The Cdc42 effectors Ste20, Cla4, and Skm1 down-regulate the expression of genes involved in sterol uptake by a mitogen-activated protein kinase-independent pathway

© 2009 by The American Society for Cell Biology. Under the License and Publishing Agreement, authors grant to the general public, effective two months after publication of (i.e.,. the appearance of) the edited manuscript in an online issue of MBoC, the nonexclusive right to copy, distribute, or display the manuscript subject to the terms of the Creative Commons–Noncommercial–Share Alike 3.0 Unported license (http://creativecommons.org/licenses/by-nc-sa/3.0).In Saccharomyces cerevisiae, the Rho-type GTPase Cdc42 regulates polarized growth through its effectors, including the p21-activated kinases (PAKs) Ste20, Cla4, and Skm1. Previously, we demonstrated that Ste20 interacts with several proteins involved in sterol synthesis that are crucial for cell polarization. Under anaerobic conditions, sterols cannot be synthesized and need to be imported into cells. Here, we show that Ste20, Cla4, and Skm1 form a complex with Sut1, a transcriptional regulator that promotes sterol uptake. All three PAKs can translocate into the nucleus and down-regulate the expression of genes involved in sterol uptake, including the Sut1 targets AUS1 and DAN1 by a novel mechanism. Consistently, deletion of either STE20, CLA4, or SKM1 results in an increased sterol influx and PAK overexpression inhibits sterol uptake. For Ste20, we demonstrate that the down-regulation of gene expression requires nuclear localization and kinase activity of Ste20. Furthermore, the Ste20-mediated control of expression of sterol uptake genes depends on SUT1 but is independent of a mitogen-activated protein kinase signaling cascade. Together, these observations suggest that PAKs translocate into the nucleus, where they modulate expression of sterol uptake genes via Sut1, thereby controlling sterol homeostasis.Deutsche Forschungsgemeinschaft and the Swiss National Science Foundation

The Rho GDI Rdi1 regulates Rho GTPases by distinct mechanisms

© 2008 by The American Society for Cell Biology. Under the License and Publishing Agreement, authors grant to the general public, effective two months after publication of (i.e.,. the appearance of) the edited manuscript in an online issue of MBoC, the nonexclusive right to copy, distribute, or display the manuscript subject to the terms of the Creative Commons–Noncommercial–Share Alike 3.0 Unported license (http://creativecommons.org/licenses/by-nc-sa/3.0).The small guanosine triphosphate (GTP)-binding proteins of the Rho family are implicated in various cell functions, including establishment and maintenance of cell polarity. Activity of Rho guanosine triphosphatases (GTPases) is not only regulated by guanine nucleotide exchange factors and GTPase-activating proteins but also by guanine nucleotide dissociation inhibitors (GDIs). These proteins have the ability to extract Rho proteins from membranes and keep them in an inactive cytosolic complex. Here, we show that Rdi1, the sole Rho GDI of the yeast Saccharomyces cerevisiae, contributes to pseudohyphal growth and mitotic exit. Rdi1 interacts only with Cdc42, Rho1, and Rho4, and it regulates these Rho GTPases by distinct mechanisms. Binding between Rdi1 and Cdc42 as well as Rho1 is modulated by the Cdc42 effector and p21-activated kinase Cla4. After membrane extraction mediated by Rdi1, Rho4 is degraded by a novel mechanism, which includes the glycogen synthase kinase 3β homologue Ygk3, vacuolar proteases, and the proteasome. Together, these results indicate that Rdi1 uses distinct modes of regulation for different Rho GTPases.Deutsche Forschungsgemeinschaf

Standardisation of synovial biopsy analyses in rheumatic diseases: a consensus of the EULAR Synovitis and OMERACT Synovial Tissue Biopsy Groups

Following publication of the original article [1], the authors reported an error in the spelling of the ninth author’s name. Incorrect spelling: Soeren Andreas Just. Correct spelling: Søren Andreas Just. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Background: The aim of this global collaboration was to develop a consensual set of items for the analysis of synovial biopsies in clinical practice and translational research through the EULAR Synovitis Study Group (ESSG) and OMERACT Synovial Tissue Biopsy Group. Methods: Participants were consulted through a modified Delphi method. Three sequential rounds occurred over 12 months. Members were sent a written questionnaire containing items divided into two parts. Items were identified and formulated based on a scoping review. The first part of the questionnaire referred to synovial biopsies in clinical practice including five subsections, and the second part to translational research with six subsections. Every participant was asked to score each item on a 5-point Likert scale. Items with a median score above 3.5 and a >70% agreement were selected for the next round. The last round was conducted orally at EULAR in June 2017. Results: Twenty-seven participants from 19 centers were contacted by email. Twenty participants from 17 centers answered. Response rates for next rounds were 100%. For the first part relating to clinical practice, 20/44 items (45.5%) were selected. For the second part relating to translational research, 18/43 items (41.9%) were selected for the final set. Conclusions: We herein propose a consensual set of analysis items to be used for synovial biopsies conducted in clinical practice and translational research. Correction: Following publication of the original article [1], the authors reported an error in the spelling of the ninth author's name

Notch signalling via RBP-J promotes myeloid differentiation

The expression of Notch receptors on hematopoietic cells and of cognate ligands on bone marrow stromal cells suggests a possible role for Notch signalling in the regulation of hematopoiesis. In order to assess the involvement of Notch1 signalling in myelopoiesis, 32D myeloid progenitor cell lines were engineered to permit the conditional induction of the constitutively active intracellular domain of murine Notch1 (mN1(IC)) by the 4-hydroxytamoxifen-inducible system. The induction of mN1(IC) resulted in accelerated and increased granulocytic differentiation. These effects were observed under growth conditions that support differentiation and, to a lesser degree, under conditions that normally promote self-renewal. Transient transfection of mN1(IC) deletion mutants showed that the differentiation promoting activity correlated with RBP-J transactivation. Furthermore, expression of a transcriptionally active derivative of RBP-J (RBP-J–VP16) increased myeloid differentiation. To test further the role of Notch signalling in a physiological context, 32D cells expressing mNotch1 were cultured on fibroblast layers that either expressed or did not express the Notch ligand Jagged1. Similar to the induction of mN1(IC), Jagged1 accelerated granulocytic differentiation of 32D cells. Taken together, our data suggest that activation of mNotch1 promotes myeloid differentiation via RBP-J transactivation

Suppression of the PI3K subunit p85α delays embryoid body development and inhibits cell adhesion

Phosphatidylinositol-3-kinases (PI3Ks) exert a variety of signaling functions in eukaryotes. We suppressed the PI3K regulatory subunit p85α using a small interfering RNA (Pik3r1 siRNA) and examined the effects on embryoid body (EB) development in hanging drop culture. We observed a 150% increase in the volume of the treated EBs within 24 h, compared to the negative controls. Fluorescence Activated Cell Sorting (FACS) assays showed that this increase in volume is not due to increased cellular proliferation. Instead, the increase in volume appears to be due to reduced cellular aggregation and adherence. This is further shown by our observation that 40% of treated EBs form twin instead of single EBs, and that they have a significantly reduced ability to adhere to culture dishes when plated. A time course over the first 96 h reveals that the impaired adherence is transient and explained by an initial 12-hour delay in EB development. Quantitative PCR expression analysis suggests that the adhesion molecule integrin-β1 (ITGB1) is transiently downregulated by the p85α suppression. In conclusion we found that suppressing p85α leads to a delay in forming compact EBs, accompanied by a transient inability of the EBs to undergo normal cell-cell and cell-substrate adhesion.</p