scholarly journals Role of plasma-membrane-bound sialidase NEU3 in clathrin-mediated endocytosis

2015 ◽  
Vol 470 (1) ◽  
pp. 131-144 ◽  
Author(s):  
Macarena Rodriguez-Walker ◽  
Aldo A. Vilcaes ◽  
Eduardo Garbarino-Pico ◽  
José L. Daniotti
Keyword(s):  
Plasma Membrane ◽  
Subcellular Distribution ◽  
Cellular Functions ◽  
Coated Pits ◽  
Key Enzyme ◽  
Membrane Bound

Sialidase NEU3 is a key enzyme in the catabolism of gangliosides. We demonstrated that NEU3 impairs cargo internalization via clathrin-coated pits, affecting AP-2 subcellular distribution. This study delineates previously unidentified cellular functions of NEU3.

scholarly journals Levels of Human Erythrocyte Membrane-Bound and Cytosolic Glycohydrolases Are Associated with Oxidative Stress in Erectile Dysfunction Patients

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
L. Massaccesi ◽  
G. V. Melzi d’Eril ◽  
G. M. Colpi ◽  
G. Tettamanti ◽  
G. Goi ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Plasma Membrane ◽  
Erectile Dysfunction ◽  
Plasma Membranes ◽  
Lag Time ◽  
Specific Marker ◽  
Human Erythrocyte Membrane ◽  
Key Enzyme ◽  
Membrane Bound

Oxidative stress (OS) and production of NO, by endothelium nitric oxide synthetase (eNOS), are involved in the pathophysiology of erectile dysfunction (ED). Moreover, OS induces modifications of the physicochemical properties of erythrocyte (RBC) plasma membranes and of the enzyme content of the same membranes. Due to their role in signalling early membrane alterations in OS-related pathologies, several plasma membrane and cytosolic glycohydrolases of human RBC have been proposed as new markers of cellular OS. In RBC, NOS can be activated and deactivated by phosphorylation/glycosylation. In this regulatory mechanism O-β-N-AcetylGlucosaminidase is a key enzyme. Cellular levels of O-GlcNAcylated proteins are related to OS; consequently dysfunctional eNOS O-GlcNAcylation seems to have a crucial role in ED. To elucidate the possible association between RBC glycohydrolases and OS, plasma hydroperoxides and antioxidant total defenses (Lag-time), cytosolic O-β-N-AcetylGlucosaminidase, cytosolic and membrane Hexosaminidase, membraneβ-D-Glucuronidase, andα-D-Glucosidase have been studied in 39 ED patients and 30 controls. In ED subjects hydroperoxides and plasma membrane glycohydrolases activities are significantly increased whereas Lag-time values and cytosolic glycohydrolases activities are significantly decreased. These data confirm the strong OS status in ED patients, the role of the studied glycohydrolases as early OS biomarker and suggest their possible use as specific marker of ED patients, particularly in those undergoing nutritional/pharmacological antioxidant therapy.

Outside or inside: role of the subcellular localization of DP4-like enzymes for substrate conversion and inhibitor effects

2011 ◽  
Vol 392 (3) ◽  
Author(s):  
Ute Bank ◽  
Anke Heimburg ◽  
Astrid Wohlfarth ◽  
Gudrun Koch ◽  
Karsten Nordhoff ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Low Molecular Weight ◽  
Immune Functions ◽  
Cellular Functions ◽  
Fluorogenic Substrates ◽  
Cellular Effects ◽  
Viable Cells ◽  
Substrate Conversion ◽  
Membrane Bound

Abstract The discovery of the DP4-related enzymes DP8 and DP9 raised controversial discussion regarding the physiological and pathophysiological function of distinct members of the DP4 family. Particularly with regard to their potential relevance in regulating immune functions, it is of interest to know which role the subcellular distribution of the enzymes play. Synthetic substrates as well as low molecular weight inhibitors are widely used as tools, but little is yet known regarding their features in cell experiments, such as their plasma membrane penetration capacity. The fluorogenic substrates Gly-Pro-AMC or (Ala-Pro)2-R110 predominantly detect plasma membrane-bound activities of viable cells (less than 0.1% of fluorochromes R110 or AMC inside viable cells after 1 h incubation). Additionally, the selective and non-selective DP8/9 inhibitors allo-Ile-isoindoline and Lys[Z(NO2)]-pyrrolidide were found to be incapable of passing the plasma membrane easily. This suggests that previously reported cellular effects are not due to inhibition of the cytosolic enzymes DP8 or DP9. Moreover, our enzymatic studies with viable cells provided evidence that DP8 and/or DP9 are also present on the surface of immune cells under certain circumstances and could gain relevance particularly in the absence of DP4 expression. In summary, in cells which do express DP4 on the surface, this archetypical member of the DP4 family is the most relevant peptidase in the regulation of cellular functions.

scholarly journals The inositol 5-phosphatase SHIP2 regulates endocytic clathrin-coated pit dynamics

2010 ◽  
Vol 190 (3) ◽  
pp. 307-315 ◽  
Author(s):  
Fubito Nakatsu ◽  
Rushika M. Perera ◽  
Louise Lucast ◽  
Roberto Zoncu ◽  
Jan Domin ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Insulin Signaling ◽  
Negative Regulator ◽  
Phosphoinositide Metabolism ◽  
Cellular Functions ◽  
Coated Pits ◽  
Positive Role ◽  
Insulin Signal ◽  
Signal Output

Phosphatidylinositol (PI) 4,5-bisphosphate (PI(4,5)P2) and its phosphorylated product PI 3,4,5-triphosphate (PI(3,4,5)P3) are two major phosphoinositides concentrated at the plasma membrane. Their levels, which are tightly controlled by kinases, phospholipases, and phosphatases, regulate a variety of cellular functions, including clathrin-mediated endocytosis and receptor signaling. In this study, we show that the inositol 5-phosphatase SHIP2, a negative regulator of PI(3,4,5)P3-dependent signaling, also negatively regulates PI(4,5)P2 levels and is concentrated at endocytic clathrin-coated pits (CCPs) via interactions with the scaffold protein intersectin. SHIP2 is recruited early at the pits and dissociates before fission. Both knockdown of SHIP2 expression and acute production of PI(3,4,5)P3 shorten CCP lifetime by enhancing the rate of pit maturation, which is consistent with a positive role of both SHIP2 substrates, PI(4,5)P2 and PI(3,4,5)P3, on coat assembly. Because SHIP2 is a negative regulator of insulin signaling, our findings suggest the importance of the phosphoinositide metabolism at CCPs in the regulation of insulin signal output.

scholarly journals EB1 binding restricts STIM1 translocation to ER–PM junctions and regulates store-operated Ca2+ entry

2018 ◽  
Vol 217 (6) ◽  
pp. 2047-2058 ◽  
Author(s):  
Chi-Lun Chang ◽  
Yu-Ju Chen ◽  
Carlo Giovanni Quintanilla ◽  
Ting-Sung Hsieh ◽  
Jen Liou
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Cellular Functions ◽  
Binding Ability ◽  
Trapping Mechanism ◽  
Spatiotemporal Regulation

The endoplasmic reticulum (ER) Ca2+ sensor STIM1 forms oligomers and translocates to ER–plasma membrane (PM) junctions to activate store-operated Ca2+ entry (SOCE) after ER Ca2+ depletion. STIM1 also interacts with EB1 and dynamically tracks microtubule (MT) plus ends. Nevertheless, the role of STIM1–EB1 interaction in regulating SOCE remains unresolved. Using live-cell imaging combined with a synthetic construct approach, we found that EB1 binding constitutes a trapping mechanism restricting STIM1 targeting to ER–PM junctions. We further showed that STIM1 oligomers retain EB1 binding ability in ER Ca2+-depleted cells. By trapping STIM1 molecules at dynamic contacts between the ER and MT plus ends, EB1 binding delayed STIM1 translocation to ER–PM junctions during ER Ca2+ depletion and prevented excess SOCE and ER Ca2+ overload. Our study suggests that STIM1–EB1 interaction shapes the kinetics and amplitude of local SOCE in cellular regions with growing MTs and contributes to spatiotemporal regulation of Ca2+ signaling crucial for cellular functions and homeostasis.

scholarly journals Morphogenic Effects of Ezrin Require a Phosphorylation-Induced Transition from Oligomers to Monomers at the Plasma Membrane

2000 ◽  
Vol 150 (1) ◽  
pp. 193-204 ◽  
Author(s):  
Alexis Gautreau ◽  
Daniel Louvard ◽  
Monique Arpin
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Wild Type ◽  
Erm Proteins ◽  
Threonine Residue ◽  
Phosphorylation Event ◽  
Membrane Bound

ERM (ezrin, radixin, moesin) proteins act as linkers between the plasma membrane and the actin cytoskeleton. An interaction between their NH2- and COOH-terminal domains occurs intramolecularly in closed monomers and intermolecularly in head-to-tail oligomers. In vitro, phosphorylation of a conserved threonine residue (T567 in ezrin) in the COOH-terminal domain of ERM proteins disrupts this interaction. Here, we have analyzed the role of this phosphorylation event in vivo, by deriving stable clones producing wild-type, T567A, and T567D ezrin from LLC-PK1 epithelial cells. We found that T567A ezrin was poorly associated with the cytoskeleton, but was able to form oligomers. In contrast, T567D ezrin was associated with the cytoskeleton, but its distribution was shifted from oligomers to monomers at the membrane. Moreover, production of T567D ezrin induced the formation of lamellipodia, membrane ruffles, and tufts of microvilli. Both T567A and T567D ezrin affected the development of multicellular epithelial structures. Collectively, these results suggest that phosphorylation of ERM proteins on this conserved threonine regulates the transition from membrane-bound oligomers to active monomers, which induce and are part of actin-rich membrane projections.

scholarly journals EB1 binding provides a diffusion trap mechanism regulating STIM1 localization and Ca2+ signaling

2017 ◽  
Author(s):  
Chi-Lun Chang ◽  
Yu-Ju Chen ◽  
Jen Liou
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Time Lapse ◽  
Cellular Functions ◽  
Binding Ability ◽  
Spatiotemporal Regulation ◽  
Time Lapse Imaging

AbstractThe endoplasmic reticulum (ER) Ca2+ sensor STIM1 forms oligomers and translocates to ER-plasma membrane (PM) junctions to activate store-operated Ca2+ entry (SOCE) following ER Ca2+ depletion. STIM1 also directly interacts with end binding protein 1 (EB1) at microtubule (MT) plus-ends and resembles comet-like structures during time-lapse imaging. Nevertheless, the role of STIM1-EB1 interaction in regulating SOCE remains unresolved. Using live-cell imaging combined with pharmacological perturbation and a reconstitution approach, we revealed that EB1 binding constitutes a diffusion trap mechanism restricting STIM1 targeting to ER-PM junctions. We further showed that STIM1 oligomers retain EB1 binding ability in ER Ca2+-depleted cells. EB1 binding delayed the translocation of STIM1 oligomers to ER-PM junctions and recaptured STIM1 to prevent excess SOCE and ER Ca2+ overload. Thus, the counterbalance of EB1 binding and PM targeting of STIM1 shapes the kinetics and amplitude of local SOCE in regions with growing MTs, and contributes to precise spatiotemporal regulation of Ca2+ signaling crucial for cellular functions and homeostasis.SummarySTIM1 activates store-operated Ca2+ entry (SOCE) by translocating to endoplasmic reticulum-plasma membrane junctions. Chang et al. revealed that STIM1 localization and SOCE are regulated by a diffusion trap mechanism mediated by STIM1 binding to EB1 at growing microtubule ends.

scholarly journals Optogenetic inhibition and activation of Rac and Rap1 using a modified iLID system

2020 ◽  
Author(s):  
Nick R. Elston ◽  
Michael Pablo ◽  
Fred Pimenta ◽  
Klaus M. Hahn ◽  
Takashi Watanabe
Keyword(s):  
Plasma Membrane ◽  
Living Cells ◽  
Small Gtpases ◽  
Spatiotemporal Dynamics ◽  
Membrane Localization ◽  
Cellular Functions ◽  
Activation Kinetics ◽  
System 1 ◽  
Control Plasma

The small GTPases Rac1 and Rap1 can fulfill multiple cellular functions because their activation kinetics and localization are precisely controlled. To probe the role of their spatiotemporal dynamics, we generated optogenetic tools that activate or inhibit endogenous Rac and Rap1 in living cells. An improved version of the light induced dimerization (iLID) system [1] was used to control plasma membrane localization of protein domains that specifically activate or inactivate Rap1 and Rac (Tiam1 and Chimerin for Rac, RasGRP2 and Rap1GAP for Rap1 [2, 3, 4, 5]). Irradiation yielded a 50% to 230% increase in the concentration of these domains at the membrane, leading to effects on cell morphodynamics consistent with the known roles of Rac1 and Rap1.

scholarly journals Quantitative FRET Microscopy Reveals a Crucial Role of Cytoskeleton in Promoting PI(4,5)P2 Confinement

2021 ◽  
Vol 22 (21) ◽  
pp. 11727
Author(s):  
Maria J. Sarmento ◽  
Luís Borges-Araújo ◽  
Sandra N. Pinto ◽  
Nuno Bernardes ◽  
Joana C. Ricardo ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Hela Cells ◽  
Membrane Trafficking ◽  
Actin Polymerization ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cellular Functions ◽  
Cytoskeleton Organization

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is an essential plasma membrane component involved in several cellular functions, including membrane trafficking and cytoskeleton organization. This function multiplicity is partially achieved through a dynamic spatiotemporal organization of PI(4,5)P2 within the membrane. Here, we use a Förster resonance energy transfer (FRET) approach to quantitatively assess the extent of PI(4,5)P2 confinement within the plasma membrane. This methodology relies on the rigorous evaluation of the dependence of absolute FRET efficiencies between pleckstrin homology domains (PHPLCδ) fused with fluorescent proteins and their average fluorescence intensity at the membrane. PI(4,5)P2 is found to be significantly compartmentalized at the plasma membrane of HeLa cells, and these clusters are not cholesterol-dependent, suggesting that membrane rafts are not involved in the formation of these nanodomains. On the other hand, upon inhibition of actin polymerization, compartmentalization of PI(4,5)P2 is almost entirely eliminated, showing that the cytoskeleton network is the critical component responsible for the formation of nanoscale PI(4,5)P2 domains in HeLa cells.

scholarly journals SCD1, autophagy and cancer: implications for therapy

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Francesca Ascenzi ◽  
Claudia De Vitis ◽  
Marcello Maugeri-Saccà ◽  
Christian Napoli ◽  
Gennaro Ciliberto ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Lipid Metabolism ◽  
Monounsaturated Fatty Acids ◽  
Cellular Functions ◽  
Cancer Heterogeneity ◽  
Intracellular Degradation ◽  
Mutual Regulation ◽  
Key Enzyme ◽  
Proliferation And Metastasis

Abstract Background Autophagy is an intracellular degradation system that removes unnecessary or dysfunctional components and recycles them for other cellular functions. Over the years, a mutual regulation between lipid metabolism and autophagy has been uncovered. Methods This is a narrative review discussing the connection between SCD1 and the autophagic process, along with the modality through which this crosstalk can be exploited for therapeutic purposes. Results Fatty acids, depending on the species, can have either activating or inhibitory roles on autophagy. In turn, autophagy regulates the mobilization of fat from cellular deposits, such as lipid droplets, and removes unnecessary lipids to prevent cellular lipotoxicity. This review describes the regulation of autophagy by lipid metabolism in cancer cells, focusing on the role of stearoyl-CoA desaturase 1 (SCD1), the key enzyme involved in the synthesis of monounsaturated fatty acids. SCD1 plays an important role in cancer, promoting cell proliferation and metastasis. The role of autophagy in cancer is more complex since it can act either by protecting against the onset of cancer or by promoting tumor growth. Mounting evidence indicates that autophagy and lipid metabolism are tightly interconnected. Conclusion Here, we discuss controversial findings of SCD1 as an autophagy inducer or inhibitor in cancer, highlighting how these activities may result in cancer promotion or inhibition depending upon the degree of cancer heterogeneity and plasticity.

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