Parameters for cellular viability and membrane function in chenopodium cells show a specific response of extracellular pH to heat shock with extreme Q10

The effect of brief heat shock on Chenopodium cells was investigated by measuring biochemical parameters for cellular vitality, membrane function and integrity: extracellular pH, release of osmotic compounds, phosphatase, protein and betalain, and cellular reduction of DCPIP and MTT. A threshold temperature was found at 45 degrees C, where release of osmotic compounds, protein and betalain, and reduction of DCPIP and MTT indicate loss of vitality. Extracellular pH and an alkaline phosphatase responded 10-20 degrees C below this threshold, suggesting that extracellular alkalinization, and probably the release of a phosphatase, are part of a specific cellular response to abiotic stress induced by heat shock. The extracellular proton concentration did not increase above 45 degrees C: this may indicate equilibration of gradients driving this process or an inactivation of cellular mechanisms responsible for extracellular alkalinization. The response of extracellular pH to heat shock in Chenopodium cell suspensions was fast, i.e., up to +1 pH in 5 min. Addition of the K+/H+ antiporter nigericin to Chenopodium cells caused an extracellular alkalinization similar to heat shock. The heat shock-induced extracellular alkalinization was characterized by Q10 values for distinct ranges of temperature (Q10 of 56 for 24-31 degrees C, 2.3 for 31-42 degrees C, and 1.0 for 42-50 degrees C). To the author's knowledge, the Q10 of 56 is the highest found up to now. These results suggest that extracellular protons are involved in temperature sensing and signalling in plant cells, probably via a channel-mediated pathway.     

The absence of direct relationship between the ability of yeasts to grow at elevated temperatures and their survival after lethal heat shock

The study of the growth of the yeasts Rhodotorula rubra, Saccharomyces cerevisiae, and Debaryomyces vanriji at elevated temperatures and their survival after transient lethal heat shock showed that the ability of these yeasts to grow at supraoptimal temperatures (i.e., their thermoresistance) and their ability to tolerate lethal heat shocks (i.e., their thermotolerance) are determined by different mechanisms. The thermotolerance of the yeasts is suggested to be mainly determined by the division rate of cells before their exposure to heat shock.     

Physiological responses of Escherichia coli exposed to different heat-stress kinetics

The effects of heat-stress kinetics on the viability of Escherichia coli were investigated. Cells were exposed to heat-stress treatments extending from 30 to 50°C, with either a slope (40 min) or a shock (10 s), both followed by a 1-h plateau at 50°C in nutritive medium. A higher survival rate was observed after the slope than after the shock, when both were followed by a plateau, so the heat slope induced a certain degree of thermotolerance. This tolerance was partly (i) linked to de novo protein synthesis during the subsequent plateau phase, and (ii) abolished after rapid cooling from 50 to 30°C, which means that cellular components with rapidly reversible thermal properties are involved in this type of thermotolerance. The heat-slope-induced thermotolerance was chiefly linked to the maintenance of the plasma membrane integrity (preservation of structure, fluidity, and permeability), and not to GroEL or DnaK overexpression. Moreover, the high level of cell mortality induced by the heat shock could be related to changes in the membrane integrity.     

Inactivation of Escherichia coli and Lactobacillus plantarum in relation to membrane permeabilization due to rapid chilling followed by cold storage

The relationship between membrane permeabilization and loss of viability by chilling depending on the chilling rate was investigated in two bacterial models: one Gram-positive bacterium, Lactobacillus plantarum, and one Gram-negative bacterium, Escherichia coli. Cells were cold shocked slowly (2°C/min) or rapidly (2,000°C/min) from physiological temperature to 0°C and maintained at this temperature for up to 1 week. Loss of membrane integrity was assessed by the uptake of the fluorescent dye propidium iodide (PI). Cell death was found to be strongly dependent on the rate of temperature downshift to 0°C. Prolonged incubation of cells after the chilling emphasized the effect of treatment on the cells, as the amount of cell death increased with the length of exposure to low temperature, particularly when cells were rapidly chilled. More than 5 and 3-log reductions in cell population were obtained with L. plantarum and E. coli after the rapid cold shock followed by 7-day storage, respectively. A correlation between cell inactivation and membrane permeabilization was demonstrated with both bacterial strains. Thus, loss of membrane integrity due to the chilling treatments was directly involved in the inactivation of vegetative bacterial cells.     

Necrosis: a specific form of programmed cell death?

For a long time necrosis was considered as an alternative to programmed cell death, apoptosis. Indeed, necrosis has distinct morphological features and it is accompanied by rapid permeabilization of plasma membrane. However, recent data indicate that, in contrast to necrosis caused by very extreme conditions, there are many examples when this form of cell death may be a normal physiological and regulated (programmed) event. Various stimuli (e.g., cytokines, ischemia, heat, irradiation, pathogens) can cause both apoptosis and necrosis in the same cell population. Furthermore, signaling pathways, such as death receptors, kinase cascades, and mitochondria, participate in both processes, and by modulating these pathways, it is possible to switch between apoptosis and necrosis. Moreover, antiapoptotic mechanisms (e.g., Bcl-2/Bcl-x proteins, heat shock proteins) are equally effective in protection against apoptosis and necrosis. Therefore, necrosis, along with apoptosis, appears to be a specific form of execution phase of programmed cell death, and there are several examples of necrosis during embryogenesis, a normal tissue renewal, and immune response. However, the consequences of necrotic and apoptotic cell death for a whole organism are quite different. In the case of necrosis, cytosolic constituents that spill into extracellular space through damaged plasma membrane may provoke inflammatory response; during apoptosis these products are safely isolated by membranes and then are consumed by macrophages. The inflammatory response caused by necrosis, however, may have obvious adaptive significance (i.e., emergence of a strong immune response) under some pathological conditions (such as cancer and infection). On the other hand, disturbance of a fine balance between necrosis and apoptosis may be a key element in development of some diseases.     

Heat shock induces apoptosis independently of any known initiator caspase-activating complex

Adaptive responses to mild heat shock are among the most widely conserved and studied in nature. More intense heat shock, however, induces apoptosis through mechanisms that remain largely unknown. Herein, we present evidence that heat shock activates an apical protease that stimulates mitochondrial outer membrane permeabilization and processing of the effector caspase-3 in a benzyloxycarbonyl-VAD-fluoromethyl ketone (polycaspase inhibitor)- and Bcl-2-inhibitable manner. Surprisingly, however, neither FADD.caspase-8 nor RAIDD.caspase-2 PIDDosome (p53-induced protein with a death domain) complexes were detected in dying cells, and neither of these initiator caspases nor the endoplasmic reticulum stress-activated caspases-4/12 were required for mitochondrial outer membrane permeabilization. Similarly, although cytochrome c was released from mitochondria following heat shock, functional Apaf-1.caspase-9 apoptosome complexes were not formed, and caspase-9 was not essential for the activation of caspase-3 or the induction of apoptosis. Thus, heat shock does not require any of the known initiator caspases or their activating complexes to promote apoptotic cell death but instead relies upon the activation of an apparently novel apical protease with caspase-like activity.     

Specificity of commercial anti-spectrin antibody in the study of fungal and Oomycete spectrin: cross-reaction with proteins other than spectrin

Spectrin was first described in erythrocytes where it forms a filamentous network in the cytoplasmic face of the plasma membrane and participates in the membrane's structural integrity in addition to controlling the lateral mobility of integral membrane proteins. In fungi, spectrin-like proteins have been described in the plasma membrane, concentrated mainly in the region of maximum apical expansion. This localization led to the idea of a spectrin based membrane skeleton in fungi participating in mechanical integrity of the plasma membrane, generating and maintaining cell polarity. The occurrence of spectrin-like proteins in filamentous fungi, yeasts and Oomycetes, however, is questionable since the presence of such proteins has only been demonstrated with immunochemical methods using antibodies whose specificity is unclear. There is no evidence of a gene coding for the high molecular weight alphabeta-spectrin in the genome of these organisms. Mass spectrometric analysis of the anti alphabeta-spectrin immunoreacting peptides from Neurospora crassa and Phytophthora infestans identified them as elongation factor 2 (NCU07700.4) and Hsp70 (PITG_13237.1), respectively. An attempt was made to correlate the reactivity of anti-spectrin antibody to a common feature of these three proteins i.e., spectrin, elongation factor 2 and heat shock protein 70, in that they all have a hydrophobic region implicated in chaperon activity.     

The Absence of a Direct Relationship between the Ability of Yeasts to Grow at Elevated Temperatures and Their Survival after Lethal Heat Shock

The study of the growth of the yeasts Rhodotorula rubra, Saccharomyces cerevisiae, and Debaryomyces vanriji at elevated temperatures and their survival after transient lethal heat shock showed that the ability of these yeasts to grow at supraoptimal temperatures (i.e., their thermoresistance) and their ability to tolerate lethal heat shocks (i.e., their thermotolerance) are determined by different mechanisms. It is suggested that the thermotolerance of the yeasts is mainly determined by the division rate of cells before their exposure to heat shock.     

Heat shock-induced apoptosis in preimplantation bovine embryos is a developmentally regulated phenomenon

Apoptosis is a form of cell death that can function to eliminate cells damaged by environmental stress. One stress that can compromise embryonic development is elevated temperature (i.e., heat shock). For the current studies, we hypothesized that heat shock induces apoptosis in bovine embryos in a developmentally regulated manner. Studies were performed to 1) determine whether heat shock can induce apoptosis in preimplantation embryos, 2) test whether heat-induced apoptosis is developmentally regulated, 3) evaluate whether heat shock-induced changes in caspase activity parallel patterns of apoptosis, and 4) ascertain whether exposure to a mild heat shock can protect embryos from heat-induced apoptosis. As determined by TUNEL reaction, exposure of bovine embryos > or =16 cells on Day 5 after insemination to 41 or 42 degrees C for 9 h increased the percentage of cells undergoing apoptosis. In addition, there was a duration-dependent increase in the proportion of blastomeres that were apoptotic when embryos were exposed to temperatures of 40 or 41 degrees C, which are more characteristic of temperatures experienced by heat-stressed cows. Heat shock also increased caspase activity in Day 5 embryos. However, heat shock did not induce apoptosis in 2- or 4-cell embryos, nor did it increase caspase activity in 2-cell embryos. The apoptotic response of 8- to 16-cell-stage bovine embryos to heat shock depended upon the day after insemination that heat shock occurred. When 8- to 16-cell embryos were collected on Day 3 after insemination, heat shock of 41 degrees C for 9 h did not induce apoptosis. In contrast, when 8- to 16-cell embryos were collected on Day 4 after insemination and exposed to heat shock, there was an increase in the percentage of cells undergoing apoptosis. Exposure of 8- to 16-cell embryos at Day 4 to a mild heat shock of 40 degrees C for 80 min blocked the apoptotic response to a subsequent, more-severe heat shock of 41 degrees C for 9 h. In conclusion, apoptosis is a developmentally acquired phenomenon that occurs in embryos exposed to elevated temperature, and it can be prevented by induced thermotolerance.     

The effects of low temperature on fertilized rabbit ova in vitro,and the normal development of ova kept at low temperature for several days

Fertilized rabbit ova at the 2-blastomere stage kept in rabbit serum were stored at low temperatures for various lengths of time. They were then cultured at 38 degrees C. for about 24 hours to determine their viability. A number of the viable ova were finally transplanted into recipient does. It was found that rapid cooling of ova to 5 degrees or to 0 degrees C. was more harmful to the subsequent viability of ova than slow cooling. Rapid cooling was not more lethal to the ova than slow cooling, but did prevent their future normal cleavage. There was no difference between those ova cooled rapidly or slowly to 10 degrees C. It was concluded that temperature shock has an adverse effect on ova, especially at the lower temperatures, though temperature shock can be remedied by acclimatization (slow cooling). Thus, the physiological significance of temperature shock would seem to be broadened. The optimal temperature for the storage of ova was investigated. It was found that 10 degrees C. was the best temperature; at this temperature viable ova were obtained after storage for 144 to 168 hours. At 0 degrees , 5 degrees , or 15 degrees C. the ova were viable for 96 to 120 hours, while at 22-24 degrees C., only for 24 to 48 hours. The percentage of dead ova was low at a favorable temperature, increasing only at the end of the storage period. At an unfavorable temperature, however, the rate of death increased steadily from beginning to end of storage. The percentage of abnormally cleaved ova (arrested cleavage and fragmentation) remained at a low level at first at a favorable temperature, but then increased just before or during death of the ova. A critical time for the viability, the abnormal cleavage, and the death of ova was characteristic of each temperature. About 24 to 28 per cent of the viable ova remaining after being stored at 0-15 degrees C. for 2 to 4 days and cultured at 38 degrees C. for 24 hours were capable of development into normal young. The compatibility of serum and ova, the absence of a correlation between the viability of the ova and the source of the fertilizing spermatozoa, and the fertilization of superovulated ova (i.e., the percentage of fertile does in follicular phase and in luteal phase, the percentage of unfertilized ova and of fertilized ova at different stages, the percentage of does that had produced a normal number of ova or had produced a large number of ova, etc.), are reported. The possibility of a more efficient utilization of the germ cells of valuable animals by means of the present techniques, and the possibility of a new approach to the experimental investigation of mammalian genetics and development, have been mentioned.     

Modulation of stability of the Escherichia coli heat shock regulatory factor sigma.

The heat shock response of Escherichia coli is under the positive control of the sigma 32 protein (the product of the rpoH gene). We found that overproduction of the sigma 32 protein led to concomitant overproduction of the heat shock proteins, suggesting that the intracellular sigma 32 levels limit heat shock gene expression. In support of this idea, the intracellular half-life of the sigma 32 protein synthesized from a multicopy plasmid was found to be extremely short, e.g., less than 1 min at 37 and 42 degrees C. The half-life increased progressively with a decrease in temperature, reaching 15 min at 22 degrees C. Finally, conditions known previously to increase the rate of synthesis of the heat shock proteins, i.e., a mutation in the dnaK gene or expression of phage lambda early proteins, were shown to simultaneously result in a three- to fivefold increase in the half-life of sigma 32.     

Cold shock response of yeast cells: induction of a 33 kDa protein and protection against freezing injury.

Cold shock (10 degrees C) treatment to Saccharomyces cerevisiae cells normally grown at 30 degrees C resulted in splitting of vacuoles and retarded membrane fluidity as detected by phase contrast microscopy and in vivo nuclear magnetic resonance (NMR) studies, respectively. The treatment was found to impart protection against subsequent freezing as studied by cell viability and colony forming efficiency. We have earlier reported similar protection and retarded membrane fluidity as a result of heat shock treatment to these cells (Obuchi et al., 1990). This suggests that cold shock and heat shock treatments to yeast cells evoke some analogous responses. However, biochemically a new 33 kDa protein (CSP 33) was detected upon cold shock treatment which is distinct from heat shock induced family of proteins (Kaul et al., 1992). We present here the first report of this kind and its practical implications for protection against freezing.     

Effect of ionophore A23187 on the membrane permeability in mouse fibroblasts.

Addition of the divalent cation ionophore A23187 to transformed mouse fibroblasts (3T6) resulted in an increase in the cell membrane permeability to normally impermeant solutes (e.g., nucleotides). The membrane permeability was assessed by following the efflux of prelabeled adenine nucleotides, the influx of p-nitrophenyl phosphate in cells attached to plastic dishes and reconstitution of intracellular protein synthesis in the presence of exogenously added normally impermeant factors required for macromolecular synthesis. The permeability change of 3T6 cells was found to be dependent on the specific presence of external calcium ion. The permeabilization was found to occur preferably in alkaline pH and specific to certain transformed cells. It is preceded by rapid efflux of K+, influx of Na+ and partial hydrolysis of cellular nucleotides in 3T6 cells. Similar ion fluxes were previously found to precede cell permeabilization by electrogenic ionophores for monovalent ions and by exogenous ATP. Our data suggest that a calcium dependent process caused the K+ release and excess Na+ entry, causing dissipation of the membrane potential and subsequent formation of aqueous channels.     

Increased thermostability of thermotolerant CHL V79 cells as determined by differential scanning calorimetry

Heat shock denatures cellular protein and induces both a state of acquired thermotolerance, defined as resistance to a subsequent heat shock, and the synthesis of a category of proteins referred to as heat-shock proteins (HSPs). Thermotolerance may be due to the stabilization of thermolabile proteins that would ordinarily denature during heat shock, either by HSPs or some other factors. We show by differential scanning calorimetry (DSC) that mild heat shock irreversibly denatures a small fraction of Chinese hamster lung V79-WNRE cell protein (i.e., the enthalpy change, which is proportional to denaturation, on scanning to 45 degrees C at 1 degree C/min is approximately 2.3% of the total calorimetric enthalpy). Thermostability, defined by the extent of denaturation during heat shock and determined from DSC scans of whole cells, increases as the V79 cells become thermotolerant. Cellular stabilization appears to be due to an increase in the denaturation temperature of the most thermolabile proteins; there is no increase in the denaturation temperatures of the most thermally resistant proteins, i.e., those denaturing above 65 degrees C. Cellular stabilization is also observed in the presence of glycerol, which is known to increase resistance to heat shock and to stabilize proteins in vitro. A model is presented, based on a direct relationship between the extent of hyperthermic killing and the denaturation or inactivation of a critical target that defines the rate-limiting step in killing, which predicts a transition temperature (Tm) of the critical target for control V79-WNRE cells of 46.0 degrees C and a Tm of 47.3 degrees C for thermotolerant cells. This shift of 1.3 degrees C is consistent with the degree of stabilization detected by DSC.     

Insulin-like growth factor-I as a survival factor for the bovine preimplantation embryo exposed to heat shock

Insulin-like growth factor-I (IGF-I) is a survival factor for preimplantation mammalian embryos exposed to stress. One stress that compromises preimplantation embryonic development is elevated temperature (i.e., heat shock). Using bovine embryos produced in vitro as a model, it was hypothesized that IGF-I would protect preimplantation embryos by reducing the effects of heat shock on total cell number, the proportion of blastomeres that undergo apoptosis, and the percentage of embryos developing to the blastocyst stage. In experiment 1, embryos were cultured with or without IGF-I; on Day 5 after insemination, embryos >or=16 cells were cultured at 38.5 degrees C for 24 h or were subjected to 41 degrees C for 9 h followed by 38.5 degrees C for 15 h. Heat shock reduced the total cell number at 24 h after initiation of heat shock and increased the percentage of blastomeres that were apoptotic. Effects of heat shock were less for IGF-I-treated embryos. Experiment 2 was conducted similarly except that embryos were allowed to develop to Day 8 after insemination. The percentage reduction in blastocyst development for heat-shocked embryos compared with those maintained at 38.5 degrees C was less for embryos cultured with IGF-I than for control embryos. Heat shock reduced the total cell number in blastocysts and increased the percentage of blastomeres that were apoptotic, whereas IGF-I-treated embryos had increased total cell number and a reduced percentage of apoptosis. Taken together, these results demonstrate that IGF-I can serve as a survival factor for preimplantation bovine embryos exposed to heat shock by reducing the effects of heat shock on development and apoptosis.     

Transport of influenza HA from the trans-Golgi network to the apical surface of MDCK cells permeabilized in their basolateral plasma membranes: energy dependence and involvement of GTP-binding proteins

A procedure employing streptolysin O to effect the selective permeabilization of either the apical or basolateral plasma membrane domains of MDCK cell monolayers grown on a filter support was developed which permeabilizes the entire monolayer, leaves the opposite cell surface domain intact, and does not abolish the integrity of the tight junctions. This procedure renders the cell interior accessible to exogenous macromolecules and impermeant reagents, permitting the examination of their effects on membrane protein transport to the intact surface. The last stages of the transport of the influenza virus hemagglutinin (HA) to the apical surface were studied in pulse-labeled, virus-infected MDCK cells that were incubated at 19.5 degrees C for 90 min to accumulate newly synthesized HA in the trans-Golgi network (TGN), before raising the temperature to 35 degrees C to allow synchronized transport to the plasma membrane. In cells permeabilized immediately after the cold block, 50% of the intracellular HA molecules were subsequently delivered to the apical surface. This transport was dependent on the presence of an exogenous ATP supply and was markedly inhibited by the addition of GTP-gamma-S at the time of permeabilization. On the other hand, the GTP analogue had no effect when it was added to cells that, after the cold block, were incubated for 15 min at 35 degrees C before permeabilization, even though at this time most HA molecules were still intracellular and their appearance at the cell surface was largely dependent on exogenous ATP. These findings indicate that GTP-binding proteins are involved in the constitutive process that effects vesicular transport from the TGN to the plasma membrane and that they are charged early in this process. Transport of HA to the cell surface could be made dependent on the addition of exogenous cytosol when, after permeabilization, cells were washed to remove endogenous cytosolic components. This opens the way towards the identification of cell components that mediate the sorting of apical and basolateral membrane components in the TGN and their polarized delivery to the cell surface.     

Heat-shock proteins in membrane vesicles of Bacillus subtilis

Fractionation of B. subtilis cells after heat shock, from 37 degrees C to 54 degrees C, shows an increase in synthesis of proteins localized in cell membranes and a decrease in synthesis of proteins localized in cytosol. There is no such effect of heat shock at temperature of 45 degrees C. Autoradiograms of electrophoretically separated proteins, labelled during heat shock at 54 degrees C, reveal 26 heat-shock proteins (hsps) in membrane vesicles and 11 hsps in cytosol, five of which are common to both fractions. Heat shock at 45 degrees C induces 18 hsps localized in membrane vesicles and 13 hsps localized in cytosol, six of which are common to both fractions. Results are interpreted as showing a relevant role of membrane proteins in cell response to shock at high temperature, pointing to two steps of defense against heat stress.     

Anthrax Lethal Toxin Induced Lysosomal Membrane Permeabilization and Cytosolic Cathepsin Release Is Nlrp1b/Nalp1b-Dependent

NOD-like receptors (NLRs) are a group of cytoplasmic molecules that recognize microbial invasion or ‘danger signals’. Activation of NLRs can induce rapid caspase-1 dependent cell death termed pyroptosis, or a caspase-1 independent cell death termed pyronecrosis. Bacillus anthracis lethal toxin (LT), is recognized by a subset of alleles of the NLR protein Nlrp1b, resulting in pyroptotic cell death of macrophages and dendritic cells. Here we show that LT induces lysosomal membrane permeabilization (LMP). The presentation of LMP requires expression of an LT-responsive allele of Nlrp1b, and is blocked by proteasome inhibitors and heat shock, both of which prevent LT-mediated pyroptosis. Further the lysosomal protease cathepsin B is released into the cell cytosol and cathepsin inhibitors block LT-mediated cell death. These data reveal a role for lysosomal membrane permeabilization in the cellular response to bacterial pathogens and demonstrate a shared requirement for cytosolic relocalization of cathepsins in pyroptosis and pyronecrosis.     

Hyperthermia-induced heat shock response and thermotolerance in postimplantation rat embryos

Postimplantation stage rat embryos (6-10 somites) undergo abnormal development after exposure to a temperature of 43 degrees C for 30 min. A heat shock of 43 degrees C for 30 min also induces the synthesis of a set of eight heat shock proteins (hsps) with molecular masses ranging from 28,000 to 82,000 Da. The synthesis of these hsps is rapidly induced after the heat shock is applied and rapidly decays after embryos are returned to 37 degrees C. A heat shock of 42 degrees C for 30 min has no effect on rat embryo growth and development, but does induce the synthesis of three hsps. The most prominent of these three is believed to be the typical mammalian 70 kDa hsp. Furthermore, a 42 degrees C, 30-min heat shock followed by a 43 degrees C 30-min heat shock leads to partial protection from the embryotoxic effects of a single exposure at 43 degrees C, i.e., thermotolerance.     

Mid2 Is a Putative Sensor for Cell Integrity Signaling in Saccharomyces cerevisiae

Hcs77 is a putative cell surface sensor for cell integrity signaling in Saccharomyces cerevisiae. Its loss of function results in cell lysis during growth at elevated temperatures (e.g., 39 degrees C) and impaired signaling to the Mpk1 mitogen-activated protein kinase in response to mild heat shock. We isolated the MID2 gene as a dosage suppressor of the cell lysis defect of an hcs77 null mutant. MID2 encodes a putative membrane protein whose function is required for survival of pheromone treatment. Mid2 possesses properties similar to those of Hcs77, including a single transmembrane domain and a long region that is rich in seryl and threonyl residues. We demonstrate that Mid2 is required for cell integrity signaling in response to pheromone. Additionally, we show that Mid2 and Hcs77 serve a redundant but essential function as cell surface sensors for cell integrity signaling during vegetative growth. Both proteins are uniformly distributed through the plasma membrane and are highly O-mannosylated on their extracellular domains. Finally, we identified a yeast homolog of MID2, designated MTL1, which provides a partially redundant function with MID2 for cell integrity signaling during vegetative growth at elevated temperature but not for survival of pheromone treatment. We conclude that Hcs77 is dedicated to signaling cell wall stress during vegetative growth and that Mid2 participates in this signaling, but its primary role is in signaling wall stress during pheromone-induced morphogenesis.