scholarly journals Massive endocytosis driven by lipidic forces originating in the outer plasmalemmal monolayer: a new approach to membrane recycling and lipid domains

2011 ◽  
Vol 137 (2) ◽  
pp. 137-154 ◽  
Author(s):  
Michael Fine ◽  
Marc C. Llaguno ◽  
Vincenzo Lariccia ◽  
Mei-Jung Lin ◽  
Alp Yaradanakul ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Atp Hydrolysis ◽  
Surface Membrane ◽  
Sodium Dodecyl ◽  
Hek293 Cells ◽  
Nitric Oxide Donor ◽  
Cytoskeleton Remodeling ◽  
Nonionic Detergents

The roles that lipids play in endocytosis are the subject of debate. Using electrical and imaging methods, we describe massive endocytosis (MEND) in baby hamster kidney (BHK) and HEK293 cells when the outer plasma membrane monolayer is perturbed by the nonionic detergents, Triton X-100 (TX100) and NP-40. Some alkane detergents, the amphipathic drugs, edelfosine and tamoxifen, and the phospholipase inhibitor, U73122, are also effective. Uptake of the membrane tracer, FM 4–64, into vesicles and loss of reversible FM 4–64 binding confirm that 40–75% of the cell surface is internalized. Ongoing MEND stops in 2–4 s when amphipaths are removed, and amphipaths are without effect from the cytoplasmic side. Thus, expansion of the outer monolayer is critical. As found for Ca-activated MEND, vesicles formed are <100 nm in diameter, membrane ruffles are lost, and β-cyclodextrin treatments are inhibitory. However, amphipath-activated MEND does not require Ca transients, adenosine triphosphate (ATP) hydrolysis, G protein cycling, dynamins, or actin cytoskeleton remodeling. With elevated cytoplasmic ATP (>5 mM), MEND can reverse completely and be repeated multiple times in BHK and HEK293 cells, but not cardiac myocytes. Reversal is blocked by N-ethylmaleimide and a nitric oxide donor, nitroprusside. Constitutively expressed Na/Ca exchangers internalize roughly in proportion to surface membrane, whereas Na/K pump activities decrease over-proportionally. Sodium dodecyl sulfate and dodecylglucoside do not cause MEND during their application, but MEND occurs rapidly when they are removed. As monitored capacitively, the binding of these detergents decreases with MEND, whereas TX100 binding does not decrease. In summary, nonionic detergents can fractionate the plasma membrane in vivo, and vesicles formed connect immediately to physiological membrane-trafficking mechanisms. We suggest that lateral and transbilayer inhomogeneities of the plasma membrane provide potential energies that, when unbridled by triggers, can drive endocytosis by lipidic forces.

scholarly journals Secretory Pathway-Dependent Localization of the Saccharomyces cerevisiae Rho GTPase-Activating Protein Rgd1p at Growth Sites

2012 ◽  
Vol 11 (5) ◽  
pp. 590-600 ◽  
Author(s):  
Fabien Lefèbvre ◽  
Valérie Prouzet-Mauléon ◽  
Michel Hugues ◽  
Marc Crouzet ◽  
Aurélie Vieillemard ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Cell Cycle Progression ◽  
Secretory Pathway ◽  
Rho Gtpase ◽  
Secretory Vesicles ◽  
Gtpase Activating Protein ◽  
Content Type

ABSTRACT Establishment and maintenance of cell polarity in eukaryotes depends upon the regulation of Rho GTPases. In Saccharomyces cerevisiae , the Rho GTPase activating protein (RhoGAP) Rgd1p stimulates the GTPase activities of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively. Consistent with the distribution of Rho3p and Rho4p, Rgd1p is found mostly in areas of polarized growth during cell cycle progression. Rgd1p was mislocalized in mutants specifically altered for Golgi apparatus-based phosphatidylinositol 4-P [PtdIns(4)P] synthesis and for PtdIns(4,5)P 2 production at the plasma membrane. Analysis of Rgd1p distribution in different membrane-trafficking mutants suggested that Rgd1p was delivered to growth sites via the secretory pathway. Rgd1p may associate with post-Golgi vesicles by binding to PtdIns(4)P and then be transported by secretory vesicles to the plasma membrane. In agreement, we show that Rgd1p coimmunoprecipitated and localized with markers specific to secretory vesicles and cofractionated with a plasma membrane marker. Moreover, in vivo imaging revealed that Rgd1p was transported in an anterograde manner from the mother cell to the daughter cell in a vectoral manner. Our data indicate that secretory vesicles are involved in the delivery of RhoGAP Rgd1p to the bud tip and bud neck.

scholarly journals Extended Synaptotagmin is a presynaptic ER Ca2+ sensor that promotes neurotransmission and synaptic growth in Drosophila

2017 ◽  
Author(s):  
Koto Kikuma ◽  
Daniel Kim ◽  
David Sutter ◽  
Xiling Li ◽  
Dion K. Dickman
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Synaptic Activity ◽  
Synaptic Growth ◽  
Synaptic Physiology ◽  
Lipid Exchange ◽  
Membrane Tethering ◽  
Conserved Gene ◽  
Neuronal Functions

ABSTRACTThe endoplasmic reticulum (ER) is an extensive presynaptic organelle, exerting important influences at synapses by responding to Ca2+ and modulating transmission, growth, lipid metabolism, and membrane trafficking. Despite intriguing evidence for these crucial functions, how presynaptic ER influences synaptic physiology remains enigmatic. To gain insight into this question, we have generated and characterized mutations in the single Extended Synaptotagmin (Esyt) ortholog in Drosophila. Esyts are evolutionarily conserved ER proteins with Ca2+ sensing domains that have recently been shown to orchestrate membrane tethering and lipid exchange between the ER and plasma membrane. We first demonstrate that Esyt localizes to an extensive ER structure that invades presynaptic terminals at the neuromuscular junction. Next, we show that synaptic growth, structure, function, and plasticity are surprisingly unperturbed at synapses lacking Esyt expression. However, presynaptic overexpression of Esyt leads to enhanced synaptic growth, neurotransmission, and sustainment of the vesicle pool during intense levels of activity, suggesting that elevated Esyt at the ER promotes constitutive membrane trafficking or lipid exchange with the plasma membrane. Finally, we find that Esyt mutants fail to maintain basal neurotransmission and short term plasticity at elevated extracellular Ca2+, consistent with Esyt functioning as an ER Ca2+ sensor that modulates synaptic activity. Thus, we identify Esyt as a presynaptic ER Ca2+ sensor that can promote neurotransmission and synaptic growth, revealing the first in vivo neuronal functions of this conserved gene family.

scholarly journals Synthesis of membrane glycoproteins in rat small-intestinal villus cells. Redistribution of l-[1,5,6-3H]-fucose-labelled membrane glycoproteins among Golgi, lateral basal and microvillus membranes in vivo

1979 ◽  
Vol 182 (1) ◽  
pp. 203-212 ◽  
Author(s):  
Andrea Quaroni ◽  
Katharina Kirsch ◽  
Milton M. Weiser
Keyword(s):  
Golgi Complex ◽  
Membrane Fraction ◽  
Surface Membrane ◽  
Sodium Dodecyl ◽  
Membrane Glycoproteins ◽  
Intestinal Villus ◽  
Microvillus Membrane ◽  
Small Intestinal ◽  
Small Intestinal Villus

The biogenesis of plasmalemma glycoproteins of rat small-intestinal villus cells was studied by following the incorporation of l-[1,5,6-3H]fucose, given intraperitoneally with and without chase, into Golgi, lateral basal and microvillus membranes. Each membrane fraction showed distinct kinetics of incorporation of labelled fucose and was differently affected by the chase, which produced a much greater decrease in incorporation of label into Golgi and microvillus than into lateral basal membranes. The kinetic data suggest a redistribution of newly synthesized glycoproteins from the site of fucosylation, the Golgi complex, directly into both lateral basal and microvillus membranes. The observed biphasic pattern of label incorporation into the microvillus membrane fraction may be evidence for a second indirect route of incorporation. The selective effect of the chase suggests the presence of two different pools of radioactive fucose in the Golgi complex that differ in (1) their accessibility to dilution with non-radioactive fucose, and (2) their utilization for the biosynthesis of membrane glycoproteins subsequently destined for either the microvillus or the lateral basal parts of the plasmalemma. The radioactively labelled glycoproteins of the different membrane fractions were separated by sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis and identified by fluorography. The patterns of labelled glycoproteins in Golgi and lateral basal membranes were identical at all times. At least 14 bands could be identified shortly after radioactive-fucose injection. Most seemed to disappear at later times, although one of them, which was never observed in microvillus membranes, increased in relative intensity. All but two of the labelled glycoproteins present in the microvillus membrane corresponded to those observed in Golgi and lateral basal membranes shortly after fucose injection. The patterns of labelled glycoproteins in all membrane fractions were little affected by the chase. These data support a flow concept for the insertion of most surface-membrane glycoproteins of the intestinal villus cells.

187 EXPLOITATION OF IN VITRO CAPACITATION FOR NANOPARTICLE INCORPORATION WITHIN MAMMALIAN SPERMATOZOA

2016 ◽  
Vol 28 (2) ◽  
pp. 224
Author(s):  
L. Myles ◽  
C. Durfey ◽  
P. Ryan ◽  
S. Willard ◽  
J. Feugang
Keyword(s):  
Plasma Membrane ◽  
Sperm Motility ◽  
Surface Membrane ◽  
Milk Powder ◽  
Noninvasive Evaluation ◽  
Control Group ◽  
Body Tissues ◽  
Sperm Plasma Membrane

Migration and interactions of mammalian gametes occur in deep body tissues after mating, rendering difficult any in situ noninvasive evaluation of their performances with current methods. In our effort to develop an effective and real-time in vivo imaging approach, we have successfully labelled porcine gametes with self-illuminating bioluminescent and red-shifted quantum dot nanoparticles (QD) in our previous studies (Feugang et al. 2012 J. Nanobiotechnol. 10, 45; Feugang et al. 2015, J. Nanobiotechnol. 13, 38). The present effort aimed at investigating whether QD could be incorporated into spermatozoa through induced in vitro capacitation, which increases sperm plasma membrane fluidity. Fresh extended boar semen was placed on top of a Percoll gradient and centrifuged. Purified motile spermatozoa were collected and washed with pre-warmed PBS. Pelleted spermatozoa were resuspended in the modified Tris-buffered medium with BSA fraction-V (1 mg mL–1; modified Tween medium B with milk powder and BSA). Sperm aliquots (108) were supplemented or not (control) with QD only (QD+; 1 nM), QD+caffeine (2 mM), or QD+heparin (10 µg mL–1); with caffeine and heparin being used as routine capacitant agents in fertilization media. All aliquots were incubated at 38.5°C, under 5% CO2 for 0.5, 1, or 3 h. Spermatozoa were then analysed for motility characteristics and imaged for confirmation of QD-sperm interactions (bioluminescence emission) and localization (transmission electron microscope; TEM). Motility data of 5 replicates were analysed with ANOVA-2, and P < 0.05 was set as threshold of significance. Total sperm motility (TSM) significantly improved with the presence of either or both QDs and capacitant agents after 0.5 and 1 h incubations. With exception of the QD+heparin, all other groups had significantly decreased TSM after 3 h of incubation, when compared with TSM at 0.5 and 1 h. Higher proportions of progressive and rapid (≥45 µm s–1) spermatozoa were observed in the presence of both capacitant agents (P < 0.05), and only QD+heparin maintained greater proportions after 3 h. Sperm straight-line velocity significantly increased in the QD+caffeine at 0.5 h and in both QD+caffeine and QD+heparin thereafter. Sperm straightness data were increased by both caffeine and heparin during incubations. Strong bioluminescence signals were observed in spermatozoa incubated with QDs compared to the background signal seen in the control group. The TEM images revealed consistent surface membrane attachment of QDs in all QD+ groups, whereas transmembrane and intra-spermatic localizations were visible in both QD+caffeine and QD+heparin groups. We concluded that supplementations of medium containing QDs with caffeine or heparin allow the crossing of sperm plasma membrane by QD. No toxic effect of QD on sperm motility was observed, which confirmed our previous report using a similar ratio of QDs over spermatozoa. Exploration of efficient incorporation of QD into spermatozoa as a promising approach for noninvasive molecular imaging is still ongoing, as well as further sperm viability assessments. Supported by the NIH grant #5T35OD010432 and USDA-ARS Biophotonics Initiative grant #58–6402–3-0120.

scholarly journals In vivo FRET analyses reveal a role of ATP hydrolysis–associated conformational changes in human P-glycoprotein

2020 ◽  
Vol 295 (15) ◽  
pp. 5002-5011 ◽  
Author(s):  
Ryota Futamata ◽  
Fumihiko Ogasawara ◽  
Takafumi Ichikawa ◽  
Atsushi Kodan ◽  
Yasuhisa Kimura ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Drug Transport ◽  
Atp Hydrolysis ◽  
Hek293 Cells ◽  
Atp Binding ◽  
Binding Domains ◽  
P Glycoprotein ◽  
Atp Binding And Hydrolysis

P-glycoprotein (P-gp; also known as MDR1 or ABCB1) is an ATP-driven multidrug transporter that extrudes various hydrophobic toxic compounds to the extracellular space. P-gp consists of two transmembrane domains (TMDs) that form the substrate translocation pathway and two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. At least two P-gp states are required for transport. In the inward-facing (pre-drug transport) conformation, the two NBDs are separated, and the two TMDs are open to the intracellular side; in the outward-facing (post-drug transport) conformation, the NBDs are dimerized, and the TMDs are slightly open to the extracellular side. ATP binding and hydrolysis cause conformational changes between the inward-facing and the outward-facing conformations, and these changes help translocate substrates across the membrane. However, how ATP hydrolysis is coupled to these conformational changes remains unclear. In this study, we used a new FRET sensor that detects conformational changes in P-gp to investigate the role of ATP binding and hydrolysis during the conformational changes of human P-gp in living HEK293 cells. We show that ATP binding causes the conformational change to the outward-facing state and that ATP hydrolysis and subsequent release of γ-phosphate from both NBDs allow the outward-facing state to return to the original inward-facing state. The findings of our study underscore the utility of using FRET analysis in living cells to elucidate the function of membrane proteins such as multidrug transporters.

Glycosylation of the osmoresponsive transient receptor potential channel TRPV4 on Asn-651 influences membrane trafficking

2006 ◽  
Vol 290 (5) ◽  
pp. F1103-F1109 ◽  
Author(s):  
Hongshi Xu ◽  
Yi Fu ◽  
Wei Tian ◽  
David M. Cohen
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Trp Channel ◽  
Hek293 Cells ◽  
Wild Type ◽  
Functional Feature ◽  
Voltage Gated ◽  
Transient Receptor

We identified a consensus N-linked glycosylation motif within the pore-forming loop between the fifth and sixth transmembrane segments of the osmoresponsive transient receptor potential (TRP) channel TRPV4. Mutation of this residue from Asn to Gln (i.e., TRPV4N651Q) resulted in loss of a slower migrating band on anti-TRPV4 immunoblots and a marked reduction in lectin-precipitable TRPV4 immunoreactivity. HEK293 cells transiently transfected with the mutant TRPV4N651Q exhibited increased calcium entry in response to hypotonic stress relative to wild-type TRPV4 transfectants. This increase in hypotonicity responsiveness was associated with an increase in plasma membrane targeting of TRPV4N651Q relative to wild-type TRPV4 in both HEK293 and COS-7 cells but had no effect on overall channel abundance in whole cell lysates. Residue N651 of TRPV4 is immediately adjacent to the pore-forming loop. Although glycosylation in this vicinity has not been reported for a TRP channel, the structurally related hexahelical hyperpolarization-activated cyclic nucleotide-gated channel, HCN2, and the voltage-gated potassium channel, human ether-a-go-go-related (HERG), share a nearly identically situated and experimentally confirmed N-linked glycosylation site which promotes rather than limits channel insertion into the plasma membrane. These data point to a potentially conserved structural and functional feature influencing membrane trafficking across diverse members of the voltage-gated-like ion channel superfamily.

scholarly journals TMEM16F activation by Ca2+triggers plasmalemma expansion and directs PD-1 trafficking

2018 ◽  
Author(s):  
Christopher Bricogne ◽  
Michael Fine ◽  
Pedro M. Pereira ◽  
Julia Sung ◽  
Maha Tijani ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Surface ◽  
T Cell Activation ◽  
Membrane Trafficking ◽  
Large Scale ◽  
Cell Activation ◽  
Transmembrane Domain ◽  
Surface Membrane ◽  
Hek293 Cells

AbstractTMEM16F, an ion channel gated by high cytoplasmic Ca2+, is required for cell surface phosphatidylserine exposure during platelet aggregation and T cell activation. Here we demonstrate in Jurkat T cells and HEK293 cells that TMEM16F activation triggers large-scale surface membrane expansion in parallel with lipid scrambling. Following TMEM16F mediated scrambling and surface expansion, cells undergo extensive membrane shedding. The membrane compartment that expands the cell surface does not involve endoplasmic reticulum or acidified lysosomes. Surprisingly, T cells lacking TMEM16F expression not only fail to expand surface membrane, but instead rapidly internalize membrane via massive endocytosis (MEND). The T cell co-receptor PD-1 is selectively shed when TMEM16F triggers membrane expansion, while it is selectively internalized in the absence of TMEM16F. Its participation in this trafficking is determined by its single transmembrane domain. Thus, we establish a fundamental role for TMEM16F as a regulator of Ca2+-activated membrane trafficking.

scholarly journals Small GTPase Rab11b regulates degradation of surface membrane L-type Cav1.2 channels

2011 ◽  
Vol 300 (5) ◽  
pp. C1023-C1033 ◽  
Author(s):  
Jabe M. Best ◽  
Jason D. Foell ◽  
Courtney R. Buss ◽  
Brian P. Delisle ◽  
Ravi C. Balijepalli ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Surface Membrane ◽  
Critical Role ◽  
Ventricular Myocardium ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Hek293 Cells ◽  
Endosomal Trafficking ◽  
Membrane Expression ◽  
Membrane Density

L-type Ca2+ channels (LTCCs) play a critical role in Ca2+-dependent signaling processes in a variety of cell types. The number of functional LTCCs at the plasma membrane strongly influences the strength and duration of Ca2+ signals. Recent studies demonstrated that endosomal trafficking provides a mechanism for dynamic changes in LTCC surface membrane density. The purpose of the current study was to determine whether the small GTPase Rab11b, a known regulator of endosomal recycling, impacts plasmalemmal expression of Cav1.2 LTCCs. Disruption of endogenous Rab11b function with a dominant negative Rab11b S25N mutant led to a significant 64% increase in peak L-type Ba2+ current ( IBa,L) in human embryonic kidney (HEK)293 cells. Short-hairpin RNA (shRNA)-mediated knockdown of Rab11b also significantly increased peak IBa,L by 66% compared when with cells transfected with control shRNA, whereas knockdown of Rab11a did not impact IBa,L. Rab11b S25N led to a 1.7-fold increase in plasma membrane density of hemagglutinin epitope-tagged Cav1.2 expressed in HEK293 cells. Cell surface biotinylation experiments demonstrated that Rab11b S25N does not significantly impact anterograde trafficking of LTCCs to the surface membrane but rather slows degradation of plasmalemmal Cav1.2 channels. We further demonstrated Rab11b expression in ventricular myocardium and showed that Rab11b S25N significantly increases peak IBa,L by 98% in neonatal mouse cardiac myocytes. These findings reveal a novel role for Rab11b in limiting, rather than promoting, the plasma membrane expression of Cav1.2 LTCCs in contrast to its effects on other ion channels including human ether-a-go-go-related gene (hERG) K+ channels and cystic fibrosis transmembrane conductance regulator. This suggests Rab11b differentially regulates the trafficking of distinct cargo and extends our understanding of how endosomal transport impacts the functional expression of LTCCs.

scholarly journals Apical targeting of syntaxin 3 is essential for epithelial cell polarity

2006 ◽  
Vol 173 (6) ◽  
pp. 937-948 ◽  
Author(s):  
Nikunj Sharma ◽  
Seng Hui Low ◽  
Saurav Misra ◽  
Bhattaram Pallavi ◽  
Thomas Weimbs
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cell ◽  
Membrane Trafficking ◽  
Cell Polarization ◽  
Apical Plasma Membrane ◽  
Membrane Expression ◽  
Tight Junction Formation ◽  
Necessary And Sufficient ◽  
Syntaxin 3

In polarized epithelial cells, syntaxin 3 localizes to the apical plasma membrane and is involved in membrane fusion of apical trafficking pathways. We show that syntaxin 3 contains a necessary and sufficient apical targeting signal centered around a conserved FMDE motif. Mutation of any of three critical residues within this motif leads to loss of specific apical targeting. Modeling based on the known structure of syntaxin 1 revealed that these residues are exposed on the surface of a three-helix bundle. Syntaxin 3 targeting does not require binding to Munc18b. Instead, syntaxin 3 recruits Munc18b to the plasma membrane. Expression of mislocalized mutant syntaxin 3 in Madin-Darby canine kidney cells leads to basolateral mistargeting of apical membrane proteins, disturbance of tight junction formation, and loss of ability to form an organized polarized epithelium. These results indicate that SNARE proteins contribute to the overall specificity of membrane trafficking in vivo, and that the polarity of syntaxin 3 is essential for epithelial cell polarization.

scholarly journals Detection of Schistosoma mansoni Membrane Antigens by Immunoblot Analysis of Sera of Patients from Low-Transmission Areas

2005 ◽  
Vol 12 (2) ◽  
pp. 280-286 ◽  
Author(s):  
Italo M. Cesari ◽  
Diana E. Ballen ◽  
Leydi Mendoza ◽  
César Matos
Keyword(s):  
Schistosoma Mansoni ◽  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Surface Membrane ◽  
Sodium Dodecyl ◽  
Mass Range ◽  
Confirmatory Test ◽  
Low Transmission ◽  
Membrane Antigens

ABSTRACT Schistosoma mansoni surface membrane components play a relevant role in the host-parasite interaction, and some are released in vivo as circulating antigens. n-Butanol extraction favors the release of membrane antigens like alkaline phosphatase, which has been shown to be specifically recognized by antibodies from S. mansoni-infected humans and animals. In the present study, components in the n-butanol extract (BE) of the adult S. mansoni worm membrane fraction were separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D SDS-PAGE [15%]) and further analyzed by immunoblotting (immunoglobulin G) using defined sera. S. mansoni-infected patient sera, but not sera of uninfected patients or sera obtained from patients infected with other parasite species, specifically and variably recognized up to 20 polypeptides in the molecular mass range of ∼8 to >80 kDa. There were some differences in the number, intensity, and frequency of recognition of the BE antigens among sera from Venezuelan sites of endemicity with a different status of schistosomiasis transmission. Antigens in the 28- to 24-kDa molecular mass range appeared as immunodominants and were recognized by S. mansoni-positive sera from all the sites, with recognition frequencies varying between 57.5 and 97.5%. Immunoblotting with BE membrane antigens resulted in a highly sensitive (98.1%), specific (96.1.0%), and confirmatory test for the immunodiagnosis of schistosomiasis in low-transmission areas.

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