Development and Validation of a Highly Sensitive Multiplex Immunoassay for SARS-CoV-2 Humoral Response Monitorization: A Study of the Antibody Response in COVID-19 Patients with Different Clinical Profiles during the First and Second Waves in Cadiz, Spain.

There is still a long way ahead regarding the COVID-19 pandemic, since emerging waves remain a daunting challenge to the healthcare system. For this reason, the development of new preventive tools and therapeutic strategies to deal with the disease have been necessary, among which serological assays have played a key role in the control of COVID-19 outbreaks and vaccine development. Here, we have developed and evaluated an immunoassay capable of simultaneously detecting multiple IgG antibodies against different SARS-CoV-2 antigens through the use of Bio-PlexTM technology. Additionally, we have analyzed the antibody response in COVID-19 patients with different clinical profiles in Cadiz, Spain. The multiplex immunoassay presented is a high-throughput and robust immune response monitoring tool capable of concurrently detecting anti-S1, anti-NC and anti-RBD IgG antibodies in serum with a very high sensitivity (94.34-97.96%) and specificity (91.84-100%). Therefore, the immunoassay proposed herein may be a useful monitoring tool for individual humoral immunity against SARS-CoV-2, as well as for epidemiological surveillance. In addition, we show the values of antibodies against multiple SARS-CoV-2 antigens and their correlation with the different clinical profiles of unvaccinated COVID-19 patients in Cadiz, Spain, during the first and second waves of the pandemic.

Divergence of cytokinesis and dimorphism control by myosin II regulatory light chain in fission yeasts.

Non-muscle myosin II activation by regulatory light chain (Rlc1Sp) phosphorylation at Ser35 is crucial for cytokinesis during respiration in the fission yeast Schizosaccharomyces pombe. We show that in the early divergent and dimorphic fission yeast S. japonicus non-phosphorylated Rlc1Sj regulates the activity of Myo2Sj and Myp2Sj heavy chains during cytokinesis. Intriguingly, Rlc1Sj-Myo2Sj nodes delay yeast to hyphae onset but are essential for mycelial development. Structure-function analysis revealed that phosphorylation-induced folding of Rlc1Sp α1 helix into an open conformation allows precise regulation of Myo2Sp during cytokinesis. Consistently, inclusion of bulky tryptophan residues in the adjacent α5 helix triggered Rlc1Sp shift and supported cytokinesis in absence of Ser35 phosphorylation. Remarkably, unphosphorylated Rlc1Sj lacking the α1 helix was competent to regulate S. pombe cytokinesis during respiration. Hence, early diversification resulted in two efficient phosphorylation-independent and -dependent modes of Rlc1 regulation of myosin II activity in fission yeasts, the latter being conserved through evolution.

Asthma control in children, socioeconomic inequality and health care.

INTRODUCTION: Socioeconomic inequality (SEI) can adversely affect asthma control. The aim of this study was to establish the association of SEI with asthma control in children and caregiver quality of life. METHODS: We assessed socioeconomic status based on the area of residence, according to the at risk of poverty rate (ARPR). After stratifying the paediatric population of Castilla y León (Spain) in ARPR tertiles, we selected participants by stratified random sampling, and identified children with asthma aged 6-14 years from the health records of primary care centres. We collected data through questionnaires completed by parents. The primary outcomes were asthma control and caregiver quality of life. We assessed their association with SEI, health care quality measures and individual factors (such as parental educational attainment) by means of multivariate regression models. RESULT: The ARPR tertile was not associated with asthma control, quality of life or health care quality. A medium or high maternal educational attainment was associated with a lower risk of making an unscheduled or urgent visit (OR = .50; 95% CI, .27-.95; P = .034) and paternal educational attainment was associated with a lower risk of uncontrolled asthma (OR = 0.51; 95% CI, .28-.94; P = .030). CONCLUSION: In the sample under study, SEI assessed at the local level was not associated with asthma control in children. Other factors, such as parental educational attainment, may have a protective effect.

Maternal Psychopathological Profile during Childbirth and Neonatal Development during the COVID-19 Pandemic: A Pre-Posttest Study.

The coronavirus SARS-CoV-2 generated an alert that became a state of emergency in health issues worldwide, a situation that affected the entire population, including pregnant women. The present study aims to understand the effect of the psychopathological profile of a sample of pregnant women at the time of the COVID-19 pandemic on themselves during childbirth (Phase 1) and after childbirth and the anthropometric measures of the neonate at birth (Phase 2). The total sample comprises 81 pregnant women aged 32.07 years (SD = 5.45) and their neonates. Sociodemographic and obstetric data of the sample were collected. During pregnancy, psychopathology was measured by means of the SCL-90, as well as other psychological measures on stress and social support. Cluster k-means techniques were used to uncover the heterogeneous profiles of psychopathology in Phase 1. Two main psychopathological profiles were found (Cluster 1: High psychopathological symptoms; Cluster 2: Low psychopathological symptoms). The clusters generated show significant differences in all the SCL-90-R subscales used and in the general index at Phase 1. After childbirth, high psychopathology profile membership was associated with a greater probability of having a non-eutocic delivery. On the other hand, the low psychopathological symptoms cluster shows higher levels of depressive symptoms, hostility, paranoid ideation, and psychotic symptoms in Phase 2. In conclusion, there seemed to exist two heterogeneous profiles of psychopathology in pregnant women during the pandemic; the stress related to the pandemic seemed uninfluential on the development of a profile of high psychopathological symptoms and the psychopathology profile may influence delivery and postpartum outcomes.

Myosin II regulatory light chain phosphorylation and formin availability modulate cytokinesis upon changes in carbohydrate metabolism.

Cytokinesis, the separation of daughter cells at the end of mitosis, relies in animal cells on a contractile actomyosin ring (CAR) composed of actin and class II myosins, whose activity is strongly influenced by regulatory light chain (RLC) phosphorylation. However, in simple eukaryotes such as the fission yeast Schizosaccharomyces pombe, RLC phosphorylation appears dispensable for regulating CAR dynamics. We found that redundant phosphorylation at Ser35 of the S. pombe RLC homolog Rlc1 by the p21-activated kinases Pak1 and Pak2, modulates myosin II Myo2 activity and becomes essential for cytokinesis and cell growth during respiration. Previously, we showed that the stress-activated protein kinase pathway (SAPK) MAPK Sty1 controls fission yeast CAR integrity by downregulating formin For3 levels (Gómez-Gil et al., 2020). Here, we report that the reduced availability of formin For3-nucleated actin filaments for the CAR is the main reason for the required control of myosin II contractile activity by RLC phosphorylation during respiration-induced oxidative stress. Thus, the restoration of For3 levels by antioxidants overrides the control of myosin II function regulated by RLC phosphorylation, allowing cytokinesis and cell proliferation during respiration. Therefore, fine-tuned interplay between myosin II function through Rlc1 phosphorylation and environmentally controlled actin filament availability is critical for a successful cytokinesis in response to a switch to a respiratory carbohydrate metabolism.

cAMP-Protein kinase A and stress-activated MAP kinase signaling mediate transcriptional control of autophagy in fission yeast during glucose limitation or starvation.

Macroautophagy/autophagy is an essential adaptive physiological response in eukaryotes induced during nutrient starvation, including glucose, the primary immediate carbon and energy source for most cells. Although the molecular mechanisms that induce autophagy during glucose starvation have been extensively explored in the budding yeast Saccharomyces cerevisiae, little is known about how this coping response is regulated in the evolutionary distant fission yeast Schizosaccharomyces pombe. Here, we show that S. pombe autophagy in response to glucose limitation relies on mitochondrial respiration and the electron transport chain (ETC), but, in contrast to S. cerevisiae, the AMP-activated protein kinase (AMPK) and DNA damage response pathway components do not modulate fission yeast autophagic flux under these conditions. In the presence of glucose, the cAMP-protein kinase A (PKA) signaling pathway constitutively represses S. pombe autophagy by downregulating the transcription factor Rst2, which promotes the expression of respiratory genes required for autophagy induction under limited glucose availability. Furthermore, the stress-activated protein kinase (SAPK) signaling pathway, and its central mitogen-activated protein kinase (MAPK) Sty1, positively modulate autophagy upon glucose limitation at the transcriptional level through its downstream effector Atf1 and by direct in vivo phosphorylation of Rst2 at S292. Thus, our data indicate that the signaling pathways that govern autophagy during glucose shortage or starvation have evolved differently in S. pombe and uncover the existence of sophisticated and multifaceted mechanisms that control this self-preservation and survival response.

Serum microRNAs targeting ACE2 and RAB14 genes distinguish asymptomatic from critical COVID-19 patients.

Despite the extraordinary advances achieved to beat COVID-19 disease, many questions remain unsolved, including the mechanisms of action of SARS-CoV-2 and which factors determine why individuals respond so differently to the viral infection. Herein, we performed an in silico analysis to identify host microRNA targeting ACE2, TMPRSS2, and/or RAB14, all genes known to participate in viral entry and replication. Next, the levels of six microRNA candidates previously linked to viral and respiratory-related pathologies were measured in the serum of COVID-19-negative controls (n = 16), IgG-positive COVID-19 asymptomatic individuals (n = 16), and critical COVID-19 patients (n = 17). Four of the peripheral microRNAs analyzed (hsa-miR-32-5p, hsa-miR-98-3p, hsa-miR-423-3p, and hsa-miR-1246) were upregulated in COVID-19 critical patients compared with COVID-19-negative controls. Moreover, hsa-miR-32-5p and hsa-miR-1246 levels were also altered in critical versus asymptomatic individuals. Furthermore, these microRNA target genes were related to viral infection, inflammatory response, and coagulation-related processes. In conclusion, SARS-CoV-2 promotes the alteration of microRNAs targeting the expression of key proteins for viral entry and replication, and these changes are associated with disease severity. The microRNAs identified could be taken as potential biomarkers of COVID-19 progression as well as candidates for future therapeutic approaches against this disease.

The Multiple Functions of Rho GTPases in Fission Yeasts.

The Rho family of GTPases represents highly conserved molecular switches involved in a plethora of physiological processes. Fission yeast Schizosaccharomyces pombe has become a fundamental model organism to study the functions of Rho GTPases over the past few decades. In recent years, another fission yeast species, Schizosaccharomyces japonicus, has come into focus offering insight into evolutionary changes within the genus. Both fission yeasts contain only six Rho-type GTPases that are spatiotemporally controlled by multiple guanine-nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), and whose intricate regulation in response to external cues is starting to be uncovered. In the present review, we will outline and discuss the current knowledge and recent advances on how the fission yeasts Rho family GTPases regulate essential physiological processes such as morphogenesis and polarity, cellular integrity, cytokinesis and cellular differentiation.

Specific Functional Features of the Cell Integrity MAP Kinase Pathway in the Dimorphic Fission Yeast Schizosaccharomyces japonicus.

Mitogen activated protein kinase (MAPK) signaling pathways execute essential functions in eukaryotic organisms by transducing extracellular stimuli into adaptive cellular responses. In the fission yeast model Schizosaccharomyces pombe the cell integrity pathway (CIP) and its core effector, MAPK Pmk1, play a key role during regulation of cell integrity, cytokinesis, and ionic homeostasis. Schizosaccharomyces japonicus, another fission yeast species, shows remarkable differences with respect to S. pombe, including a robust yeast to hyphae dimorphism in response to environmental changes. We show that the CIP MAPK module architecture and its upstream regulators, PKC orthologs Pck1 and Pck2, are conserved in both fission yeast species. However, some of S. pombe's CIP-related functions, such as cytokinetic control and response to glucose availability, are regulated differently in S. japonicus. Moreover, Pck1 and Pck2 antagonistically regulate S. japonicus hyphal differentiation through fine-tuning of Pmk1 activity. Chimeric MAPK-swapping experiments revealed that S. japonicus Pmk1 is fully functional in S. pombe, whereas S. pombe Pmk1 shows a limited ability to execute CIP functions and promote S. japonicus mycelial development. Our findings also suggest that a modified N-lobe domain secondary structure within S. japonicus Pmk1 has a major influence on the CIP signaling features of this evolutionarily diverged fission yeast.

The Fission Yeast Cell Integrity Pathway: A Functional Hub for Cell Survival upon Stress and Beyond.

The survival of eukaryotic organisms during environmental changes is largely dependent on the adaptive responses elicited by signal transduction cascades, including those regulated by the Mitogen-Activated Protein Kinase (MAPK) pathways. The Cell Integrity Pathway (CIP), one of the three MAPK pathways found in the simple eukaryote fission of yeast Schizosaccharomyces pombe, shows strong homology with mammalian Extracellular signal-Regulated Kinases (ERKs). Remarkably, studies over the last few decades have gradually positioned the CIP as a multi-faceted pathway that impacts multiple functional aspects of the fission yeast life cycle during unperturbed growth and in response to stress. They include the control of mRNA-stability through RNA binding proteins, regulation of calcium homeostasis, and modulation of cell wall integrity and cytokinesis. Moreover, distinct evidence has disclosed the existence of sophisticated interplay between the CIP and other environmentally regulated pathways, including Stress-Activated MAP Kinase signaling (SAPK) and the Target of Rapamycin (TOR). In this review we present a current overview of the organization and underlying regulatory mechanisms of the CIP in S. pombe, describe its most prominent functions, and discuss possible targets of and roles for this pathway. The evolutionary conservation of CIP signaling in the dimorphic fission yeast S. japonicus will also be addressed.

Stress-activated MAPK signaling controls fission yeast actomyosin ring integrity by modulating formin For3 levels.

Cytokinesis, which enables the physical separation of daughter cells once mitosis has been completed, is executed in fungal and animal cells by a contractile actin- and myosin-based ring (CAR). In the fission yeast Schizosaccharomyces pombe, the formin For3 nucleates actin cables and also co-operates for CAR assembly during cytokinesis. Mitogen-activated protein kinases (MAPKs) regulate essential adaptive responses in eukaryotic organisms to environmental changes. We show that the stress-activated protein kinase pathway (SAPK) and its effector, MAPK Sty1, downregulates CAR assembly in S. pombe when its integrity becomes compromised during cytoskeletal damage and stress by reducing For3 levels. Accurate control of For3 levels by the SAPK pathway may thus represent a novel regulatory mechanism of cytokinesis outcome in response to environmental cues. Conversely, SAPK signaling favors CAR assembly and integrity in its close relative Schizosaccharomyces japonicus, revealing a remarkable evolutionary divergence of this response within the fission yeast clade.

HIV antibodies level as a marker of HIV persistence: the role of hepatitis C virus coinfection.

Human immunodeficiency virus (HIV) antibodies have been proposed as a measure of the size of the HIV reservoir. The aim of our study is to quantify the anti-HIV antibodies level in a cohort of people living with HIV (PLWH), stratified based on the presence of continuous undetectable HIV viral load and the co-existence of hepatitis C virus infection. A sample of 229 HIV-monoinfected (n = 114) or HIV/HCV-coinfected [either with resolved HCV infection (n = 75) or active HCV coinfection (n = 40)] patients, followed up a median of 34 (IQR 20-44) months, was studied. Anti-HIV index was obtained as the 1:800 dilution of HIV antibodies. CD4+ T cell count, time with undetectable HIV viral load, annual increase of CD4+ T cell count, anti-HCV therapy, and diagnosis of cirrhosis were analyzed. Patients with a continued suppressed HIV viral load had significant lower anti-HIV index compared with those with virologic failure during the follow-up. Significant higher CD4+ T cell increase was observed in those with a lower anti-HIV index. HIV-monoinfected patients showed an anti-HIV index significantly lower than patients with HCV coinfection. Resolved HCV infection after interferon-based therapy, but not with direct acting antivirals, was associated with a lower anti-HIV index. HIV/HCV-coinfected patients showed higher HIV antibodies level when compared with HIV-monoinfected individuals. A decrease in anti-HIV index in HIV/HCV-coinfected patients was detected when a sustained virological HCV response was obtained after interferon-based therapy, in possible relation with the direct or indirect effect of interferon on PLWH CD4 T cells.

RNA-Binding Protein Rnc1 Regulates Cell Length at Division and Acute Stress Response in Fission Yeast through Negative Feedback Modulation of the Stress-Activated Mitogen-Activated Protein Kinase Pathway.

RNA-binding proteins (RBPs) play a major role during control of mRNA localization, stability, and translation and are central to most cellular processes. In the fission yeast Schizosaccharomyces pombe, the multiple K homology (KH) domain RBP Rnc1 downregulates the activity of the cell integrity pathway (CIP) via stabilization of pmp1+ mRNA, which encodes the Pmp1 phosphatase that inactivates Pmk1, the mitogen-activated protein kinase (MAPK) component of this signaling cascade. However, Rnc1 likely regulates the half-life/stability of additional mRNAs. We show that Rnc1 downregulates the activity of Sty1, the MAPK of the stress-activated MAPK pathway (SAPK), during control of cell length at division and recovery in response to acute stress. Importantly, this control strictly depends on Rnc1's ability to bind mRNAs encoding activators (Wak1 MAPKKK, Wis1 MAPKK) and downregulators (Atf1 transcription factor, Pyp1 and Pyp2 phosphatases) of Sty1 phosphorylation through its KH domains. Moreover, Sty1 is responsible for Rnc1 phosphorylation in vivo at multiple phosphosites during growth and stress, and these modifications trigger Rnc1 for proper binding and destabilization of the above mRNA targets. Phosphorylation by Sty1 prompts Rnc1-dependent mRNA destabilization to negatively control SAPK signaling, thus revealing an additional feedback mechanism that allows precise tuning of MAPK activity during unperturbed cell growth and stress.IMPORTANCE Control of mRNA localization, stability, turnover, and translation by RNA-binding proteins (RBPs) influences essential processes in all eukaryotes, including signaling by mitogen-activated protein kinase (MAPK) pathways. We describe that in the fission yeast Schizosaccharomyces pombe the RBP Rnc1 negatively regulates cell length at division during unperturbed growth and recovery after acute stress by reducing the activity of the MAPK Sty1, which regulates cell growth and differentiation during environmental cues. This mechanism relies on Rnc1 binding to specific mRNAs encoding both enhancers and negative regulators of Sty1 activity. Remarkably, multiple phosphorylation of Rnc1 by Sty1 favors RBP binding and destabilization of the above mRNAs. Thus, posttranscriptional modulation of MAP kinase signaling by RNA-binding proteins emerges as a major regulatory mechanism that dictates the growth cycle and cellular adaptation in response to the changing environment in eukaryotic organisms.

Functional interaction between Cdc42 and the stress MAPK signaling pathway during the regulation of fission yeast polarized growth.

Cell polarization can be defined as the generation and maintenance of directional cellular organization. The spatial distribution and protein or lipid composition of the cell are not symmetric but organized in specialized domains which allow cells to grow and acquire a certain shape that is closely linked to their physiological function. The establishment and maintenance of polarized growth requires the coordination of diverse processes including cytoskeletal dynamics, membrane trafficking, and signaling cascade regulation. Some of the major players involved in the selection and maintenance of sites for polarized growth are Rho GTPases, which recognize the polarization site and transmit the signal to regulatory proteins of the cytoskeleton. Additionally, cytoskeletal organization, polarized secretion, and endocytosis are controlled by signaling pathways including those mediated by mitogen-activated protein kinases (MAPKs). Rho GTPases and the MAPK signaling pathways are strongly conserved from yeast to mammals, suggesting that the basic mechanisms of polarized growth have been maintained throughout evolution. For this reason, the study of how polarized growth is established and regulated in simple organisms such as the fission yeast Schizosaccharomyces pombe has contributed to broaden our knowledge about these processes in multicellular organisms. We review here the function of the Cdc42 GTPase and the stress activated MAPK (SAPK) signaling pathways during fission yeast polarized growth, and discuss the relevance of the crosstalk between both pathways.

Correction: Quorum sensing and stress-activated MAPK signaling repress yeast to hypha transition in the fission yeast Schizosaccharomyces japonicus.

[This corrects the article DOI: 10.1371/journal.pgen.1008192.].

Quorum sensing and stress-activated MAPK signaling repress yeast to hypha transition in the fission yeast Schizosaccharomyces japonicus.

Quorum sensing (QS), a mechanism of microbial communication dependent on cell density, governs developmental decisions in many bacteria and in some pathogenic and non-pathogenic fungi including yeasts. In these simple eukaryotes this response is mediated by the release into the growth medium of quorum-sensing molecules (QSMs) whose concentration increases proportionally to the population density. To date the occurrence of QS is restricted to a few yeast species. We show that a QS mediated by the aromatic alcohols phenylethanol and tryptophol represses the dimorphic yeast to hypha differentiation in the fission yeast S. japonicus in response to an increased population density. In addition, the stress activated MAPK pathway (SAPK), which controls cell cycle progression and adaptation to environmental changes in this organism, constitutively represses yeast to hypha differentiation both at transcriptional and post-translational levels. Moreover, deletion of its main effectors Sty1 MAPK and Atf1 transcription factor partially suppressed the QS-dependent block of hyphal development under inducing conditions. RNAseq analysis showed that the expression of nrg1+, which encodes a putative ortholog of the transcription factor Nrg1 that represses yeast to hypha dimorphism in C. albicans, is downregulated both by QS and the SAPK pathway. Remarkably, Nrg1 may act in S. japonicus as an activator of hyphal differentiation instead of being a repressor. S. japonicus emerges as an attractive and amenable model organism to explore the QS mechanisms that regulate cellular differentiation in fungi.

Co-Occurrence of mcr-1 and qnrS1 on an IncHI2 Plasmid in Clinical Isolates of Salmonella Typhimurium in Spain.

Salmonella enterica is a well-adapted zoonotic bacterium associated to cases of gastroenteritis and bacteremia with increased morbidity and mortality. In this study, three isolates of Salmonella Typhimurium obtained from human clinical samples, showing colistin resistance and low-level resistance to quinolones, have been genetically characterized. We detected the co-occurrence of mcr-1 and qnrS1 on a single IncHI2 plasmid in isolates of Salmonella Typhimurium obtained from Spanish children without a travel history. The multiresistant region contained numerous resistance genes. Isolates were clonally related, which suggests the presence of these clones in the community and the potential to cause outbreaks affecting the most susceptible population. It is necessary to monitor the presence of these plasmid-mediated resistance genes in human European strains of Salmonella spp. because of the risk of producing outbreaks of community-acquired infections.

Staphylococcus aureus carriage in older populations in community residential care homes: Prevalence and molecular characterization of MRSA isolates.

INTRODUCTION: The epidemiology of S. aureus depends on conditions in specific populations. Few studies of S. aureus colonization in the older population have been performed in Spain. The aim of this study was to determine the prevalence of methicillin-resistant S. aureus (MRSA) colonization and its molecular epidemiological characteristics in an institutionalized population in community residential care homes in Cadiz, Spain. METHODS: A cross-sectional epidemiological study was conducted in three residential care homes for older people. Axilla and nostril samples were tested. Identification of S. aureus and antimicrobial susceptibility testing were by MALDI-TOF and MicroScan panels. MRSA strains were subjected to SCCmec typing, multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). The presence of Panton-Valentine leukocidin (PVL) genes was determined by PCR in all S. aureus strains. RESULTS: A total of 293 residents were included. Fifty-one residents (17.4%) were colonized with methicillin-sensitive S. aureus (MSSA) and 11 (3.8%) with MRSA. Resistance to at least two aminoglycosides was observed in 25.4% of MSSA and 90.9% and of MRSA isolates, and resistance to levofloxacin in 80.3% of MSSA and 100% of MRSA isolates. SCCmecIV was detected in all isolates and all except one (ST-125) were ST-8. None of the S. aureus isolates were positive for PVL. CONCLUSIONS: A low rate of S. aureus carriage was detected and the prevalence of MRSA was very low. ST8-MRSA-IVc was the dominant clone, and only one strain belonged to ST125-MRSA-IVc. We found MRSA transmission within the residential care homes and a very high rate of quinolone resistance in MSSA and MRSA.

Distinct functional relevance of dynamic GTPase cysteine methylation in fission yeast.

The final step in post-translational processing of Ras and Rho GTPases involves methylation of the prenylated cysteine residue by an isoprenylcysteine-O-carboxyl methyltransferase (ICMT). ICMT activity is essential for cell growth and development in higher eukaryotes, and inhibition of GTPase methylation has become an attractive target in cancer therapy to inactivate prenylated oncoproteins. However, the specificity and dynamics of the GTPase methylation process remain to be fully clarified. Notably, cells lacking Mam4, the ICMT ortholog in the fission yeast Schizosaccharomyces pombe, are viable. We have exploited this feature to analyze the role of methylation on GTPase localization and function. We show that methylation differentially affects GTPase membrane localization, being particularly relevant for plasma membrane tethering and downstream signaling of palmitoylated and farnesylated GTPases Ras1 and Rho2 lacking C-terminal polybasic motifs. Indeed, Ras1 and Rho2 cysteine methylation is required for proper regulation of differentiation elicited by MAPK Spk1 and for stress-dependent activation of the cell integrity pathway (CIP) and its main effector MAPK Pmk1. Further, Mam4 negatively regulates TORC2 signaling by a cross-inhibitory mechanism relying on Rho GTPase methylation. These results highlight the requirement for a tight control of GTPase methylation in vivo to allow adequate GTPase function.