However, culture of cells in scaffolds sufficiently large to bridge critical-sized defects is a challenge for tissue engineers. Diffusion may not be sufficient to supply nutrients into large scaffolds and consequently cells may grow preferentially at the periphery under static culture conditions. Three alternative culturing schemes that convect media were considered: a spinner flask, a rotary vessel, and a perfusion flow system. Poly(DL-lactic-co-glycolic acid) (PLGA) foam discs (12.7 mm diameter, 6.0 mm thick, 78.8% porous) were seeded with osteoblastic marrow stromal cells and cultured in the presence of dexamethasone and L-ascorbic acid for 7 and 14 days. Cell numbers per foam were found to be similar with all culturing schemes indicating that cell growth could not be enhanced by convection, but histological analysis indicated that the rotary vessel and flow system produced a more uniform distribution of cells throughout the foams. Alkaline phosphatase (ALP) activity per cell was higher with culture in the flow system and spinner flask after 7 days, while no differences in osteocalcin (OC) activity per cell were observed among culturing methods after 14 days in culture. Based on the higher ALP activity and better cell uniformity throughout the cultured foams, the flow system appears to be the superior culturing method, although equally important is the fact that in none of the tests did any of the alternative culturing techniques underperform the static controls. Thus, this study demonstrates that culturing techniques that utilize fluid flow, and in particular the flow perfusion system, improve the properties of the seeded cells over those maintained in static culture.

Regulation of cell cycle progression by Swe1p and Hog1p following hypertonic stress.

Exposure of yeast cells to an increase in external osmolarity induces a temporary growth arrest. Recovery from this stress is mediated by the accumulation of intracellular glycerol and the transcription of several stress response genes. Increased external osmolarity causes a transient accumulation of 1N and 2N cells and a concomitant depletion of S phase cells. Hypertonic stress triggers a cell cycle delay in G2 phase cells that appears distinct from the morphogenesis checkpoint, which operates in early S phase cells. Hypertonic stress causes a decrease in CLB2 mRNA, phosphorylation of Cdc28p, and inhibition of Clb2p-Cdc28p kinase activity, whereas Clb2 protein levels are unaffected. Like the morphogenesis checkpoint, the osmotic stress-induced G2 delay is dependent upon the kinase Swe1p, but is not tightly correlated with inhibition of Clb2p-Cdc28p kinase activity. Thus, deletion of SWE1 does not prevent the hypertonic stress-induced inhibition of Clb2p-Cdc28p kinase activity. Mutation of the Swe1p phosphorylation site on Cdc28p (Y19) does not fully eliminate the Swe1p-dependent cell cycle delay, suggesting that Swe1p may have functions independent of Cdc28p phosphorylation. Conversely, deletion of the mitogen-activated protein kinase HOG1 does prevent Clb2p-Cdc28p inhibition by hypertonic stress, but does not block Cdc28p phosphorylation or alleviate the cell cycle delay. However, Hog1p does contribute to proper nuclear segregation after hypertonic stress in cells that lack Swe1p. These results suggest a hypertonic stress-induced cell cycle delay in G2 phase that is mediated in a novel way by Swe1p in cooperation with Hog1p.

Activation of the Saccharomyces cerevisiae filamentation/invasion pathway by osmotic stress in high-osmolarity glycogen pathway mutants.

Mitogen-activated protein kinase (MAPK) cascades are frequently used signal transduction mechanisms in eukaryotes. Of the five MAPK cascades in Saccharomyces cerevisiae, the high-osmolarity glycerol response (HOG) pathway functions to sense and respond to hypertonic stress. We utilized a partial loss-of-function mutant in the HOG pathway, pbs2-3, in a high-copy suppressor screen to identify proteins that modulate growth on high-osmolarity media. Three high-copy suppressors of pbs2-3 osmosensitivity were identified: MSG5, CAK1, and TRX1. Msg5p is a dual-specificity phosphatase that was previously demonstrated to dephosphorylate MAPKs in yeast. Deletions of the putative MAPK targets of Msg5p revealed that kss1delta could suppress the osmosensitivity of pbs2-3. Kss1p is phosphorylated in response to hyperosmotic shock in a pbs2-3 strain, but not in a wild-type strain nor in a pbs2-3 strain overexpressing MSG5. Both TEC1 and FRE::lacZ expressions are activated in strains lacking a functional HOG pathway during osmotic stress in a filamentation/invasion-pathway-dependent manner. Additionally, the cellular projections formed by a pbs2-3 mutant on high osmolarity are absent in strains lacking KSS1 or STE7. These data suggest that the loss of filamentation/invasion pathway repression contributes to the HOG mutant phenotype.

MAP kinase pathways in the yeast Saccharomyces cerevisiae.

A cascade of three protein kinases known as a mitogen-activated protein kinase (MAPK) cascade is commonly found as part of the signaling pathways in eukaryotic cells. Almost two decades of genetic and biochemical experimentation plus the recently completed DNA sequence of the Saccharomyces cerevisiae genome have revealed just five functionally distinct MAPK cascades in this yeast. Sexual conjugation, cell growth, and adaptation to stress, for example, all require MAPK-mediated cellular responses. A primary function of these cascades appears to be the regulation of gene expression in response to extracellular signals or as part of specific developmental processes. In addition, the MAPK cascades often appear to regulate the cell cycle and vice versa. Despite the success of the gene hunter era in revealing these pathways, there are still many significant gaps in our knowledge of the molecular mechanisms for activation of these cascades and how the cascades regulate cell function. For example, comparison of different yeast signaling pathways reveals a surprising variety of different types of upstream signaling proteins that function to activate a MAPK cascade, yet how the upstream proteins actually activate the cascade remains unclear. We also know that the yeast MAPK pathways regulate each other and interact with other signaling pathways to produce a coordinated pattern of gene expression, but the molecular mechanisms of this cross talk are poorly understood. This review is therefore an attempt to present the current knowledge of MAPK pathways in yeast and some directions for future research in this area.

The osmoregulatory pathway represses mating pathway activity in Saccharomyces cerevisiae: isolation of a FUS3 mutant that is insensitive to the repression mechanism.

Mitogen-activated protein (MAP) kinase cascades are conserved signal transduction pathways that are required for eukaryotic cells to respond to a variety of stimuli. Multiple MAP kinase pathways can function within a single cell type; therefore, mechanisms that insulate one MAP kinase pathway from adventitious activations by parallel pathways may exist. We have studied interactions between the mating pheromone response and the osmoregulatory (high-osmolarity glycerol response [HOG]) pathways in Saccharomyces cerevisiae which utilize the MAP kinases Fus3p and Hog1p, respectively. Inactivating mutations in HOG pathway kinases cause an increase in the phosphotyrosine content of Fus3p, greater expression of pheromone-responsive genes, and increased sensitivity to growth arrest by pheromone. Therefore, the HOG pathway represses mating pathway activity. In a HOG1+ strain, Fus3p phosphotyrosine increases modestly and transiently following an increase in the extracellular osmolarity; however, it increases to a greater extent and for a sustained duration in a hog1-delta strain. Thus, the HOG-mediated repression of mating pathway activity may insulate the mating pathway from activation by osmotic stress. A FUS3 allele whose gene product is resistant to the HOG-mediated repression of its phosphotyrosine content has been isolated. This mutant encodes an amino acid substitution in the highly conserved DPXDEP motif in subdomain XI. Other investigators have shown that the corresponding amino acid is also mutated in a gain-of-function allele of the MAP kinase encoded by the rolled locus in Drosophila melanogaster. These data suggest that the DPXDEP motif plays a role in the negative regulation of MAP kinases.

A second osmosensing signal transduction pathway in yeast. Hypotonic shock activates the PKC1 protein kinase-regulated cell integrity pathway.

Yeast cells respond to hypertonic shock by activation of a (MAP) mitogen-activated protein kinase cascade called the (HOG) high osmolarity glycerol response pathway. How yeast respond to hypotonic shock is unknown. Results of this investigation show that a second MAP kinase cascade in yeast called the protein kinase C1 (PKC1) pathway is activated by hypotonic shock. Tyrosine phosphorylation of the PKC1 pathway MAP kinase increased rapidly in cells following a shift of the external medium to lower osmolarity. The intensity of the response was proportional to the magnitude of the decrease in extracellular osmolarity. This response to hypotonic shock required upstream protein kinases of the PKC1 pathway. Increasing external osmolarity inhibited tyrosine phosphorylation of the PKC1 pathway MAP kinase, a response that was blocked by BCK1-20, a constitutively active mutant in an upstream protein kinase. These results indicate that yeast contain two osmosensing signal transduction pathways, the HOG pathway and the PKC1 pathway, that respond to hypertonic and hypotonic shock, respectively.

The HOG pathway controls osmotic regulation of transcription via the stress response element (STRE) of the Saccharomyces cerevisiae CTT1 gene.

The HOG signal pathway of the yeast Saccharomyces cerevisiae is defined by the PBS2 and HOG1 genes encoding members of the MAP kinase kinase and of the MAP kinase family, respectively. Mutations in this pathway (deletions of PBS2 or HOG1, or point mutations in HOG1) almost completely abolish the induction of transcription by osmotic stress that is mediated by stress response elements (STREs). We have demonstrated previously that STREs also mediate induction of transcription by heat shock, nitrogen starvation and oxidative stress. This study shows that they are also activated by low external pH, sorbate, benzoate or ethanol stress. Induction by these other stress signals appears to be HOG pathway independent. HOG1-dependent osmotic induction of transcription of the CTT1 gene encoding the cytosolic catalase T occurs in the presence of a protein synthesis inhibitor and can be detected rapidly after an increase of tyrosine phosphorylation of Hog1p triggered by high osmolarity. Consistent with a role of STREs in the induction of stress resistance, a number of other stress protein genes (e.g. HSP104) are regulated like CTT1. Furthermore, catalase T was shown to be important for viability under severe osmotic stress, and heat shock was demonstrated to provide cross-protection against osmotic stress.

Positioning of cell growth and division after osmotic stress requires a MAP kinase pathway.

The yeast Saccharomyces cerevisiae has a genetic program for selecting and assembling a bud site on the cell cortex. Yeast cells confine their growth to the emerging bud, a process directed by cortical patches of actin filaments within the bud. We have investigated how cells regulate budding in response to osmotic stress, focusing on the role of the high osmolarity glycerol response (HOG) pathway in mediating this regulation. An increase in external osmolarity induces a growth arrest in which actin filaments are lost from the bud. This is followed by a recovery phase in which actin filaments return to their original locations and growth of the original bud resumes. After recovery from osmotic stress, haploid cells retain an axial pattern of bud site selection while diploids change their bipolar budding pattern to an increased bias for forming a bud on the opposite side of the cell from the previous bud site. Mutants lacking the mitogen-activated protein (MAP) kinase encoded by HOG1 or the MAP kinase kinase encoded by PBS2 (previously HOG4) show a similar growth arrest after osmotic stress. However, in the recovery phase, the mutant cells (a) do not restart growth of the original bud but rather start a new bud, (b) fail to restore actin filaments to the original bud but move them to the new one, and (c) show a more random budding pattern. These defects are elicited by an increase in osmolarity and not by other environmental stresses (e.g., heat shock or change in carbon source) that also cause a temporary growth arrest and shift in actin distribution. Thus, the HOG pathway is required for repositioning of the actin cytoskeleton and the normal spatial patterns of cell growth after recovery from osmotic stress.

An osmosensing signal transduction pathway in yeast.

Yeast genes were isolated that are required for restoring the osmotic gradient across the cell membrane in response to increased external osmolarity. Two of these genes, HOG1 and PBS2, encode members of the mitogen-activated protein kinase (MAP kinase) and MAP kinase kinase gene families, respectively. MAP kinases are activated by extracellular ligands such as growth factors and function as intermediate kinases in protein phosphorylation cascades. A rapid, PBS2-dependent tyrosine phosphorylation of HOG1 protein occurred in response to increases in extracellular osmolarity. These data define a signal transduction pathway that is activated by changes in the osmolarity of the extracellular environment.

Osmotic stress and the yeast cytoskeleton: phenotype-specific suppression of an actin mutation.

In the yeast Saccharomyces cerevisiae, actin filaments function to direct cell growth to the emerging bud. Yeast has a single essential actin gene, ACT1. Diploid cells containing a single copy of ACT1 are osmosensitive (Osms), i.e., they fail to grow in high osmolarity media (D. Shortle, unpublished observations cited by Novick, P., and D. Botstein. 1985. Cell. 40:415-426). This phenotype suggests that an underlying physiological process involving actin is osmosensitive. Here, we demonstrate that this physiological process is a rapid and reversible change in actin filament organization in cells exposed to osmotic stress. Filamentous actin was stained using rhodamine phalloidin. Increasing external osmolarity caused a rapid loss of actin filament cables, followed by a slower redistribution of cortical actin filament patches. In the recovery phase, cables and patches were restored to their original levels and locations. Strains containing an act1-1 mutation are both Osms and temperature-sensitive (Ts) (Novick and Botstein, 1985). To identify genes whose products functionally interact with actin in cellular responses to osmotic stress, we have isolated extragenic suppressors which revert only the Osms but not the Ts phenotype of an act1-1 mutant. These suppressors identify three genes, RAH1-RAH3. Morphological and genetic properties of a dominant suppressor mutation suggest that the product of the wild-type allele, RAH3+, is an actin-binding protein that interacts with actin to allow reassembly of the cytoskeleton following osmotic stress.

A mechanosensitive ion channel in the yeast plasma membrane.

Mechanosensitive ion channels use mechanical energy to gate the dissipation of electrochemical gradients across cell membranes. This function is fundamental to physiological processes such as hearing and touch. In electrophysiological studies of ion channels in the plasma membrane of the yeast Saccharomyces cerevisiae, channels were observed that were activated by, and adapted to, stretching of the membrane. Adaptation of channel activity to mechanical stimuli was voltage-dependent. Because these mechanosensitive channels pass both cations and anions, they may play a role in turgor regulation in this walled organism.

Regulation of ciliary adenylate cyclase by Ca2+ in Paramecium.

In the ciliated protozoan Paramecium, Ca2+ and cyclic nucleotides are believed to act as second messengers in the regulation of the ciliary beat. Ciliary adenylate cyclase was activated 20-30-fold (half-maximal at 0.8 microM) and inhibited by higher concentrations (10-20 microM) of free Ca2+ ion. Ca2+ activation was the result of an increase in Vmax., not a change in Km for ATP. The activation by Ca2+ was seen only with Mg2+ATP as substrate; with Mn2+ATP the basal adenylate cyclase activity was 10-20-fold above that with Mg2+ATP, and there was no further activation by Ca2+. The stimulation by Ca2+ of the enzyme in cilia and ciliary membranes was blocked by the calmodulin antagonists calmidazolium (half-inhibition at 5 microM), trifluoperazine (70 microM) and W-7 (50-100 microM). When ciliary membranes (which contained most of the ciliary adenylate cyclase) were prepared in the presence of Ca2+, their adenylate cyclase was insensitive to Ca2+ in the assay. However, the inclusion of EGTA in buffers used for fractionation of cilia resulted in full retention of Ca2+-sensitivity by the ciliary membrane adenylate cyclase. The membrane-active agent saponin specifically suppressed the Ca2+-dependent adenylate cyclase without inhibiting basal activity with Mg2+ATP or Mn2+ATP. The ciliary adenylate cyclase was shown to be distinct from the Ca2+-dependent guanylate cyclase; the two activities had different kinetic parameters and different responses to added calmodulin and calmodulin antagonists. Our results suggest that Ca2+ influx through the voltage-sensitive Ca2+ channels in the ciliary membrane may influence intraciliary cyclic AMP concentrations by regulating adenylate cyclase.

Ion channels in yeast.

Voltage-dependent ion channels have been found in the plasma membrane of the yeast Saccharomyces cerevisiae. Ion channel activities were recorded from spheroplasts or patches of plasma membrane with the patch-clamp technique. The most prominent activities came from a set of potassium channels with the properties of activation by positive but not negative voltages, high selectivity for potassium over sodium ion, unit conductance of 20 picosiemens, inhibition by tetraethylammonium or barium ions, and bursting kinetics.

Regulation of ciliary motility by membrane potential in Paramecium: a role for cyclic AMP.

The membrane potential of Paramecium controls the frequency and direction of the ciliary beat, thus determining the cell's swimming behavior. Stimuli that hyperpolarize the membrane potential increase the ciliary beat frequency and therefore increase forward swimming speed. We have observed that 1) drugs that elevate intracellular cyclic AMP increased swimming speed 2-3-fold, 2) hyperpolarizing the membrane potential by manipulation of extracellular cations (e.g., K+) induced both a transient increase in, and a higher sustained level of cyclic AMP compared to the control, and 3) the swimming speed of detergent-permeabilized cells in MgATP was stimulated 2-fold by the addition of cyclic AMP. Our results suggest that the membrane potential can regulate intracellular cAMP in Paramecium and that control of swimming speed by membrane potential may in part be mediated by cAMP.

Characterization of the phosphorylated intermediate of the K+-ouabain-insensitive ATPase of the rabbit colon brush-border membrane.

The rabbit colon brush-border membrane possesses a unique K+-stimulated, ouabain-insensitive ATPase. This enzyme is similar to previously described potassium-transporting enzymes such as the ubiquitous (Na + K)-ATPase and the gastric (H+ + K+)-ATPase in forming a phosphorylated intermediate whose rate of dephosphorylation is accelerated by K+. The molecular weight of the phosphorylated polypeptide subunit of the colon membrane enzyme is 114,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The release of 32Pi from the 32P-labeled ATPase at alkaline pH and in the presence of hydroxylamine at pH 5.0 suggests that the phosphorylated enzyme intermediate is an acyl phosphate. These results indicate that the phosphorylated intermediate of the colon brush-border membrane is similar in chemical nature and molecular weight to the phosphorylated intermediates of other cation-transporting ATPases.