Cell cycle-dependent and independent mating blocks ensure fungal zygote survival and ploidy maintenance.

To ensure genome stability, sexually reproducing organisms require that mating brings together exactly 2 haploid gametes and that meiosis occurs only in diploid zygotes. In the fission yeast Schizosaccharomyces pombe, fertilization triggers the Mei3-Pat1-Mei2 signaling cascade, which represses subsequent mating and initiates meiosis. Here, we establish a degron system to specifically degrade proteins postfusion and demonstrate that mating blocks not only safeguard zygote ploidy but also prevent lysis caused by aberrant fusion attempts. Using long-term imaging and flow-cytometry approaches, we identify previously unrecognized and independent roles for Mei3 and Mei2 in zygotes. We show that Mei3 promotes premeiotic S-phase independently of Mei2 and that cell cycle progression is both necessary and sufficient to reduce zygotic mating behaviors. Mei2 not only imposes the meiotic program and promotes the meiotic cycle, but also blocks mating behaviors independently of Mei3 and cell cycle progression. Thus, we find that fungi preserve zygote ploidy and survival by at least 2 mechanisms where the zygotic fate imposed by Mei2 and the cell cycle reentry triggered by Mei3 synergize to prevent zygotic mating

Optogenetics reveals Cdc42 local activation by scaffold-mediated positive feedback and Ras GTPase.

Local activity of the small GTPase Cdc42 is critical for cell polarization. Whereas scaffold-mediated positive feedback was proposed to break symmetry of budding yeast cells and produce a single zone of Cdc42 activity, the existence of similar regulation has not been probed in other organisms. Here, we address this problem using rod-shaped cells of fission yeast Schizosaccharomyces pombe, which exhibit zones of active Cdc42-GTP at both cell poles. We implemented the CRY2-CIB1 optogenetic system for acute light-dependent protein recruitment to the plasma membrane, which allowed to directly demonstrate positive feedback. Indeed, optogenetic recruitment of constitutively active Cdc42 leads to co-recruitment of the guanine nucleotide exchange factor (GEF) Scd1 and endogenous Cdc42, in a manner dependent on the scaffold protein Scd2. We show that Scd2 function is dispensable when the positive feedback operates through an engineered interaction between the GEF and a Cdc42 effector, the p21-activated kinase 1 (Pak1). Remarkably, this rewired positive feedback confers viability and allows cells to form 2 zones of active Cdc42 even when otherwise essential Cdc42 activators are lacking. These cells further revealed that the small GTPase Ras1 plays a role in both localizing the GEF Scd1 and promoting its activity, which potentiates the positive feedback. We conclude that scaffold-mediated positive feedback, gated by Ras activity, confers robust polarization for rod-shape formation

Cell patterning by secretion-induced plasma membrane flows.

Cells self-organize using reaction-diffusion and fluid-flow principles. Whether bulk membrane flows contribute to cell patterning has not been established. Here, using mathematical modeling, optogenetics, and synthetic probes, we show that polarized exocytosis causes lateral membrane flows away from regions of membrane insertion. Plasma membrane-associated proteins with sufficiently low diffusion and/or detachment rates couple to the flows and deplete from areas of exocytosis. In rod-shaped fission yeast cells, zones of Cdc42 GTPase activity driving polarized exocytosis are limited by GTPase activating proteins (GAPs). We show that membrane flows pattern the GAP Rga4 distribution and that coupling of a synthetic GAP to membrane flows is sufficient to establish the rod shape. Thus, membrane flows induced by Cdc42-dependent exocytosis form a negative feedback restricting the zone of Cdc42 activity

Variable Expression of Neural Cell Adhesion Molecule Isoforms in Renal Tissue: Possible Role in Incipient Renal Fibrosis.

Rare neural cell adhesion molecule (NCAM) positive cells have been previously described within the normal human adult kidney interstitium, speculating that they could increase in the interstitium with incipient interstitial renal fibrosis (IRF). In the present study, among 93 biopsy samples of various kidney diseases, NCAM+ interstitial cells were detected in 62.4% cases. An increased number of NCAM+ cells was significantly observed only in incipient IRF compared to normal renal tissues and advanced IRF stages (p<0.001), independently of underlying diseases (p = 0.657). All three major NCAM isoforms' RT-PCR bands were visible either in normal or in kidneys with incipient IRF, albeit their mRNA expression levels measured by qRT-PCR were different. Applying qRT-PCR on pure NCAM+ cells population, obtained by laser capture microdissection, significant mRNA over-expression of NCAM140kD isoform was found in NCAM+ cells within incipient IRF (p = 0.004), while NCAM120kD and NCAM180kD isoforms were not changed significantly (p = 0.750; p = 0.704; respectively). Simultaneously, qRT-PCR also showed significant αSMA (p = 0.014) and SLUG (p = 0.004) mRNAs up-regulation within the NCAM+ cells of incipient IRF, as well as highly decreased matrix metalloproteinases (MMP) -2 and -9 mRNAs (p = 0.028; p = 0.036; respectively). However, using double immunofluorescence MMP-9 could still be detectable on the protein level in rare NCAM+ cells within the incipient IRF. Further characterization of NCAM+ cells by double immunofluorescent labeling revealed their association with molecules involved in fibrosis. Fibroblast growth factor receptor 1 (FGFR1) and α5β1 integrin were extensively expressed on NCAM+ cells within the incipient IRF areas, whereas human epididymis protein-4 (HE4) was found to be present in few NCAM+ cells of both normal and interstitium with incipient fibrosis. Heterogeneity of NCAM+ interstitial cells in normal and incipient IRF, concerning molecules related to fibrosis and variable expression of NCAM isoforms, could suggest diverse role of NCAM+ cells in homeostasis and in regulation of renal fibrosis in diseased kidneys

Expression of FGFR1, HE4 and α5β1 integrin on NCAM positive cells in incipient renal fibrosis.

<p>(<b>A</b>) Double immunofluorescent labeling of NCAM and FGFR1; merge of these two markers clearly shows that all NCAM+ cells coexpressed FGFR1 (white arrows); diffuse NCAM expression on interstitial cells; strong FGFR1 expression on bold vessels (white stars) and diffuse expression on interstitial cells; x200. (<b>B</b>) Double immunofluorescent labeling of NCAM and HE4; merge of NCAM and HE4 revealed single cells coexpressing both markers (white arrow); x400. (<b>C</b>) Double immunofluorescence labeling of NCAM and α5β1; merge of these two markers clearly shows co-expression of NCAM and α5β1 on renal interstitial cells in area of incipient fibrosis (white arrows); x600. <b>(D)</b> Double immunofluorescence labeling of NCAM and αSMA; merge of these two markers showed no overlapping of NCAM and αSMA on renal interstitial cells in area of incipient fibrosis, although areas of NCAM⁺ and SMA⁺ interstitial cells are close to each other; x100. Staining techniques are described in detail under Material and Methods.</p

Primer sequences used for qRT-PCR procedures.

<p>Primer sequences used for qRT-PCR procedures.</p

NCAM positive interstitial staining among various kidney diseases and their relationship to severity of renal interstitial fibrosis.

<p><b>(A)</b> Frequency of interstitial NCAM positivity among various kidney diseases. <b>(B)</b> Number of detected NCAM⁺ cells per field of view on ×400 magnification in controls and in diseased kidneys with regard to severity of interstitial renal fibrosis (IRF); p values after applying Mann-Whiteny U test. <b>(C-D)</b> FSGS with slight interstitial fibrosis (IRF-1) without tubular atrophy exhibiting an increased diffuse NCAM interstitial positivity detected on slides from paraffin-embedded tissue. (C) PAS, x400. (D) Immunoperoxidase staining, NCAM clone 123C3.D5, x400. <b>(E-G)</b> Lupus nephritis with NCAM positive interstitial cells detected focally around tubuli in the area with slight IRF (IRF-1). (E) PAS, x400. (F) Massone trichrome staining, x400. (G) Immunoperoxidase staining, NCAM clone 123C3.D5, x400.</p

Double immunofluorescent labeling of NCAM with erythropoietin (EPO) and granzyme B.

<p><b>(A-C)</b> NCAM positive interstitial cells did not express EPO. Merge of NCAM (clone Eric-1) and EPO, cryostat sections, double immunofluorescent labeling, x400. <b>(D)</b> Diffuse NCAM (clone EP257Y) staining in peritubular incipient interstitial fibrosis of FSGS case, without any granzyme B positivity, cryostat section, double immunofluorescene, x400. <b>(E)</b> Overlapping of NCAM (clone EP257Y) and granzyme B in a single cell within the whole biopsy core of the case illustrated in previous picture, cryostat section, double immunofluorescence, x400. <b>(F)</b> Mononuclear interstitial inflammatory infiltrate of lupus nephritis, arrow indicates two NCAM+ cells without overlapping with granzyme B, cryostat section, double immunofluorescence, x400.</p

Differences in relative mRNA expression levels of molecules relevant for renal fibrosis among NCAM positive cells from normal and renal interstitium with incipient fibrosis, obtained by laser capture microdissection.

<p><b>(A)</b> Relative mRNA expression levels of various molecules relevant for renal fibrosis; data are presented with mean values and standard error bars; *- indicate statistically significant difference, p<0.05; graph is made of mean values in order to unify variable presentations, although only αSMA, SLUG and ALK3 followed normal distribution; thus, some variables differed extremely among cases and consequently these data did not display normal distribution; due to influence of these extreme values on the mean value presented in the graph, bars are high but without statistical significance (such as BMP7); Student’s t test was used for variables with normal distribution both in control and kidneys with fibrosis: αSMA, SLUG and ALK3; due to high variability of other variables, exclusively in diseased kidneys, we applied nonparametric Mann Whitney U test to assess the difference in mRNA levels between controls and diseased kidneys; there were 6 samples (2 cases in triplicates) of control cases and 42 (14 cases in triplicates) samples of cases with incipient renal fibrosis. <b>(B)</b> NCAM positivity in peritubular incipient interstitial fibrosis shown on cryostat section, immunofluorescene, clone EP257Y, x400. <b>(C)</b> MMP-9 positivity in peritubular incipient interstitial fibrosis shown on cryostat section, immunofluorescence, clone 6-6B, x400. <b>(D)</b> arrows indicate the overlapping of NCAM and MMP-9 in interstitial cells.</p

Relative mRNA levels of molecules related to renal fibrosis in NCAM positive renal interstitial cells laser captured from normal and renal interstitium with incipient fibrosis.

<p>95% CI- 95% Confidence Inteval for Mean;</p><p>Relative mRNA levels of molecules related to renal fibrosis in NCAM positive renal interstitial cells laser captured from normal and renal interstitium with incipient fibrosis.</p