scholarly journals Drosophila PLP forms centriolar-clouds that promote centriole stability, cohesion and MT nucleation

2017 ◽  
Author(s):  
Helio Roque ◽  
Metta Pratt ◽  
Errin Johnson ◽  
Jordan W. Raff
Keyword(s):  
Plasma Membrane ◽  
Transition Zone ◽  
Dense Material ◽  
Structural Defects ◽  
Partial Loss ◽  
Centrosomal Protein ◽  
Electron Dense Material ◽  
Mother Centriole ◽  
Mutant Cells

AbstractPericentrin is a conserved centrosomal protein whose dysfunction has been linked to several human diseases. The precise function of Pericentrin, however, is controversial. Here, we examine Drosophila Pericentrin-like- protein (PLP) function in vivo, in tissues that form both centrosomes and cilia. PLP mutant centrioles exhibit four major defects: (1) They are too short and have subtle structural defects; (2) They separate prematurely, and so overduplicate; (3) They organise fewer MTs during interphase; (4) They fail to establish and/or maintain a proper connection to the plasma membrane— although, surprisingly, mutant centrioles can still form an axoneme and recruit transition zone (TZ) proteins. We show that PLP helps to form “ pericentriolar clouds” of electron-dense material that emanate from the central cartwheel spokes and spread outward to surround the mother centriole. The partial loss of these structures may explain the complex centriole, centrosome and cilium defects we observe in PLP mutant cells.

scholarly journals Loss of CEP162 function at the primary cilium delays ciliogenesis and causes retinal ciliopathy in humans

2021 ◽  
Author(s):  
Nafisa Nuzhat ◽  
Kristof Van Schil ◽  
Sandra Liakopoulos ◽  
Miriam Bauwens ◽  
Alfredo Dueñas Rey ◽  
...  
Keyword(s):  
Transition Zone ◽  
Primary Cilium ◽  
Outer Segment ◽  
Retinal Dystrophy ◽  
Mrna Levels ◽  
Loss Of Function ◽  
Photoreceptor Outer Segment ◽  
Zone Formation ◽  
Mother Centriole

Ciliopathies often comprise retinal degeneration since the photoreceptor outer segment is an adapted primary cilium. CEP162 is a distal end centriolar protein required for proper transition zone assembly during ciliogenesis and whose loss causes ciliopathy in zebrafish. CEP162 has so far not been implicated in human disease. Here, we identified a homozygous CEP162 frameshift variant, c.1935dupA (p.(E646R*5)), in retinitis pigmentosa patients from two unrelated Moroccan families, likely representing a founder allele. We found that even though mRNA levels were reduced, the truncated CEP162-E646R*5 protein was expressed and localized to the mitotic spindle during mitosis, but not at the basal body of the cilium. In CEP162 knockdown cells, expression of the truncated CEP162-E646R*5 protein is unable to restore ciliation indicating its loss of function at the cilium. In patient fibroblasts, cilia overcome the absence of CEP162 from the primary cilium by delaying ciliogenesis through the persistence of CP110 at the mother centriole. The patient fibroblasts are ultimately able to extend some abnormally long cilia that are missing key transition zone components. Defective transition zone formation likely disproportionately affects the long-living ciliary outer segment of photoreceptors resulting in retinal dystrophy. CEP162 is expressed in human retina, and we show that wild-type CEP162, but not truncated CEP162-E646R*5, specifically localizes to the distal end of centrioles of mouse photoreceptor cilia. Together, our genetic, cell-based, and in vivo modeling establish that CEP162 deficiency causes retinal ciliopathy in humans.

scholarly journals Elimination of defective alpha-factor pheromone receptors.

1997 ◽  
Vol 17 (11) ◽  
pp. 6236-6245 ◽  
Author(s):  
D D Jenness ◽  
Y Li ◽  
C Tipper ◽  
P Spatrick
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Cell Surface ◽  
Half Life ◽  
Structural Defects ◽  
Receptor Protein ◽  
Mutant Form ◽  
Wild Type ◽  
Alpha Factor ◽  
Mutant Cells

This report compares trafficking routes of a plasma membrane protein that was misfolded either during its synthesis or after it had reached the cell surface. A temperature-sensitive mutant form of the yeast alpha-factor pheromone receptor (ste2-3) was found to provide a model substrate for quality control of plasma membrane proteins. We show for the first time that a misfolded membrane protein is recognized at the cell surface and rapidly removed. When the ste2-3 mutant cells were cultured continuously at 34 degrees C, the mutant receptor protein (Ste2-3p) failed to accumulate at the plasma membrane and was degraded with a half-life of 4 min, compared with a half-life of 33 min for wild-type receptor protein (Ste2p). Degradation of both Ste2-3p and Ste2p required the vacuolar proteolytic activities controlled by the PEP4 gene. At 34 degrees C, Ste2-3p comigrated with glycosylated Ste2p on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that Ste2-3p enters the secretory pathway. Degradation of Ste2-3p did not require delivery to the plasma membrane as the sec1 mutation failed to block rapid turnover. Truncation of the C-terminal cytoplasmic domain of the mutant receptors did not permit accumulation at the plasma membrane; thus, the endocytic signals contained in this domain are unnecessary for intracellular retention. In the pep4 mutant, Ste2-3p accumulated as series of high-molecular-weight species, suggesting a potential role for ubiquitin in the elimination process. When ste2-3 mutant cells were cultured continuously at 22 degrees C, Ste2-3p accumulated in the plasma membrane. When the 22 degrees C culture was shifted to 34 degrees C, Ste2-3p was removed from the plasma membrane and degraded by a PEP4-dependent mechanism with a 24-min half-life; the wild-type Ste2p displayed a 72-min half-life. Thus, structural defects in Ste2-3p synthesized at 34 degrees C are recognized in transit to the plasma membrane, leading to rapid degradation, and Ste2-3p that is preassembled at the plasma membrane is also removed and degraded following a shift to 34 degrees C.

Deletion of the PDR16 gene influences the plasma membrane properties of the yeast Kluyveromyces lactis

2015 ◽  
Vol 61 (4) ◽  
pp. 273-279 ◽  
Author(s):  
Nora Toth Hervay ◽  
Eduard Goffa ◽  
Alexandra Svrbicka ◽  
Zuzana Simova ◽  
Peter Griac ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Kluyveromyces Lactis ◽  
Lipid Synthesis ◽  
Membrane Properties ◽  
First Line ◽  
Cell Defense ◽  
Transfer Protein ◽  
Phosphatidylinositol Transfer ◽  
Mutant Cells

The plasma membrane is the first line of cell defense against changes in external environment, thus its integrity and functionality are of utmost importance. The plasma membrane properties depend on both its protein and lipid composition. The PDR16 gene is involved in the control of Kluyveromyces lactis susceptibility to drugs and alkali metal cations. It encodes the homologue of the major K. lactis phosphatidylinositol transfer protein Sec14p. Sec14p participates in protein secretion, regulation of lipid synthesis, and turnover in vivo. We report here that the plasma membrane of the Klpdr16Δ mutant is hyperpolarized and its fluidity is lower than that of the parental strain. In addition, protoplasts prepared from the Klpdr16Δ cells display decreased stability when subjected to hypo-osmotic conditions. These changes in membrane properties lead to an accumulation of radiolabeled fluconazole and lithium cations inside mutant cells. Our results point to the fact that the PDR16 gene of K. lactis (KlPDR16) influences the plasma membrane properties in K. lactis that lead to subsequent changes in susceptibility to a broad range of xenobiotics.

Comparative Morphology of Intercellular Bridges Between Developing Germ Cells of the Ovary

1970 ◽  
Vol 28 ◽  
pp. 328-329
Author(s):  
J. R. Ruby ◽  
R. F. Dyer ◽  
R. G. Skalko ◽  
R. F. Gasser ◽  
E. P. Volpe
Keyword(s):  
Germ Cells ◽  
Rana Pipiens ◽  
Comparative Morphology ◽  
Dense Material ◽  
Individual Species ◽  
Microscope Examination ◽  
Electron Dense Material ◽  
Intercellular Bridges ◽  
Cytoplasmic Side ◽  
Intercellular Connections

An electron microscope examination of fetal ovaries has revealed that developing germ cells are connected by intercellular bridges. In this investigation several species have been studied including human, mouse, chicken, and tadpole (Rana pipiens). These studies demonstrate that intercellular connections are similar in morphology regardless of the species.Basically, all bridges are characterized by a band of electron-dense material on the cytoplasmic side of the tri-laminar membrane surrounding the connection (Fig.l). This membrane is continuous with the plasma membrane of the conjoined cells. The dense material, however, never extends beyond the limits of the bridge. Variations in the configuration of intercellular connections were noted in all ovaries studied. However, the bridges in each individual species usually exhibits one structural characteristic seldom found in the others. For example, bridges in the human ovary very often have large blebs projecting from the lateral borders whereas the sides of the connections in the mouse gonad merely demonstrate a slight convexity.

scholarly journals Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

1999 ◽  
Vol 339 (2) ◽  
pp. 299-307 ◽  
Author(s):  
Arthur L. KRUCKEBERG ◽  
Ling YE ◽  
Jan A. BERDEN ◽  
Karel van DAM
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Glucose Transport ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Hexose Transporter ◽  
High Concentrations ◽  
Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

scholarly journals RNF141 interacts with KRAS to promote colorectal cancer progression

Oncogene ◽  
2021 ◽  
Author(s):  
Jiuna Zhang ◽  
Xiaoyu Jiang ◽  
Jie Yin ◽  
Shiying Dou ◽  
Xiaoli Xie ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Plasma Membrane ◽  
Tumor Growth ◽  
Cancer Progression ◽  
Critical Role ◽  
Ring Finger ◽  
Immunohistochemical Analysis ◽  
Bimolecular Fluorescence Complementation

AbstractRING finger proteins (RNFs) play a critical role in cancer initiation and progression. RNF141 is a member of RNFs family; however, its clinical significance, roles, and mechanism in colorectal cancer (CRC) remain poorly understood. Here, we examined the expression of RNF141 in 64 pairs of CRC and adjacent normal tissues by real-time PCR, Western blot, and immunohistochemical analysis. We found that there was more expression of RNF141 in CRC tissue compared with its adjacent normal tissue and high RNF141 expression associated with T stage. In vivo and in vitro functional experiments were conducted and revealed the oncogenic role of RNF141 in CRC. RNF141 knockdown suppressed proliferation, arrested the cell cycle in the G1 phase, inhibited migration, invasion and HUVEC tube formation but promoted apoptosis, whereas RNF141 overexpression exerted the opposite effects in CRC cells. The subcutaneous xenograft models showed that RNF141 knockdown reduced tumor growth, but its overexpression promoted tumor growth. Mechanistically, liquid chromatography-tandem mass spectrometry indicated RNF141 interacted with KRAS, which was confirmed by Co-immunoprecipitation, Immunofluorescence assay. Further analysis with bimolecular fluorescence complementation (BiFC) and Glutathione-S-transferase (GST) pull-down assays showed that RNF141 could directly bind to KRAS. Importantly, the upregulation of RNF141 increased GTP-bound KRAS, but its knockdown resulted in a reduction accordingly. Next, we demonstrated that RNF141 induced KRAS activation via increasing its enrichment on the plasma membrane not altering total KRAS expression, which was facilitated by the interaction with LYPLA1. Moreover, KRAS silencing partially abolished the effect of RNF141 on cell proliferation and apoptosis. In addition, our findings presented that RNF141 functioned as an oncogene by upregulating KRAS activity in a manner of promoting KRAS enrichment on the plasma membrane in CRC.

scholarly journals Roles for RNA export factor, Nxt1, in ensuring muscle integrity and normal RNA expression in Drosophila

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Kevin van der Graaf ◽  
Katia Jindrich ◽  
Robert Mitchell ◽  
Helen White-Cooper
Keyword(s):  
Mrna Export ◽  
Rna Stability ◽  
Muscle Degeneration ◽  
Loss Of Function ◽  
Cellular Functions ◽  
Partial Loss ◽  
Export Pathway ◽  
Pathway Gene ◽  
Rna Export

Abstract The mRNA export pathway is responsible for the transport of mRNAs from the nucleus to the cytoplasm, and thus is essential for protein production and normal cellular functions. A partial loss of function allele of the mRNA export factor Nxt1 in Drosophila shows reduced viability and sterility. A previous study has shown that the male fertility defect is due to a defect in transcription and RNA stability, indicating the potential for this pathway to be implicated in processes beyond the known mRNA transport function. Here we investigate the reduced viability of Nxt1 partial loss of function mutants, and describe a defect in growth and maintenance of the larval muscles, leading to muscle degeneration. RNA-seq revealed reduced expression of a set of mRNAs, particularly from genes with long introns in Nxt1 mutant carcass. We detected differential expression of circRNA, and significantly fewer distinct circRNAs expressed in the mutants. Despite the widespread defects in gene expression, muscle degeneration was rescued by increased expression of the costamere component tn (abba) in muscles. This is the first report of a role for the RNA export pathway gene Nxt1 in the maintenance of muscle integrity. Our data also links the mRNA export pathway to a specific role in the expression of mRNA and circRNA from common precursor genes, in vivo.

scholarly journals Synaptotagmins at the endoplasmic reticulum–plasma membrane contact sites maintain diacylglycerol homeostasis during abiotic stress

2021 ◽  
Author(s):  
Noemi Ruiz-Lopez ◽  
Jessica Pérez-Sancho ◽  
Alicia Esteban del Valle ◽  
Richard P Haslam ◽  
Steffen Vanneste ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Abiotic Stress ◽  
Fluorescent Protein ◽  
Mechanical Damage ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Contact ◽  
Green Fluorescent

Abstract Endoplasmic reticulum-plasma membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis thaliana mutants lacking the ER-PM protein tether synaptotagmin1 (SYT1) exhibit decreased plasma membrane (PM) integrity under multiple abiotic stresses such as freezing, high salt, osmotic stress and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER-PM tether that also functions in maintaining PM integrity. The ER-PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to wild type while the levels of most glycerolipid species remain unchanged. Additionally, the SYT1-green fluorescent protein (GFP) fusion preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress.

scholarly journals Norepinephrine Protects against Methamphetamine Toxicity through β2-Adrenergic Receptors Promoting LC3 Compartmentalization

2021 ◽  
Vol 22 (13) ◽  
pp. 7232
Author(s):  
Gloria Lazzeri ◽  
Carla L. Busceti ◽  
Francesca Biagioni ◽  
Cinzia Fabrizi ◽  
Gabriele Morucci ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Light Chain ◽  
Adrenergic Receptors ◽  
Cell Damage ◽  
Protective Effects ◽  
Autophagy Flux ◽  
Brain Areas ◽  
Indirect Consequence

Norepinephrine (NE) neurons and extracellular NE exert some protective effects against a variety of insults, including methamphetamine (Meth)-induced cell damage. The intimate mechanism of protection remains difficult to be analyzed in vivo. In fact, this may occur directly on target neurons or as the indirect consequence of NE-induced alterations in the activity of trans-synaptic loops. Therefore, to elude neuronal networks, which may contribute to these effects in vivo, the present study investigates whether NE still protects when directly applied to Meth-treated PC12 cells. Meth was selected based on its detrimental effects along various specific brain areas. The study shows that NE directly protects in vitro against Meth-induced cell damage. The present study indicates that such an effect fully depends on the activation of plasma membrane β2-adrenergic receptors (ARs). Evidence indicates that β2-ARs activation restores autophagy, which is impaired by Meth administration. This occurs via restoration of the autophagy flux and, as assessed by ultrastructural morphometry, by preventing the dissipation of microtubule-associated protein 1 light chain 3 (LC3) from autophagy vacuoles to the cytosol, which is produced instead during Meth toxicity. These findings may have an impact in a variety of degenerative conditions characterized by NE deficiency along with autophagy impairment.

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