Streptococcus pyogenes Cas9 ribonucleoprotein delivery for efficient, rapid and marker-free gene editing in Trypanosoma and Leishmania.

Kinetoplastids are unicellular eukaryotic flagellated parasites found in a wide range of hosts within the animal and plant kingdoms. They are known to be responsible in humans for African sleeping sickness (Trypanosoma brucei), Chagas disease (Trypanosoma cruzi), and various forms of leishmaniasis (Leishmania spp.), as well as several animal diseases with important economic impact (African trypanosomes, including Trypanosoma congolense). Understanding the biology of these parasites necessarily implies the ability to manipulate their genomes. In this study, we demonstrate that transfection of a ribonucleoprotein complex, composed of recombinant Streptococcus pyogenes Cas9 (SpCas9) and an in vitro-synthesized guide RNA, results in rapid and efficient genetic modifications of trypanosomatids, in marker-free conditions. This approach was successfully developed to inactivate, delete, and mutate candidate genes in various stages of the life cycle of T. brucei and T. congolense, and Leishmania promastigotes. The functionality of SpCas9 in these parasites now provides, to the research community working on these parasites, a rapid and efficient method of genome editing, without requiring plasmid construction and selection by antibiotics but requires only cloning and PCR screening of the clones. Importantly, this approach is adaptable to any wild-type parasite.

Annexin-A5 and annexin-A6 silencing prevents metastasis of breast cancer cells in zebrafish.

BACKGROUND INFORMATION: During tumor invasion and metastasis processes, cancer cells are exposed to major compressive and shearing forces, due to their migration through extracellular matrix, dense cell areas, and complex fluids, which may lead to numerous plasma membrane damages. Cancer cells may survive to these mechanical stresses thanks to an efficient membrane repair machinery. Consequently, this machinery may constitute a relevant target to inhibit cancer cell dissemination. RESULTS: We show here that annexin-A5 (ANXA5) and ANXA6 participate in membrane repair of MDA-MB-231 cells, a highly invasive triple-negative breast cancer cell line. These crucial components of the membrane repair machinery are substantially expressed in breast cancer cells in correlation with their invasive properties. In addition, high expression of ANXA5 and ANXA6 predict poor prognosis in high-grade lung, gastric, and breast cancers. In zebrafish, the genetic inhibition of ANXA5 and ANXA6 leads to drastic reduction of tumor cell dissemination. CONCLUSION: We conclude that the inhibition of ANXA5 and ANXA6 prevents membrane repair in cancer cells, which are thus unable to survive to membrane damage during metastasis. SIGNIFICANCE: This result opens a new therapeutic strategy based on targeting membrane repair machinery to inhibit tumor invasion and metastasis.

Specific High-Sensitivity Enzymatic Reporter UnLOCKing-Mediated Detection of Oncogenic BCR::ABL1 and EGFR Rearrangements.

Advances in molecular medicine have placed nucleic acid detection methods at the center of an increasing number of clinical applications. Polymerase chain reaction (PCR)-based diagnostics have been widely adopted for their versatility, specificity, and sensitivity. However, recently reported clustered regularly interspaced short palindromic repeats-based methods have demonstrated equivalent to superior performance, with increased portability and reduced processing time and cost. In this study, we applied Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) technology to the detection of oncogenic rearrangements. We implemented SHERLOCK for the detection of BCR::ABL1 mRNA, a hallmark of chronic myeloid leukemia (CML), and EGFR DNA oncogenic alleles, frequently detected in glioblastoma and non-small cell lung cancer (NSCLC). SHERLOCK enabled rapid, sensitive, and variant-specific detection of BCR::ABL1 and EGFR alterations. Compared with the gold-standard PCR-based methods currently used in clinic, SHERLOCK achieved equivalent to greater sensitivity, suggesting it could be a new tool in CML and NSCLC, to detect low level of molecular residual disease.

Inhibition of Autophagy Negates Radiofrequency-Induced Adaptive Response in SH-SY5Y Neuroblastoma Cells.

In the last years, radiofrequency (RF) has demonstrated that it can reduce DNA damage induced by a subsequent treatment with chemical or physical agents in different cell types, resembling the adaptive response, a phenomenon well documented in radiobiology. Such an effect has also been reported by other authors both in vitro and in vivo, and plausible hypotheses have been formulated, spanning from the perturbation of the cell redox status, to DNA repair mechanisms, and stress response machinery, as possible cellular mechanisms activated by RF pre-exposure. These mechanisms may underpin the observed phenomenon, and require deeper investigations. The present study aimed to determine whether autophagy contributes to RF-induced adaptive response. To this purpose, SH-SY5Y human neuroblastoma cells were exposed for 20 h to 1950 MHz, UMTS signal, and then treated with menadione. The results obtained indicated a reduction in menadione-induced DNA damage, assessed by applying the comet assay. Such a reduction was negated when autophagy was inhibited by bafilomycin A1 and E64d. Moreover, CRISPR SH-SY5Y cell lines defective for ATG7 or ATG5 genes did not show an adaptive response. These findings suggest the involvement of autophagy in the RF-induced adaptive response in human neuroblastoma cells; although, further investigation is required to extend such observation at the molecular level.

TRAIL Triggers CRAC-Dependent Calcium Influx and Apoptosis through the Recruitment of Autophagy Proteins to Death-Inducing Signaling Complex.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively kills various cancer cell types, but also leads to the activation of signaling pathways that favor resistance to cell death. Here, we investigated the as yet unknown roles of calcium signaling and autophagy regulatory proteins during TRAIL-induced cell death in leukemia cells. Taking advantage of the Gene Expression Profiling Interactive Analysis (GEPIA) project, we first found that leukemia patients present a unique TRAIL receptor gene expression pattern that may reflect their resistance to TRAIL. The exposure of NB4 acute promyelocytic leukemia cells to TRAIL induces intracellular Ca2+ influx through a calcium release-activated channel (CRAC)-dependent mechanism, leading to an anti-apoptotic response. Mechanistically, we showed that upon TRAIL treatment, two autophagy proteins, ATG7 and p62/SQSTM1, are recruited to the death-inducing signaling complex (DISC) and are essential for TRAIL-induced Ca2+ influx and cell death. Importantly, the treatment of NB4 cells with all-trans retinoic acid (ATRA) led to the upregulation of p62/SQSTM1 and caspase-8 and, when added prior to TRAIL stimulation, significantly enhanced DISC formation and the apoptosis induced by TRAIL. In addition to uncovering new pleiotropic roles for autophagy proteins in controlling the calcium response and apoptosis triggered by TRAIL, our results point to novel therapeutic strategies for sensitizing leukemia cells to TRAIL.

Inhibition of DDR1 enhances in vivo chemosensitivity in KRAS-mutant lung adenocarcinoma.

Platinum-based chemotherapy in combination with immune-checkpoint inhibitors is the current standard of care for patients with advanced lung adenocarcinoma (LUAD). However, tumor progression evolves in most cases. Therefore, predictive biomarkers are needed for better patient stratification and for the identification of new therapeutic strategies, including enhancing the efficacy of chemotoxic agents. Here, we hypothesized that discoidin domain receptor 1 (DDR1) may be both a predictive factor for chemoresistance in patients with LUAD and a potential target positively selected in resistant cells. By using biopsies from patients with LUAD, KRAS-mutant LUAD cell lines, and in vivo genetically engineered KRAS-driven mouse models, we evaluated the role of DDR1 in the context of chemotherapy treatment. We found that DDR1 is upregulated during chemotherapy both in vitro and in vivo. Moreover, analysis of a cohort of patients with LUAD suggested that high DDR1 levels in pretreatment biopsies correlated with poor response to chemotherapy. Additionally, we showed that combining DDR1 inhibition with chemotherapy prompted a synergistic therapeutic effect and enhanced cell death of KRAS-mutant tumors in vivo. Collectively, this study suggests a potential role for DDR1 as both a predictive and prognostic biomarker, potentially improving the chemotherapy response of patients with LUAD.

The Nuclear Remodeling Induced by Helicobacter Cytolethal Distending Toxin Involves MAFB Oncoprotein.

Enterohepatic Helicobacters, such as Helicobacter hepaticus and Helicobacter pullorum, are associated with several intestinal and hepatic diseases. Their main virulence factor is the cytolethal distending toxin (CDT). In the present study, whole genome microarray-based identification of differentially expressed genes was performed in vitro in HT-29 intestinal cells while following the ectopic expression of the active CdtB subunit of H. hepaticus CDT. A CdtB-dependent upregulation of the V-maf musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB) gene encoding the MAFB oncoprotein was found, as well as the CdtB-dependent regulation of several MAFB target genes. The transduction and coculture experiments confirmed MAFB mRNA and protein induction in response to CDT and its CdtB subunit in intestinal and hepatic cell lines. An analysis of MAFB protein subcellular localization revealed a strong nuclear and perinuclear localization in the CdtB-distended nuclei in intestinal and hepatic cells. MAFB was also detected at the cell periphery of the CdtB-induced lamellipodia in some cells. The silencing of MAFB changed the cellular response to CDT with the formation of narrower lamellipodia, a reduction of the increase in nucleus size, and the formation of less γH2AX foci, the biomarker for DNA double-strand breaks. Taken together, these data show that the CDT of enterohepatic Helicobacters modulates the expression of the MAFB oncoprotein, which is translocated in the nucleus and is associated with the remodeling of the nuclei and actin cytoskeleton.

A genome-scale CRISPR knock-out screen in chronic myeloid leukemia identifies novel drug resistance mechanisms along with intrinsic apoptosis and MAPK signaling.

Understanding resistance mechanisms in cancer is of utmost importance for the discovery of novel "druggable" targets. Efficient genetic screening, now even more possible with CRISPR-Cas9 gene-editing technology, next-generation sequencing and bioinformatics, is an important tool for deciphering novel cellular processes, such as resistance to treatment in cancer. Imatinib specifically eliminates chronic myeloid leukemia (CML) cells by targeting and blocking the kinase activity of BCR-ABL1; however, resistance to treatment exists. In order to discover BCR-ABL1 independent mechanisms of imatinib resistance, we utilized the genome-scale CRISPR knock-out library to screen for imatinib-sensitizing genes in vitro on K562 cells. We revealed genes that seem essential for imatinib-induced cell death, such as proapoptotic genes (BIM, BAX) or MAPK inhibitor SPRED2. Specifically, reestablishing apoptosis in BIM knock-out (KO) cells with BH3 mimetics, or inhibiting MAPK signaling in SPRED2 KO cells with MEK inhibitors restores sensitivity to imatinib. In this work, we discovered previously identified pathways and novel pathways that modulate response to imatinib in CML cell lines, such as the implication of the Mediator complex, mRNA processing and protein ubiquitinylation. Targeting these specific genetic lesions with combinational therapy can overcome resistance phenotypes and paves the road for the use of precision oncology.

The Expression of Myeloproliferative Neoplasm-Associated Calreticulin Variants Depends on the Functionality of ER-Associated Degradation.

BACKGROUND: Mutations in CALR observed in myeloproliferative neoplasms (MPN) were recently shown to be pathogenic via their interaction with MPL and the subsequent activation of the Janus Kinase - Signal Transducer and Activator of Transcription (JAK-STAT) pathway. However, little is known on the impact of those variant CALR proteins on endoplasmic reticulum (ER) homeostasis. METHODS: The impact of the expression of Wild Type (WT) or mutant CALR on ER homeostasis was assessed by quantifying the expression level of Unfolded Protein Response (UPR) target genes, splicing of X-box Binding Protein 1 (XBP1), and the expression level of endogenous lectins. Pharmacological and molecular (siRNA) screens were used to identify mechanisms involved in CALR mutant proteins degradation. Coimmunoprecipitations were performed to define more precisely actors involved in CALR proteins disposal. RESULTS: We showed that the expression of CALR mutants alters neither ER homeostasis nor the sensitivity of hematopoietic cells towards ER stress-induced apoptosis. In contrast, the expression of CALR variants is generally low because of a combination of secretion and protein degradation mechanisms mostly mediated through the ER-Associated Degradation (ERAD)-proteasome pathway. Moreover, we identified a specific ERAD network involved in the degradation of CALR variants. CONCLUSIONS: We propose that this ERAD network could be considered as a potential therapeutic target for selectively inhibiting CALR mutant-dependent proliferation associated with MPN, and therefore attenuate the associated pathogenic outcomes.

CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations.

CRISPR-Cas9 is a promising technology for genome editing. Here we use Cas9 nuclease-induced double-strand break DNA (DSB) at the UROS locus to model and correct congenital erythropoietic porphyria. We demonstrate that homology-directed repair is rare compared with NHEJ pathway leading to on-target indels and causing unwanted dysfunctional protein. Moreover, we describe unexpected chromosomal truncations resulting from only one Cas9 nuclease-induced DSB in cell lines and primary cells by a p53-dependent mechanism. Altogether, these side effects may limit the promising perspectives of the CRISPR-Cas9 nuclease system for disease modeling and gene therapy. We show that the single nickase approach could be safer since it prevents on- and off-target indels and chromosomal truncations. These results demonstrate that the single nickase and not the nuclease approach is preferable, not only for modeling disease but also and more importantly for the safe management of future CRISPR-Cas9-mediated gene therapies.

MiR-10a and HOXB4 are overexpressed in atypical myeloproliferative neoplasms.

BACKGROUND: Atypical Myeloproliferative Neoplasms (aMPN) share characteristics of MPN and Myelodysplastic Syndromes. Although abnormalities in cytokine signaling are common in MPN, the pathophysiology of atypical MPN still remains elusive. Since deregulation of microRNAs is involved in the biology of various cancers, we studied the miRNome of aMPN patients. METHODS: MiRNome and mutations in epigenetic regulator genes ASXL1, TET2, DNMT3A, EZH2 and IDH1/2 were explored in aMPN patients. Epigenetic regulation of miR-10a and HOXB4 expression was investigated by treating hematopoietic cell lines with 5-aza-2'deoxycytidine, valproic acid and retinoic acid. Functional effects of miR-10a overexpression on cell proliferation, differentiation and self-renewal were studied by transducing CD34+ cells with lentiviral vectors encoding the pri-miR-10a precursor. RESULTS: MiR-10a was identified as the most significantly up-regulated microRNA in aMPN. MiR-10a expression correlated with that of HOXB4, sitting in the same genomic locus. The transcription of these two genes was increased by DNA demethylation and histone acetylation, both necessary for optimal expression induction by retinoic acid. Moreover, miR-10a and HOXB4 overexpression seemed associated with DNMT3A mutation in hematological malignancies. However, overexpression of miR-10a had no effect on proliferation, differentiation or self-renewal of normal hematopoietic progenitors. CONCLUSIONS: MiR-10a and HOXB4 are overexpressed in aMPN. This overexpression seems to be the result of abnormalities in epigenetic regulation mechanisms. Our data suggest that miR-10a could represent a simple marker of transcription at this genomic locus including HOXB4, widely recognized as involved in stem cell expansion.

Assignment of 1H, 13C and 15N resonances and secondary structure of the Rgd1-RhoGAP domain.

The protein Rgd1 is involved in the regulation of cytoskeleton formation and in signalling pathways that control cell polarity and growth in Saccharomyces cerevisiae. Rgd1p is composed of a F-BAR domain required for membrane binding and a RhoGAP domain responsible for activating Rho3p and Rho4p, two GTPases respectively involved in bud growth and cytokinesis. Rgd1p is recruited to the membrane through interactions with phosphoinositide lipids, which bind the two isolated domains and stimulate the RhoGAP activity on Rho4p. As previously shown by crystallography, the membrane-binding F-BAR domain contains a conserved inositol phosphate binding site, which explains the preferential binding of phosphoinositides. In contrast, RhoGAP domains are not expected to bind lipids. In order to unravel this puzzling feature, we solved the three-dimensional structure of the isolated protein and found a cryptic phosphoinositide binding site involving non conserved residues (Martinez et al. 2017). The assignment of the resonances and secondary structure of Rgd1-RhoGAP (aa 450-666) is presented here.

A tyrosine kinase-STAT5-miR21-PDCD4 regulatory axis in chronic and acute myeloid leukemia cells.

MicroRNAs (miRNAs) are regulators of several key patho-physiological processes, including cell cycle and apoptosis. Using microarray-based miRNA profiling in K562 cells, a model of chronic myeloid leukemia (CML), we found that the oncoprotein BCR-ABL1 regulates the expression of miR-21, an "onco-microRNA", found to be overexpressed in several cancers. This effect relies on the presence of two STAT binding sites on the promoter of miR-21, and on the phosphorylation status of STAT5, a transcription factor activated by the kinase activity of BCR-ABL1. Mir-21 regulates the expression of PDCD4 (programmed cell death protein 4), a tumor suppressor identified through a proteomics approach. The phosphoSTAT5 - miR-21 - PDCD4 pathway was active in CML primary CD34+ cells, but also in acute myeloid leukemia (AML) models like MV4.11 and MOLM13, where the constitutively active tyrosine kinase FLT3-ITD plays a similar role to BCR-ABL1 in the K562 cell line.

Structural evidence of a phosphoinositide-binding site in the Rgd1-RhoGAP domain.

Phosphoinositide lipids recruit proteins to the plasma membrane involved in the regulation of cytoskeleton organization and in signalling pathways that control cell polarity and growth. Among those, Rgd1p is a yeast GTPase-activating protein (GAP) specific for Rho3p and Rho4p GTPases, which control actin polymerization and stress signalling pathways. Phosphoinositides not only bind Rgd1p, but also stimulate its GAP activity on the membrane-anchored form of Rho4p. Both F-BAR (F-BAR FCH, and BAR) and RhoGAP domains of Rgd1p are involved in lipid interactions. In the Rgd1p-F-BAR domain, a phosphoinositide-binding site has been recently characterized. We report here the X-ray structure of the Rgd1p-RhoGAP domain, identify by NMR spectroscopy and confirm by docking simulations, a new but cryptic phosphoinositide-binding site, comprising contiguous A1, A1' and B helices. The addition of helix A1', unusual among RhoGAP domains, seems to be crucial for lipid interactions. Such a site was totally unexpected inside a RhoGAP domain, as it was not predicted from either the protein sequence or its three-dimensional structure. Phosphoinositide-binding sites in RhoGAP domains have been reported to correspond to polybasic regions, which are located at the unstructured flexible termini of proteins. Solid-state NMR spectroscopy experiments confirm the membrane interaction of the Rgd1p-RhoGAP domain upon the addition of PtdIns(4,5)P2 and indicate a slight membrane destabilization in the presence of the two partners.

MYB-GATA1 fusion promotes basophilic leukaemia: involvement of interleukin-33 and nerve growth factor receptors.

Acute basophilic leukaemia (ABL) is a rare subtype of acute myeloblastic leukaemia. We previously described a recurrent t(X;6)(p11;q23) translocation generating an MYB-GATA1 fusion gene in male infants with ABL. To better understand its role, the chimeric MYB-GATA1 transcription factor was expressed in CD34-positive haematopoietic progenitors, which were transplanted into immunodeficient mice. Cells expressing MYB-GATA1 showed increased expression of markers of immaturity (CD34), of granulocytic lineage (CD33 and CD117), and of basophilic differentiation (CD203c and FcϵRI). UT-7 cells also showed basophilic differentiation after MYB-GATA1 transfection. A transcriptomic study identified nine genes deregulated by both MYB-GATA1 and basophilic differentiation. Induction of three of these genes (CCL23, IL1RL1, and NTRK1) was confirmed in MYB-GATA1-expressing CD34-positive cells by reverse transcription quantitative polymerase chain reaction. Interleukin (IL)-33 and nerve growth factor (NGF), the ligands of IL-1 receptor-like 1 (IL1RL1) and neurotrophic receptor tyrosine kinase 1 (NTRK1), respectively, enhanced the basophilic differentiation of MYB-GATA1-expressing UT-7 cells, thus demonstrating the importance of this pathway in the basophilic differentiation of leukaemic cells and CD34-positive primary cells. Finally, gene reporter assays confirmed that MYB and MYB-GATA1 directly activated NTRK1 and IL1RL1 transcription, leading to basophilic skewing of the blasts. MYB-GATA1 is more efficient than MYB, because of better stability. Our results highlight the role of IL-33 and NGF receptors in the basophilic differentiation of normal and leukaemic cells. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The GyrA encoded gene: A pertinent marker for the phylogenetic revision of Helicobacter genus.

Phylogeny of Epsilonproteobacteria is based on sequencing of the 16S rRNA gene. However, this gene is not sufficiently discriminatory in Helicobacter species and alternative markers would be useful. In this study, the 16S rRNA, gyrA, hsp60, gyrB, and ureA-ureB gene sequences, as well as GyrA, HSP60 and GyrB protein sequences were analyzed as tools to support Helicobacter species phylogeny: 72 Helicobacter strains, belonging to 41 species of which 36 are validated species, were included. Results of the phylogenetic reconstructions of the GyrA gene encoded protein (approximately 730 residues) indicated the most stable trees to bootstrap resampling with a good separation of Helicobacter taxa, especially between gastric and enterohepatic species. Moreover, the GyrA tree revealed high similarity with that of the gyrB and ureA-ureB genes (restricted to urease-positive Helicobacter species). However, some differences in clustering were observed when compared to the hsp60 and 23S rRNA gene trees. Altogether, these revised phylogenies (except the 16S rRNA gene for enterohepatic Helicobacters) enabled reliable clustering of Helicobacter cinaedi and 'Flexispira' strains, determined a reliable position for Helicobacter mustelae (except the hsp60 gene) and for novel Helicobacter species proposed such as 'Helicobacter sanguini', 'Helicobacter apodemus' or 'Helicobacter winghamensis', and suggest that Helicobacter species MIT 09-6949 and MIT 05-5293 isolated from rodents constitute novel species. Although they are not commonly used to study the phylogeny of Epsilonproteobacteria, protein sequences and, in particular, the GyrA protein sequence may constitute pertinent phylogenetic markers for Helicobacter genus.

Synergistic cooperation between ABT-263 and MEK1/2 inhibitor: effect on apoptosis and proliferation of acute myeloid leukemia cells.

In spite of intensive research to improve treatment of acute myeloid leukemia (AML) more than half of all patients continue to develop a refractory disease. Therefore there is need to improve AML treatment. The overexpression of the BCL-2 family anti-apoptotic members, like BCL-2 or BCL-xL has been largely reported in lymphoid tumors but also in AML and other tumors. To counteract the anti-apoptotic effect of BCL-2, BH3 mimetics have been developed to target cancer cells. An increase in activity of ERK1/2 mitogen activated protein (MAP) kinase has also been reported in AML and might be targeted by MEK1/2 inhibitors. Hence, in the current work, we investigated whether the association of a BH3 mimetic such ABT-263 and the MEK1/2 inhibitor pimasertib (MEKI), was efficient to target AML cells. A synergistic increasing of apoptosis was observed in AML cell lines and in primary cells without affecting normal bone marrow cells. Such cooperation was confirmed on tumor growth in a mouse xenograft model of AML. In addition we demonstrated that MEKI sensitized the cells to apoptosis through its ability to promote a G1 cell cycle arrest. So, this combination of a MAP Kinase pathway inhibitor and a BH3 mimetic could be a promising strategy to improve the treatment of AML.

CBL controls a tyrosine kinase network involving AXL, SYK and LYN in nilotinib-resistant chronic myeloid leukaemia.

A tyrosine kinase network composed of the TAM receptor AXL and the cytoplasmic kinases LYN and SYK is involved in nilotinib-resistance of chronic myeloid leukaemia (CML) cells. Here, we show that the E3-ubiquitin ligase CBL down-regulation occurring during prolonged drug treatment plays a critical role in this process. Depletion of CBL in K562 cells increases AXL and LYN protein levels, promoting cell resistance to nilotinib. Conversely, forced expression of CBL in nilotinib-resistant K562 cells (K562-rn) dramatically reduces AXL and LYN expression and resensitizes K562-rn cells to nilotinib. A similar mechanism was found to operate in primary CML CD34(+) cells. Mechanistically, the E3-ligase CBL counteracts AXL/SYK signalling, promoting LYN transcription by controlling AXL protein stability. Surprisingly, the role of AXL in resistance was independent of its ligand GAS6 binding and its TK activity, in accordance with a scaffold activity for this receptor being involved in this cellular process. Collectively, our results demonstrate a pivotal role for CBL in the control of a tyrosine kinase network mediating resistance to nilotinib treatment in CML cells.

The Saccharomyces cerevisiae RhoGAP Rgd1 is phosphorylated by the Aurora B like kinase Ipl1.

Polarized growth of the yeast Saccharomyces cerevisiae depends on different biological processes and requires several signaling pathways. Signaling is mediated through a set of proteins, which include Rho3p and Rho4p GTPases. Although these two proteins are involved in the control of distinct aspects of polarized growth in yeast, they have a common regulator: the Rgd1 RhoGAP protein. Here we demonstrate that Rgd1p is phosphorylated by the Aurora B like kinase Ipl1 and we observe that loss of Ipl1 function leads to a new Rgd1p distribution in a small part of the cell population.