scholarly journals The septin cytoskeleton facilitates membrane retraction during motility and blebbing

2012 ◽  
Vol 196 (1) ◽  
pp. 103-114 ◽  
Author(s):  
Julia K. Gilden ◽  
Sebastian Peck ◽  
Yi-Chun M. Chen ◽  
Matthew F. Krummel
Keyword(s):  
Plasma Membrane ◽  
Dendritic Spines ◽  
Membrane Trafficking ◽  
Shape Change ◽  
Critical Role ◽  
Regulatory Volume Decrease ◽  
Physiological Processes ◽  
Amoeboid Motility ◽  
Dynamic Shape ◽  
Volume Decrease

Increasing evidence supports a critical role for the septin cytoskeleton at the plasma membrane during physiological processes including motility, formation of dendritic spines or cilia, and phagocytosis. We sought to determine how septins regulate the plasma membrane, focusing on this cytoskeletal element’s role during effective amoeboid motility. Surprisingly, septins play a reactive rather than proactive role, as demonstrated during the response to increasing hydrostatic pressure and subsequent regulatory volume decrease. In these settings, septins were required for rapid cortical contraction, and SEPT6-GFP was recruited into filaments and circular patches during global cortical contraction and also specifically during actin filament depletion. Recruitment of septins was also evident during excessive blebbing initiated by blocking membrane trafficking with a dynamin inhibitor, providing further evidence that septins are recruited to facilitate retraction of membranes during dynamic shape change. This function of septins in assembling on an unstable cortex and retracting aberrantly protruding membranes explains the excessive blebbing and protrusion observed in septin-deficient T cells.

Hyposmotic activation of ICl,swell in rabbit nonpigmented ciliary epithelial cells involves increased ClC-3 trafficking to the plasma membrane

2004 ◽  
Vol 82 (6) ◽  
pp. 708-718 ◽  
Author(s):  
John P Vessey ◽  
Chanjuan Shi ◽  
Christine AB Jollimore ◽  
Kelly T Stevens ◽  
Miguel Coca-Prados ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Kinase Inhibitor ◽  
Regulatory Volume Decrease ◽  
Membrane Dynamics ◽  
Cell Swelling ◽  
Cl Channels ◽  
Phosphoinositide 3 Kinase ◽  
Nonpigmented Ciliary Epithelial ◽  
Volume Decrease

In mammalian nonpigmented ciliary epithelial (NPE) cells, hyposmotic stimulation leading to cell swelling activates an outwardly rectifying Cl– conductance (ICl,swell), which, in turn, results in regulatory volume decrease. The aim of this study was to determine whether increased trafficking of intracellular ClC-3 Cl channels to the plasma membrane could contribute to the ICl,swell following hyposmotic stimulation. Our results demonstrate that hyposmotic stimulation reversibly activates an outwardly rectifying Cl– current that is inhibited by phorbol-12-dibutyrate and niflumic acid. Transfection with ClC-3 antisense, but not sense, oligonucleotides reduced ClC-3 expression as well as ICl,swell. Intracellular dialysis with 2 different ClC-3 antibodies abolished activation of ICl,swell. Immunofluorescence microscopy showed that hyposmotic stimulation increased ClC-3 immunoreactivity at the plasma membrane. To determine whether this increased expression of ClC-3 at the plasma membrane could be due to increased vesicular trafficking, we examined membrane dynamics with the fluorescent membrane dye FM1-43. Hyposmotic stimulation rapidly increased the rate of exocytosis, which, along with ICl,swell, was inhibited by the phosphoinositide-3-kinase inhibitor wortmannin and the microtubule disrupting agent, nocodazole. These findings suggest that ClC-3 channels contribute to ICl,swell following hyposmotic stimulation through increased trafficking of channels to the plasma membrane.Key words: ClC-3, NPE, cell swelling, membrane trafficking, ciliary body epithelium.

scholarly journals Synbindin, a Novel Syndecan-2–Binding Protein in Neuronal Dendritic Spines

2000 ◽  
Vol 151 (1) ◽  
pp. 53-68 ◽  
Author(s):  
Iryna M. Ethell ◽  
Kazuki Hagihara ◽  
Yoshiaki Miura ◽  
Fumitoshi Irie ◽  
Yu Yamaguchi
Keyword(s):  
Dendritic Spines ◽  
Membrane Trafficking ◽  
Hippocampal Neurons ◽  
Critical Role ◽  
Postsynaptic Membrane ◽  
Synaptic Membrane ◽  
Excitatory Synapses ◽  
Spine Formation ◽  
Intracellular Vesicles ◽  
Spine Morphogenesis

Dendritic spines are small protrusions on the surface of dendrites that receive the vast majority of excitatory synapses. We previously showed that the cell-surface heparan sulfate proteoglycan syndecan-2 induces spine formation upon transfection into hippocampal neurons. This effect requires the COOH-terminal EFYA sequence of syndecan-2, suggesting that cytoplasmic molecules interacting with this sequence play a critical role in spine morphogenesis. Here, we report a novel protein that binds to the EFYA motif of syndecan-2. This protein, named synbindin, is expressed by neurons in a pattern similar to that of syndecan-2, and colocalizes with syndecan-2 in the spines of cultured hippocampal neurons. In transfected hippocampal neurons, synbindin undergoes syndecan-2–dependent clustering. Synbindin is structurally related to yeast proteins known to be involved in vesicle transport. Immunoelectron microscopy localized synbindin on postsynaptic membranes and intracellular vesicles within dendrites, suggesting a role in postsynaptic membrane trafficking. Synbindin coimmunoprecipitates with syndecan-2 from synaptic membrane fractions. Our results show that synbindin is a physiological syndecan-2 ligand on dendritic spines. We suggest that syndecan-2 induces spine formation by recruiting intracellular vesicles toward postsynaptic sites through the interaction with synbindin.

The volume-regulated anion channel is formed by LRRC8 heteromers – molecular identification and roles in membrane transport and physiology

2015 ◽  
Vol 396 (9-10) ◽  
pp. 975-990 ◽  
Author(s):  
Tobias Stauber
Keyword(s):  
Cell Biology ◽  
Regulatory Volume Decrease ◽  
Anion Channel ◽  
Cell Swelling ◽  
Transepithelial Transport ◽  
Organic Osmolytes ◽  
Physiological Processes ◽  
Starting Point ◽  
And Migration ◽  
Volume Decrease

Abstract Cellular volume regulation is fundamental for numerous physiological processes. The volume-regulated anion channel, VRAC, plays a crucial role in regulatory volume decrease. This channel, which is ubiquitously expressed in vertebrates, has been vastly characterized by electrophysiological means. It opens upon cell swelling and conducts chloride and arguably organic osmolytes. VRAC has been proposed to be critically involved in various cellular and organismal functions, including cell proliferation and migration, apoptosis, transepithelial transport, swelling-induced exocytosis and intercellular communication. It may also play a role in pathological states like cancer and ischemia. Despite many efforts, the molecular identity of VRAC had remained elusive for decades, until the recent discovery of heteromers of LRRC8A with other LRRC8 family members as an essential VRAC component. This identification marks a starting point for studies on the structure-function relation, for molecular biological investigations of its cell biology and for re-evaluating the physiological roles of VRAC. This review recapitulates the identification of LRRC8 heteromers as VRAC components, depicts the similarities between LRRC8 proteins and pannexins, and discussed whether VRAC conducts larger osmolytes. Furthermore, proposed physiological functions of VRAC and the present knowledge about the physiological significance of LRRC8 proteins are summarized and collated.

scholarly journals Ubiquitination-dependent quality control of hERG K+ channel with acquired and inherited conformational defect at the plasma membrane

2013 ◽  
Vol 24 (24) ◽  
pp. 3787-3804 ◽  
Author(s):  
Pirjo M. Apaja ◽  
Brian Foo ◽  
Tsukasa Okiyoneda ◽  
William C. Valinsky ◽  
Herve Barriere ◽  
...  
Keyword(s):  
Quality Control ◽  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Critical Role ◽  
Sudden Cardiac Arrest ◽  
K Channel ◽  
Missense Mutations ◽  
Drug Induced ◽  
Interacting Protein ◽  
Expression Phenotype

Membrane trafficking in concert with the peripheral quality control machinery plays a critical role in preserving plasma membrane (PM) protein homeostasis. Unfortunately, the peripheral quality control may also dispose of partially or transiently unfolded polypeptides and thereby contribute to the loss-of-expression phenotype of conformational diseases. Defective functional PM expression of the human ether-a-go-go–related gene (hERG) K+ channel leads to the prolongation of the ventricular action potential that causes long QT syndrome 2 (LQT2), with increased propensity for arrhythmia and sudden cardiac arrest. LQT2 syndrome is attributed to channel biosynthetic processing defects due to mutation, drug-induced misfolding, or direct channel blockade. Here we provide evidence that a peripheral quality control mechanism can contribute to development of the LQT2 syndrome. We show that PM hERG structural and metabolic stability is compromised by the reduction of extracellular or intracellular K+ concentration. Cardiac glycoside–induced intracellular K+ depletion conformationally impairs the complex-glycosylated channel, which provokes chaperone- and C-terminal Hsp70-interacting protein–dependent polyubiquitination, accelerated internalization, and endosomal sorting complex required for transport–dependent lysosomal degradation. A similar mechanism contributes to the down-regulation of PM hERG harboring LQT2 missense mutations, with incomplete secretion defect. These results suggest that PM quality control plays a determining role in the loss-of-expression phenotype of hERG in certain hereditary and acquired LTQ2 syndromes.

scholarly journals LRRC8/VRAC Channels and the Redox Balance: A Complex Relationship

2021 ◽  
Vol 55 (S1) ◽  
pp. 106-118
Keyword(s):  
Regulatory Volume Decrease ◽  
Redox System ◽  
Redox Balance ◽  
Research Field ◽  
Complex Relationship ◽  
Anion Channels ◽  
Molecular Identity ◽  
Physiological Processes ◽  
Main Pathway ◽  
Volume Decrease

More than three decades after their first biophysical description, Volume Regulated Anion Channels (VRACs) still remain challenging to understand. Initially, VRACs were identified as the main pathway for the cell to extrude Cl- ions during the regulatory volume decrease (RVD) mechanism contributing in fine to the recovery of normal cell volume. For years, scientists have tried unsuccessfully to find their molecular identity, leading to controversy within the field that only ended in 2014 when two independent groups demonstrated that VRACs were formed by heteromers of LRRC8 proteins. This breakthrough gave a second breath to the research field and was followed by many publications regarding LRRC8/VRACs structure/ function, physiological roles and 3D structures. Nevertheless, far from simplifying the field, these discoveries have instead exponentially increased its complexity. Indeed, the channel's biophysical properties seem to be dependent on the LRRC8 subunits composition with each heteromer showing different ion/molecule permeabilities and regulatory mechanisms. One clear example of this complexity is the intricate relationship between LRRC8/VRACs and the redox system. On one hand, VRACs appear to be directly regulated by oxidation or reduction depending on their subunit composition. On the other hand, VRACs can also impact the redox balance within the cells, through their permeability to reduced glutathione or through other as yet uncharacterized pathways. Unravelling this issue is particularly crucial as LRRC8/VRACs play an important role in a wide variety of physiological processes involving oxidative stress signaling. In this regard, we have tried to systematically identify in the literature both preand post-LRRC8 discovery as well as the interplay between VRACs and the redox system to provide new insights into this complex relationship.

scholarly journals Isolation and characterization of multi-protein complexes enriched in the K-Cl co-transporter 2 from brain plasma membranes

2020 ◽  
Author(s):  
Joshua L. Smalley ◽  
Georgina Kontou ◽  
Catherine Choi ◽  
Qiu Ren ◽  
David Albrecht ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Plasma Membranes ◽  
Quaternary Structure ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Critical Role ◽  
Inhibitory Synapses ◽  
Isolation And Characterization

ABSTRACTKcc2 plays a critical role in determining the efficacy of synaptic inhibition, however, the cellular mechanism neurons use to regulate its membrane trafficking, stability and activity are ill-defined. To address these issues, we used affinity purification to isolate stable multi-protein complexes of Kcc2 from the plasma membrane of murine forebrain. We resolved these using Blue-native polyacrylamide gel electrophoresis (BN-PAGE) coupled to LC-MS/MS. Purified Kcc2 migrated as distinct molecular species of 300, 600 and 800 kDa following BN-PAGE. In excess of 90% coverage of the soluble N and C-termini of Kcc2 was obtained. The 300kDa species largely contained Kcc2, which is consistent with a dimeric quaternary structure for this transporter. Intriguingly, lower levels of Kcc1 were also found in this species suggesting the existence of “mixed” Kcc2/Kcc1 heterodimers. The 600 and 800 kDa species represented stable multi-protein complexes of Kcc2. We identified a set of novel structural, ion transporting and signaling protein interactors, that are present at both excitatory and inhibitory synapses, consistent with the proposed association of Kcc2. These included spectrins, ankyrins, and the IP3 receptor. We also identified interactors more directly associated with phosphorylation; Akap5 and Lmtk3. Finally, we used LC-MS/MS on highly purified endogenous plasma membrane Kcc2 to detect phosphorylation sites. We detected 11 sites with high confidence, including known and novel sites. Collectively our experiments demonstrate that Kcc2 is associated with components of the neuronal cytoskeleton and signaling molecules that may act to regulate transporter membrane trafficking, stability, and activity.

scholarly journals TRIM21-regulated Annexin A2 plasma membrane trafficking facilitates osteosarcoma cell differentiation through the TFEB-mediated autophagy

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Huan-Tian Zhang ◽  
Qingzhong Zeng ◽  
Baomeng Wu ◽  
Junlei Lu ◽  
Kui-Leung Tong ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Differentiation ◽  
Membrane Trafficking ◽  
Critical Role ◽  
Annexin A2 ◽  
Osteosarcoma Cell ◽  
Functional Link ◽  
Signaling Axis ◽  
Poor Differentiation ◽  
Transcription Factor Eb

AbstractOsteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents, which is characterized by dysfunctional autophagy and poor differentiation. Our recent studies have suggested that the tripartite motif containing-21 (TRIM21) plays a crucial role in regulating OS cell senescence and proliferation via interactions with several proteins. Yet, its implication in autophagy and differentiation in OS is largely unknown. In the present study, we first showed that TRIM21 could promote OS cell autophagy, as determined by the accumulation of LC3-II, and the degradation of cargo receptor p62. Further, we were able to identify that Annexin A2 (ANXA2), as a novel interacting partner of TRIM21, was critical for TIRM21-induced OS cell autophagy. Although TRIM21 had a negligible effect on the mRNA and protein expressions of ANXA2, we did find that TRIM21 facilitated the translocation of ANXA2 toward plasma membrane (PM) in OS cells through a manner relying on TRIM21-mediated cell autophagy. This functional link has been confirmed by observing a nice co-expression of TRIM21 and ANXA2 (at the PM) in the OS tissues. Mechanistically, we demonstrated that TRIM21, via facilitating the ANXA2 trafficking at the PM, enabled to release the transcription factor EB (TFEB, a master regulator of autophagy) from the ANXA2-TFEB complex, which in turn entered into the nucleus for the regulation of OS cell autophagy. In accord with previous findings that autophagy plays a critical role in the control of differentiation, we also demonstrated that autophagy inhibited OS cell differentiation, and that the TRIM21/ANXA2/TFEB axis is implicated in OS cell differentiation through the coordination with autophagy. Taken together, our results suggest that the TRIM21/ANXA2/TFEB axis is involved in OS cell autophagy and subsequent differentiation, indicating that targeting this signaling axis might lead to a new clue for OS treatment.

Localization of Adenosine Triphosphatase Activity in the Phleom of Nicotiana Tabacum

1972 ◽  
Vol 30 ◽  
pp. 230-231
Author(s):  
James Cronshaw ◽  
Jamison E. Gilder
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Adenosine Triphosphatase ◽  
Higher Plants ◽  
High Surface ◽  
Root Tip ◽  
Animal Cells ◽  
Physiological Processes ◽  
Tip Cells ◽  
Atpase Localization

Adenosine triphosphatase (ATPase) activity has been shown to be associated with numerous physiological processes in both plants and animal cells. Biochemical studies have shown that in higher plants ATPase activity is high in cell wall preparations and is associated with the plasma membrane, nuclei, mitochondria, chloroplasts and lysosomes. However, there have been only a few ATPase localization studies of higher plants at the electron microscope level. Poux (1967) demonstrated ATPase activity associated with most cellular organelles in the protoderm cells of Cucumis roots. Hall (1971) has demonstrated ATPase activity in root tip cells of Zea mays. There was high surface activity largely associated with the plasma membrane and plasmodesmata. ATPase activity was also demonstrated in mitochondria, dictyosomes, endoplasmic reticulum and plastids.

Human Platelets Exposed to Mechanical Stresses Express a Potent Lipoxygenase Product

1992 ◽  
Vol 68 (05) ◽  
pp. 589-594 ◽  
Author(s):  
Alon Margalit ◽  
Avinoam A Livne
Keyword(s):  
Regulatory Volume Decrease ◽  
Human Platelets ◽  
Mechanical Stresses ◽  
Arterial Stenosis ◽  
Platelet Suspension ◽  
Suspension Flows ◽  
Lipoxygenase Product ◽  
Pathological Conditions ◽  
K Permeability ◽  
Volume Decrease

SummaryHuman platelets exposed to hypotonicity undergo regulatory volume decrease (RVD), controlled by a potent, yet labile, lipoxygenase product (LP). LP is synthesized and excreted during RVD affecting selectively K+ permeability. LP is assayed by its capacity to reconstitute RVD when lipoxygenase is blocked. Centrifugation for preparing washed platelets (1,550 × g, 10 min) is sufficient to express LP activity, with declining potency in repeated centrifugations, indicating that it is not readily replenish-able. When platelet suspension flows in a vinyl tubing (1 mm i.d.), at physiological velocity, controlled at 90–254 cm/s, LP formation increases as a function of velocity but declines as result of increasing the tubing length. Stirring the platelets in an aggregometer cuvette for 30 s, yields no LP unless the stirring is intermittent. No associated platelet lysis or aggregation are observed following the mechanical stress applications. These results demonstrate that although mechanical stresses result in LP production, the mode of its application plays a major role. These results may indicate that LP is synthesized under pathological conditions and could be of relevance to platelets behavior related to arterial stenosis.

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