scholarly journals A Novel Ras Inhibitor, Eri1, Engages Yeast Ras at the Endoplasmic Reticulum

2003 ◽  
Vol 23 (14) ◽  
pp. 4983-4990 ◽  
Author(s):  
Andrew K. Sobering ◽  
Martin J. Romeo ◽  
Heather A. Vay ◽  
David E. Levin
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Regulatory Function ◽  
Ras Signaling ◽  
Cell Proliferation And Differentiation ◽  
Acid Protein ◽  
Cytoplasmic Face ◽  
Proliferation And Differentiation ◽  
Mutant Forms

ABSTRACT Ras oncoproteins are monomeric GTPases that link signals from the cell surface to pathways that regulate cell proliferation and differentiation. Constitutively active mutant forms of Ras are found in ca. 30% of human tumors. Here we report the isolation of a novel gene from Saccharomyces cerevisiae, designated ERI1 (for endoplasmic reticulum-associated Ras inhibitor 1), which behaves genetically as an inhibitor of Ras signaling. ERI1 encodes a 68-amino-acid protein that associates in vivo with GTP-bound Ras in a manner that requires an intact Ras-effector loop, suggesting that Eri1 competes for the same binding site as Ras target proteins. We show that Eri1 localizes primarily to the membrane of the endoplasmic reticulum (ER), where it engages Ras. The recent demonstration that signaling from mammalian Ras is not restricted to the cell surface but can also proceed from the cytoplasmic face of the ER suggests a regulatory function for Eri1 at that membrane.

scholarly journals C. elegansparaoxonase-like proteins control the functional expression of DEG/ENaC mechanosensory proteins

2016 ◽  
Author(s):  
Yushu Chen ◽  
Shashank Bharill ◽  
Zeynep Altun ◽  
Robert O'Hagan ◽  
Brian Coblitz ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Caenorhabditis Elegans ◽  
Cell Surface ◽  
Functional Expression ◽  
Similar Protein ◽  
Touch Sensitivity ◽  
Additional Protein

Caenorhabditis eleganssenses gentle touch via a mechanotransduction channel formed from the DEG/ENaC proteins MEC-4 and MEC-10. An additional protein, the paraoxonase-like protein MEC-6, is essential for transduction, and previous work suggested that MEC-6 was part of the transduction complex. We found that MEC-6 and a similar protein, POML-1, reside primarily in the endoplasmic reticulum and do not colocalize with MEC-4 on the plasma membrane in vivo. As with MEC-6, POML-1 is needed for touch sensitivity, for the neurodegeneration caused by themec-4(d)mutation, and for the expression and distribution of MEC-4 in vivo. Both proteins are likely needed for the proper folding or assembly of MEC-4 channels in vivo as measured by FRET. MEC-6 detectably increases the rate of MEC-4 accumulation on theXenopusoocyte plasma membrane. These results suggest that MEC-6 and POML-1 interact with MEC-4 to facilitate expression and localization of MEC-4 on the cell surface. Thus, MEC-6 and POML-1 act more like chaperones for MEC-4 than channel components.

scholarly journals Mechanics of the cellular microenvironment as perceived by cells in vivo

2021 ◽  
Author(s):  
Alessandro Mongera ◽  
Marie Pochitaloff ◽  
Hannah J. Gustafson ◽  
Georgina A. Stooke-Vaughan ◽  
Payam Rowghanian ◽  
...  
Keyword(s):  
Stress Relaxation ◽  
Mechanical Parameters ◽  
Cellular Microenvironment ◽  
Stem Cell Maintenance ◽  
Quantitative Studies ◽  
Cell Proliferation And Differentiation ◽  
3D Microenvironment ◽  
Proliferation And Differentiation

Tissue morphogenesis and repair, as well as organ homeostasis, require cells to constantly monitor their 3D microenvironment and adapt their behaviors in response to local biochemical and mechanical cues1-6. In vitro studies have shown that substrate stiffness and stress relaxation are important mechanical parameters in the control of cell proliferation and differentiation, stem cell maintenance, cell migration 7-11, as well as tumor progression and metastasis12,13. Yet, the mechanical parameters of the microenvironment that cells perceive in vivo, within 3D tissues, remain unknown. In complex materials with strain- and time-dependent material properties, the perceived mechanical parameters depend both on the strain and timescales at which the material is mechanically probed14. Here, we quantify in vivo and in situ the mechanics of the cellular microenvironment that cells probe during vertebrate presomitic mesoderm (PSM) specification. By analyzing the magnitude and dynamics of endogenous, cell-generated strains, we show that individual cells preferentially probe the stiffness associated with deformations of the supracellular, foam-like tissue architecture. We reveal how stress relaxation leads to a perceived microenvironment stiffness that decreases over time, with cells probing the softest regime. While stress relaxation timescales are spatially uniform in the tissue, most mechanical parameters, including those probed by cells, vary along the anteroposterior axis, as mesodermal progenitors commit to different lineages. Understanding the mechanical parameters that cells probe in their native 3D environment is important for quantitative studies of mechanosensation in vivo2-4,6,15 and can help design scaffolds for tissue engineering applications16-18.

scholarly journals Apolipoprotein L1-Specific Antibodies Detect Endogenous APOL1 inside the Endoplasmic Reticulum and on the Plasma Membrane of Podocytes

2020 ◽  
Vol 31 (9) ◽  
pp. 2044-2064 ◽  
Author(s):  
Suzie J. Scales ◽  
Nidhi Gupta ◽  
Ann M. De Mazière ◽  
George Posthuma ◽  
Cecilia P. Chiu ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Cell Surface ◽  
High Frequency ◽  
Lipid Droplets ◽  
Human Kidney ◽  
Cytoplasmic Face ◽  
Large Panel ◽  
Surface Localization ◽  
Apolipoprotein L1

BackgroundAPOL1 is found in human kidney podocytes and endothelia. Variants G1 and G2 of the APOL1 gene account for the high frequency of nondiabetic CKD among African Americans. Proposed mechanisms of kidney podocyte cytotoxicity resulting from APOL1 variant overexpression implicate different subcellular compartments. It is unclear where endogenous podocyte APOL1 resides, because previous immunolocalization studies utilized overexpressed protein or commercially available antibodies that crossreact with APOL2. This study describes and distinguishes the locations of both APOLs.MethodsImmunohistochemistry, confocal and immunoelectron microscopy, and podocyte fractionation localized endogenous and transfected APOL1 using a large panel of novel APOL1-specific mouse and rabbit monoclonal antibodies.ResultsBoth endogenous podocyte and transfected APOL1 isoforms vA and vB1 (and a little of isoform vC) localize to the luminal face of the endoplasmic reticulum (ER) and to the cell surface, but not to mitochondria, endosomes, or lipid droplets. In contrast, APOL2, isoform vB3, and most vC of APOL1 localize to the cytoplasmic face of the ER and are consequently absent from the cell surface. APOL1 knockout podocytes do not stain for APOL1, attesting to the APOL1-specificity of the antibodies. Stable re-transfection of knockout podocytes with inducible APOL1-G0, -G1, and -G2 showed no differences in localization among variants.ConclusionsAPOL1 is found in the ER and plasma membrane, consistent with either the ER stress or surface cation channel models of APOL1-mediated cytotoxicity. The surface localization of APOL1 variants potentially opens new therapeutic targeting avenues.

scholarly journals Critical role of integrin CD11c in splenic dendritic cell capture of missing-self CD47 cells to induce adaptive immunity

2018 ◽  
Vol 115 (26) ◽  
pp. 6786-6791 ◽  
Author(s):  
Jiaxi Wu ◽  
Huaizhu Wu ◽  
Jinping An ◽  
Christie M. Ballantyne ◽  
Jason G. Cyster
Keyword(s):  
Critical Role ◽  
T Cell Proliferation ◽  
Cell Capture ◽  
Antigen Uptake ◽  
Cell Proliferation And Differentiation ◽  
Proliferation And Differentiation ◽  
Missing Self

CD11c, also known as integrin alpha X, is the most widely used defining marker for dendritic cells (DCs). CD11c can bind complement iC3b and mediate phagocytosis in vitro, for which it is also referred to as complement receptor 4. However, the functions of this prominent marker protein in DCs, especially in vivo, remain poorly defined. Here, in the process of studying DC activation and immune responses induced by cells lacking self-CD47, we found that DC capture of CD47-deficient cells and DC activation was dependent on the integrin-signaling adaptor Talin1. Specifically, CD11c and its partner Itgb2 were required for DC capture of CD47-deficient cells. CD11b was not necessary for this process but could partially compensate in the absence of CD11c. Mice with DCs lacking Talin1, Itgb2, or CD11c were defective in supporting T-cell proliferation and differentiation induced by CD47-deficient cell associated antigen. These findings establish a critical role for CD11c in DC antigen uptake and activation in vivo. They may also contribute to understanding the functional mechanism of CD47-blockade therapies.

scholarly journals Two Endoplasmic Reticulum (ER) Membrane Proteins That Facilitate ER-to-Golgi Transport of Glycosylphosphatidylinositol-anchored Proteins

1999 ◽  
Vol 10 (4) ◽  
pp. 1043-1059 ◽  
Author(s):  
Wolfgang P. Barz ◽  
Peter Walter
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Protein Translocation ◽  
Sequence Similarity ◽  
Surface Proteins ◽  
Golgi Transport ◽  
Er Membrane

Many eukaryotic cell surface proteins are anchored in the lipid bilayer through glycosylphosphatidylinositol (GPI). GPI anchors are covalently attached in the endoplasmic reticulum (ER). The modified proteins are then transported through the secretory pathway to the cell surface. We have identified two genes inSaccharomyces cerevisiae, LAG1 and a novel gene termed DGT1 (for “delayed GPI-anchored protein transport”), encoding structurally related proteins with multiple membrane-spanning domains. Both proteins are localized to the ER, as demonstrated by immunofluorescence microscopy. Deletion of either gene caused no detectable phenotype, whereas lag1Δ dgt1Δ cells displayed growth defects and a significant delay in ER-to-Golgi transport of GPI-anchored proteins, suggesting thatLAG1 and DGT1 encode functionally redundant or overlapping proteins. The rate of GPI anchor attachment was not affected, nor was the transport rate of several non–GPI-anchored proteins. Consistent with a role of Lag1p and Dgt1p in GPI-anchored protein transport, lag1Δ dgt1Δ cells deposit abnormal, multilayered cell walls. Both proteins have significant sequence similarity to TRAM, a mammalian membrane protein thought to be involved in protein translocation across the ER membrane. In vivo translocation studies, however, did not detect any defects in protein translocation in lag1Δ dgt1Δcells, suggesting that neither yeast gene plays a role in this process. Instead, we propose that Lag1p and Dgt1p facilitate efficient ER-to-Golgi transport of GPI-anchored proteins.

scholarly journals Klf5 regulates muscle differentiation by directly targeting muscle-specific genes in cooperation with MyoD in mice

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Shinichiro Hayashi ◽  
Ichiro Manabe ◽  
Yumi Suzuki ◽  
Frédéric Relaix ◽  
Yumiko Oishi
Keyword(s):  
Skeletal Muscle ◽  
Muscle Regeneration ◽  
Myogenic Differentiation ◽  
Muscle Differentiation ◽  
Skeletal Muscle Regeneration ◽  
Biological Processes ◽  
Cell Proliferation And Differentiation ◽  
Proliferation And Differentiation ◽  
Acting In Concert

Krüppel-like factor 5 (Klf5) is a zinc-finger transcription factor that controls various biological processes, including cell proliferation and differentiation. We show that Klf5 is also an essential mediator of skeletal muscle regeneration and myogenic differentiation. During muscle regeneration after injury (cardiotoxin injection), Klf5 was induced in the nuclei of differentiating myoblasts and newly formed myofibers expressing myogenin in vivo. Satellite cell-specific Klf5 deletion severely impaired muscle regeneration, and myotube formation was suppressed in Klf5-deleted cultured C2C12 myoblasts and satellite cells. Klf5 knockdown suppressed induction of muscle differentiation-related genes, including myogenin. Klf5 ChIP-seq revealed that Klf5 binding overlaps that of MyoD and Mef2, and Klf5 physically associates with both MyoD and Mef2. In addition, MyoD recruitment was greatly reduced in the absence of Klf5. These results indicate that Klf5 is an essential regulator of skeletal muscle differentiation, acting in concert with myogenic transcription factors such as MyoD and Mef2.

scholarly journals A role for GPx3 in activity of normal and leukemia stem cells

2012 ◽  
Vol 209 (5) ◽  
pp. 895-901 ◽  
Author(s):  
Olivier Herault ◽  
Kristin J. Hope ◽  
Eric Deneault ◽  
Nadine Mayotte ◽  
Jalila Chagraoui ◽  
...  
Keyword(s):  
Stem Cells ◽  
Low Frequency ◽  
Leukemia Stem Cells ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Proliferation And Differentiation ◽  
Proliferation And Differentiation ◽  
Progenitor Cell Proliferation ◽  
Novel Target

The determinants of normal and leukemic stem cell self-renewal remain poorly characterized. We report that expression of the reactive oxygen species (ROS) scavenger glutathione peroxidase 3 (GPx3) positively correlates with the frequency of leukemia stem cells (LSCs) in Hoxa9+Meis1-induced leukemias. Compared with a leukemia with a low frequency of LSCs, a leukemia with a high frequency of LSCs showed hypomethylation of the Gpx3 promoter region, and expressed high levels of Gpx3 and low levels of ROS. LSCs and normal hematopoietic stem cells (HSCs) engineered to express Gpx3 short hairpin RNA (shRNA) were much less competitive in vivo than control cells. However, progenitor cell proliferation and differentiation was not affected by Gpx3 shRNA. Consistent with this, HSCs overexpressing Gpx3 were significantly more competitive than control cells in long-term repopulation experiments, and overexpression of the self-renewal genes Prdm16 or Hoxb4 boosted Gpx3 expression. In human primary acute myeloid leukemia samples, GPX3 expression level directly correlated with adverse prognostic outcome, revealing a potential novel target for the eradication of LSCs.

scholarly journals Maximal Induction of a Subset of Interferon Target Genes Requires the Chromatin-Remodeling Activity of the BAF Complex

2002 ◽  
Vol 22 (18) ◽  
pp. 6471-6479 ◽  
Author(s):  
Hong Liu ◽  
Hyeog Kang ◽  
Rui Liu ◽  
Xin Chen ◽  
Keji Zhao
Keyword(s):  
Chromatin Remodeling ◽  
Target Genes ◽  
Transcription Activator ◽  
Baf Complex ◽  
Antiproliferative Effects ◽  
Cell Proliferation And Differentiation ◽  
Proliferation And Differentiation ◽  
Maximal Induction ◽  
Antiproliferative Activities

ABSTRACT The mammalian SWI/SNF-like chromatin-remodeling BAF complex plays several important roles in controlling cell proliferation and differentiation. Interferons (IFNs) are key mediators of cellular antiviral and antiproliferative activities. In this report, we demonstrate that the BAF complex is required for the maximal induction of a subset of IFN target genes by alpha IFN (IFN-α). The BAF complex is constitutively associated with the IFITM3 promoter in vivo and facilitates the chromatin remodeling of the promoter upon IFN-α induction. Furthermore, we show that the ubiquitous transcription activator Sp1 interacts with the BAF complex in vivo and augments the BAF-mediated activation of the IFITM3 promoter. Sp1 binds constitutively to the IFITM3 promoter in the absence of the BAF complex, suggesting that it may recruit and/or stabilize the BAF complex binding to the IFITM3 promoter. Our results bring new mechanistic insights into the antiproliferative effects of the chromatin-remodeling BAF complex.

Dynein and plus-end microtubule-dependent motors are associated with specialized Sertoli cell junction plaques (ectoplasmic specializations)

2000 ◽  
Vol 113 (12) ◽  
pp. 2167-2176
Author(s):  
J.A. Guttman ◽  
G.H. Kimel ◽  
A.W. Vogl
Keyword(s):  
Endoplasmic Reticulum ◽  
Sertoli Cell ◽  
Motor Proteins ◽  
Ultrastructural Level ◽  
Cytoplasmic Dynein ◽  
Cell Junction ◽  
Cytoplasmic Face ◽  
Microtubule Transport ◽  
Type Motor

The mechanism responsible for spermatid translocation in the mammalian seminiferous epithelium was proposed to be the microtubule-based transport of specialized junction plaques (ectoplasmic specializations) that occur in Sertoli cell regions attached to spermatid heads. These plaques each consist of a cistern of endoplasmic reticulum, a layer of actin filaments and the adjacent plasma membrane. It is predicted that motor proteins function to move the junction plaques, and hence the attached spermatids, first towards the base and then back to the apex of the epithelium, along microtubules. If this hypothesis is true, motor proteins should be associated with the cytoplasmic face of the endoplasmic reticulum component of ectoplasmic specializations. In addition, isolated junction plaques should support microtubule movement both in the plus and minus directions to account for the bidirectional translocation of spermatids in vivo. To determine if cytoplasmic dynein is localized to the endoplasmic reticulum of the plaques, perfusion-fixed rat testes were immunologically probed, at the ultrastructural level, for the intermediate chain of cytoplasmic dynein (IC74). In addition, testicular fractions enriched for spermatid/junction complexes were incubated with and without gelsolin, centrifuged and the supernatants compared, by western blot analysis, for Glucose Regulated Protein 94 (a marker for endoplasmic reticulum) and IC74. At the ultrastructural level, the probe for IC74 clearly labelled material associated with the cytoplasmic face of the endoplasmic reticulum component of the junction plaques. In the gelsolin experiments, both probes reacted more strongly with appropriate bands from the gelsolin-treated supernatants than with corresponding bands from controls. To determine if the junction plaques support microtubule transport in both directions, polarity-labelled microtubules were bound to isolated spermatid/junction complexes and then assessed for motility in the presence of ATP and testicular cytosol (2 mg/ml). Of 25 recorded motility events, 17 were in a direction consistent with a plus-end directed motor being present, and 8 were in the minus-end direction. The results are consistent with the conclusion that the junction plaques have the potential for moving along microtubules in both the plus and minus directions and that both a kinesin-type and a dynein-type motor may be associated with the junction plaques. The data also indicate that cytoplasmic dynein is localized to the cytoplasmic face of the endoplasmic reticulum component of the plaques.

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