scholarly journals CAPS and Munc13 utilize distinct PIP2-linked mechanisms to promote vesicle exocytosis

2014 ◽  
Vol 25 (4) ◽  
pp. 508-521 ◽  
Author(s):  
Greg Kabachinski ◽  
Masaki Yamaga ◽  
D. Michelle Kielar-Grevstad ◽  
Stephen Bruinsma ◽  
Thomas F. J. Martin
Keyword(s):  
Spatial Distribution ◽  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Total Internal Reflection ◽  
Vesicle Fusion ◽  
Live Cells ◽  
Membrane Domains ◽  
Direct View ◽  
Vesicle Exocytosis ◽  
Dependent Protein

Phosphoinositides provide compartment-specific signals for membrane trafficking. Plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2) is required for Ca2+-triggered vesicle exocytosis, but whether vesicles fuse into PIP2-rich membrane domains in live cells and whether PIP2 is metabolized during Ca2+-triggered fusion were unknown. Ca2+-dependent activator protein in secretion 1 (CAPS-1; CADPS/UNC31) and ubMunc13-2 (UNC13B) are PIP2-binding proteins required for Ca2+-triggered vesicle exocytosis in neuroendocrine PC12 cells. These proteins are likely effectors for PIP2, but their localization during exocytosis had not been determined. Using total internal reflection fluorescence microscopy in live cells, we identify PIP2-rich membrane domains at sites of vesicle fusion. CAPS is found to reside on vesicles but depends on plasma membrane PIP2 for its activity. Munc13 is cytoplasmic, but Ca2+-dependent translocation to PIP2-rich plasma membrane domains is required for its activity. The results reveal that vesicle fusion into PIP2-rich membrane domains is facilitated by sequential PIP2-dependent activation of CAPS and PIP2-dependent recruitment of Munc13. PIP2 hydrolysis only occurs under strong Ca2+ influx conditions sufficient to activate phospholipase Cη2 (PLCη2). Such conditions reduce CAPS activity and enhance Munc13 activity, establishing PLCη2 as a Ca2+-dependent modulator of exocytosis. These studies provide a direct view of the spatial distribution of PIP2 linked to vesicle exocytosis via regulation of lipid-dependent protein effectors CAPS and Munc13.

Membrane microdomains: lessons from microscopy

1995 ◽  
Vol 53 ◽  
pp. 776-777
Author(s):  
J.M. Robinson ◽  
J.M Oliver
Keyword(s):  
Spatial Distribution ◽  
Plasma Membrane ◽  
Epithelial Cells ◽  
Plasma Membranes ◽  
Cell Types ◽  
Imaging Modalities ◽  
Membrane Microdomains ◽  
Membrane Domains ◽  
Lateral Heterogeneity ◽  
Functional Importance

Specialized regions of plasma membranes displaying lateral heterogeneity are the focus of this Symposium. Specialized membrane domains are known for certain cell types such as differentiated epithelial cells where lateral heterogeneity in lipids and proteins exists between the apical and basolateral portions of the plasma membrane. Lateral heterogeneity and the presence of microdomains in membranes that are uniform in appearance have been more difficult to establish. Nonetheless a number of studies have provided evidence for membrane microdomains and indicated a functional importance for these structures.This symposium will focus on the use of various imaging modalities and related approaches to define membrane microdomains in a number of cell types. The importance of existing as well as emerging imaging technologies for use in the elucidation of membrane microdomains will be highlighted. The organization of membrane microdomains in terms of dimensions and spatial distribution is of considerable interest and will be addressed in this Symposium.

Visualizing membrane trafficking using total internal reflection fluorescence microscopy

2003 ◽  
Vol 31 (4) ◽  
pp. 819-823 ◽  
Author(s):  
V. Beaumont
Keyword(s):  
Fluorescence Microscopy ◽  
Membrane Trafficking ◽  
Cell Biology ◽  
Total Internal Reflection ◽  
Optical Resolution ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Live Cells ◽  
Excitable Cells ◽  
Cellular Processes

There is a dizzying array of fluorescent probes now commercially available to monitor cellular processes, and advances in molecular biology have highlighted the ease with which proteins can now be labelled with fluorophores without loss of functionality. This has led to an explosion in the popularity of fluorescence microscopy techniques. One such specialized technique, total internal reflection fluorescence microscopy (TIR-FM), is ideally suited to gaining insight into events occurring at, or close to, the plasma membrane of live cells with excellent optical resolution. In the last few years, the application of TIR-FM to membrane trafficking events in both non-excitable and excitable cells has been an area of notable expansion and fruition. This review gives a brief overview of that literature, with emphasis on the study of the regulation of exocytosis and endocytosis in excitable cells using TIR-FM. Finally, recent applications of TIR-FM to the study of cellular processes at the molecular level are discussed briefly, providing promise that the future of TIR-FM in cell biology will only get brighter.

scholarly journals Detergent Resistant Membrane Domains in Broccoli Plasma Membrane Associated to the Response to Salinity Stress

2020 ◽  
Vol 21 (20) ◽  
pp. 7694
Author(s):  
Lucía Yepes-Molina ◽  
Micaela Carvajal ◽  
Maria Carmen Martínez-Ballesta
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Lower Amount ◽  
Membrane Domains ◽  
Cellular Functions ◽  
Osmotic Water ◽  
Fatty Acid Saturation ◽  
Protein Partitioning ◽  
Detergent Resistant Membranes ◽  
Detergent Resistant Membrane

Detergent-resistant membranes (DRMs) microdomains, or “raft lipids”, are key components of the plasma membrane (PM), being involved in membrane trafficking, signal transduction, cell wall metabolism or endocytosis. Proteins imbibed in these domains play important roles in these cellular functions, but there are few studies concerning DRMs under abiotic stress. In this work, we determine DRMs from the PM of broccoli roots, the lipid and protein content, the vesicles structure, their water osmotic permeability and a proteomic characterization focused mainly in aquaporin isoforms under salinity (80 mM NaCl). Based on biochemical lipid composition, higher fatty acid saturation and enriched sterol content under stress resulted in membranes, which decreased osmotic water permeability with regard to other PM vesicles, but this permeability was maintained under control and saline conditions; this maintenance may be related to a lower amount of total PIP1 and PIP2. Selective aquaporin isoforms related to the stress response such as PIP1;2 and PIP2;7 were found in DRMs and this protein partitioning may act as a mechanism to regulate aquaporins involved in the response to salt stress. Other proteins related to protein synthesis, metabolism and energy were identified in DRMs independently of the treatment, indicating their preference to organize in DMRs.

scholarly journals Confinement of β1- and β2-adrenergic receptors in the plasma membrane of cardiomyocyte-like H9c2 cells is mediated by selective interactions with PDZ domain and A-kinase anchoring proteins but not caveolae

2011 ◽  
Vol 22 (16) ◽  
pp. 2970-2982 ◽  
Author(s):  
Cathleen D. Valentine ◽  
Peter M. Haggie
Keyword(s):  
Plasma Membrane ◽  
Adrenergic Receptors ◽  
Protein Complexes ◽  
Pdz Domain ◽  
Single Particle Tracking ◽  
Live Cells ◽  
Membrane Domains ◽  
Anchoring Proteins ◽  
Differential Signaling ◽  
Receptor Dynamics

The sympathetic nervous system regulates cardiac output by activating adrenergic receptors (ARs) in cardiac myocytes. The predominant cardiac ARs, β1- and β2AR, are structurally similar but mediate distinct signaling responses. Scaffold protein–mediated compartmentalization of ARs into discrete, multiprotein complexes has been proposed to dictate differential signaling responses. To test the hypothesis that βARs integrate into complexes in live cells, we measured receptor diffusion and interactions by single-particle tracking. Unstimulated β1- and β2AR were highly confined in the membrane of H9c2 cardiomyocyte-like cells, indicating that receptors are tethered and presumably integrated into protein complexes. Selective disruption of interactions with postsynaptic density protein 95/disks large/zonula occludens-1 (PDZ)–domain proteins and A-kinase anchoring proteins (AKAPs) increased receptor diffusion, indicating that these scaffold proteins participate in receptor confinement. In contrast, modulation of interactions between the putative scaffold caveolae and β2AR did not alter receptor dynamics, suggesting that these membrane domains are not involved in β2AR confinement. For both β1- and β2AR, the receptor carboxy-terminus was uniquely responsible for scaffold interactions. Our data formally demonstrate that distinct and stable protein complexes containing β1- or β2AR are formed in the plasma membrane of cardiomyocyte-like cells and that selective PDZ and AKAP interactions are responsible for the integration of receptors into complexes.

scholarly journals Polarized Sphingolipid Transport from the Subapical Compartment: Evidence for Distinct Sphingolipid Domains

1999 ◽  
Vol 10 (10) ◽  
pp. 3449-3461 ◽  
Author(s):  
Sven C. D. van IJzendoorn ◽  
Dick Hoekstra
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Apical Membrane ◽  
Apical Plasma Membrane ◽  
Membrane Domains ◽  
Dibutyryl Camp ◽  
Calmodulin Antagonists ◽  
Luminal Side ◽  
Basolateral Plasma Membrane ◽  
Recycling Pathway

In polarized HepG2 cells, the sphingolipids glucosylceramide and sphingomyelin (SM), transported along the reverse transcytotic pathway, are sorted in subapical compartments (SACs), and subsequently targeted to either apical or basolateral plasma membrane domains, respectively. In the present study, evidence is provided that demonstrates that these sphingolipids constitute separate membrane domains at the luminal side of the SAC membrane. Furthermore, as revealed by the use of various modulators of membrane trafficking, such as calmodulin antagonists and dibutyryl-cAMP, it is shown that the fate of these separate sphingolipid domains is regulated by different signals, including those that govern cell polarity development. Thus under conditions that stimulate apical plasma membrane biogenesis, SM is rerouted from a SAC-to-basolateral to a SAC-to-apical pathway. The latter pathway represents the final leg in the transcytotic pathway, followed by the transcytotic pIgR–dIgA protein complex. Interestingly, this pathway is clearly different from the apical recycling pathway followed by glucosylceramide, further indicating that randomization of these pathways, which are both bound for the apical membrane, does not occur. The consequence of the potential coexistence of separate sphingolipid domains within the same compartment in terms of “raft” formation and apical targeting is discussed.

scholarly journals Quantitative characterization of Tetraspanin 8 homointeractions in the plasma membrane

2021 ◽  
Author(s):  
Daniel Wirth ◽  
Ece Özdemir ◽  
Christopher King ◽  
Lena Ahlswede ◽  
Dirk Schneider ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Dissociation Constant ◽  
Membrane Trafficking ◽  
Dissociation Constants ◽  
Cell Communication ◽  
Live Cells ◽  
Functional Protein ◽  
Quantitative Fluorescence ◽  
Dissociation Constant Kd

The spatial distribution of proteins in cell membranes is crucial for signal transduction, cell communication and membrane trafficking. Members of the Tetraspanin family organize functional protein clusters within the plasma membrane into so-called Tetraspanin-enriched microdomains (TEMs). Direct interactions between Tetraspanins are believed to be important for this organization. However, studies thus far have utilized mainly co-immunoprecipitation methods that cannot distinguish between direct and indirect, through common partners, interactions. Here we study Tetraspanin 8 homointeractions in living cells via quantitative fluorescence microscopy. We demonstrate that Tetraspanin 8 exists in a monomer-dimer equilibrium in the plasma membrane. Tetraspanin 8 dimerization is described by a high dissociation constant (Kd = 14700 ± 1100 Tspan/μm2), one of the highest dissociation constants measured for membrane proteins in live cells. We propose that this high dissociation constant, and thus the short lifetime of the Tetraspanin 8 dimer, is critical for Tetraspanin 8 functioning as a master regulator of cell signaling.

scholarly journals Dynein-mediated transport and membrane trafficking control PAR3 polarised distribution

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Julie Jouette ◽  
Antoine Guichet ◽  
Sandra B Claret
Keyword(s):  
Plasma Membrane ◽  
Cell Polarity ◽  
Membrane Trafficking ◽  
Control Cell ◽  
Vesicular Trafficking ◽  
Second Phase ◽  
Membrane Domains ◽  
Anterior Cortex ◽  
Essential Proteins ◽  
Dynein Motor

The scaffold protein PAR3 and the kinase PAR1 are essential proteins that control cell polarity. Their precise opposite localisations define plasma membrane domains with specific functions. PAR3 and PAR1 are mutually inhibited by direct or indirect phosphorylations, but their fates once phosphorylated are poorly known. Through precise spatiotemporal quantification of PAR3 localisation in the Drosophila oocyte, we identify several mechanisms responsible for its anterior cortex accumulation and its posterior exclusion. We show that PAR3 posterior plasma membrane exclusion depends on PAR1 and an endocytic mechanism relying on RAB5 and PI(4,5)P2. In a second phase, microtubules and the dynein motor, in connection with vesicular trafficking involving RAB11 and IKK-related kinase, IKKε, are required for PAR3 transport towards the anterior cortex. Altogether, our results point to a connection between membrane trafficking and dynein-mediated transport to sustain PAR3 asymmetry.

scholarly journals Role of phosphatidylinositol 3,4,5-trisphosphate in regulating the activity and localization of 3-phosphoinositide-dependent protein kinase-1

1999 ◽  
Vol 337 (3) ◽  
pp. 575-583 ◽  
Author(s):  
Richard A. CURRIE ◽  
Kay S. WALKER ◽  
Alex GRAY ◽  
Maria DEAK ◽  
Antonio CASAMAYOR ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Fluorescent Protein ◽  
Lipid Vesicles ◽  
Live Cells ◽  
Ph Domain ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Green Fluorescent

3-Phosphoinositide-dependent protein kinase-1 (PDK1) interacts stereoselectively with the d-enantiomer of PtdIns(3,4,5)P3 (KD 1.6 nM) and PtdIns(3,4)P2 (KD 5.2 nM), but binds with lower affinity to PtdIns3P or PtdIns(4,5)P2. The binding of PtdIns(3,4,5)P3 to PDK1 was greatly decreased by making specific mutations in the pleckstrin homology (PH) domain of PDK1 or by deleting it. The same mutations also greatly decreased the rate at which PDK1 activated protein kinase Bα (PKBα) in vitro in the presence of lipid vesicles containing PtdIns(3,4,5)P3, but did not affect the rate at which PDK1 activated a PKBα mutant lacking the PH domain in the absence of PtdIns(3,4,5)P3. When overexpressed in 293 or PAE cells, PDK1 was located at the plasma membrane and in the cytosol, but was excluded from the nucleus. Mutations that disrupted the interaction of PtdIns(3,4,5)P3 or PtdIns(4,5)P2 with PDK1 abolished the association of PDK1 with the plasma membrane. Growth-factor stimulation promoted the translocation of transfected PKBα to the plasma membrane, but had no effect on the subcellular distribution of PDK1 as judged by immunoelectron microscopy of fixed cells. This conclusion was also supported by confocal microscopy of green fluorescent protein–PDK1 in live cells. These results, together with previous observations, indicate that PtdIns(3,4,5)P3 plays several roles in the PDK1-induced activation of PKBα. First, it binds to the PH domain of PKB, altering its conformation so that it can be activated by PDK1. Secondly, interaction with PtdIns(3,4,5)P3 recruits PKB to the plasma membrane with which PDK1 is localized constitutively by virtue of its much stronger interaction with PtdIns(3,4,5)P3 or PtdIns(4,5)P2. Thirdly, the interaction of PDK1 with PtdIns(3,4,5)P3 facilitates the rate at which it can activate PKB.

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