scholarly journals Free diffusion of PI(4,5)P2 in the plasma membrane in the presence of high density effector protein complexes

2022 ◽  
Author(s):  
Jonathan Pacheco ◽  
Anna C Cassidy ◽  
James P Zewe ◽  
Rachel C Wills ◽  
Gerald R Hammond
Keyword(s):  
Plasma Membrane ◽  
Protein Complexes ◽  
Focal Adhesions ◽  
Single Particle Tracking ◽  
Effector Proteins ◽  
Coated Pits ◽  
Single Class ◽  
And Function ◽  
Cytoskeletal Elements ◽  
Open Question

The lipid phosphatidyl-D-myo-inositol-4,5-bisphosphate [PI(4,5)P2] is a master regulator of plasma membrane (PM) function. It engages effector proteins that regulate diverse traffic, transport, signaling and cytoskeletal processes that define PM structure and function. How a single class of lipid molecules independently regulate so many parallel processes remains an open question. We tested the hypothesis that spatially segregated pools of PI(4,5)P2 are associated with, and thus reserved for regulation of, different functional complexes in the PM. The mobility of PI(4,5)P2 in the membrane was measured using lipid biosensors by single particle tracking photoactivation localization microscopy (sptPALM). We found that PI(4,5)P2, and several other classes of inner PM lipids, diffuse rapidly at approximately 0.3 microns squared per second with largely Brownian motion, although they spend approximately a third of their time diffusing much more slowly. Surprisingly, areas of the PM occupied by PI(4,5)P2-dependent complexes, such endoplasmic-reticulum:PM contact sites, clathrin-coated structures, and several actin cytoskeletal elements including focal adhesions, did not cause a change in PI(4,5)P2 lateral mobility. Only the spectrin and septin cytoskeletons were observed to produce a slowing of PI(4,5)P2 diffusion. We conclude that even structures with high densities of PI(4,5)P2-engaging effector proteins, such as clathrin coated pits and focal adhesions, do not corral free PI(4,5)P2, questioning a role for spatially segregated PI(4,5)P2 pools in organizing and regulating parallel PM functions.

Costameres, focal adhesions, and cardiomyocyte mechanotransduction

2005 ◽  
Vol 289 (6) ◽  
pp. H2291-H2301 ◽  
Author(s):  
Allen M. Samarel
Keyword(s):  
Growth Factor ◽  
Focal Adhesions ◽  
Growth Factor Signaling ◽  
Cellular Mechanisms ◽  
Growth And Survival ◽  
Specific Proteins ◽  
And Function ◽  
Cytoskeletal Elements ◽  
Biochemical Signals ◽  
Cellular Components

Mechanotransduction refers to the cellular mechanisms by which load-bearing cells sense physical forces, transduce the forces into biochemical signals, and generate appropriate responses leading to alterations in cellular structure and function. This process affects the beat-to-beat regulation of cardiac performance but also affects the proliferation, differentiation, growth, and survival of the cellular components that comprise the human myocardium. This review focuses on the experimental evidence indicating that the costamere and its structurally related structure the focal adhesion complex are critical cytoskeletal elements involved in cardiomyocyte mechanotransduction. Biochemical signals originating from the extracellular matrix-integrin-costameric protein complex share many common features with those signals generated by growth factor receptors. The roles of key regulatory kinases and other muscle-specific proteins involved in mechanotransduction and growth factor signaling are discussed, and issues requiring further study in this field are outlined.

scholarly journals Aggregation state determines the localization and function of M1– and M23–aquaporin-4 in astrocytes

2014 ◽  
Vol 204 (4) ◽  
pp. 559-573 ◽  
Author(s):  
Alex J. Smith ◽  
Byung-Ju Jin ◽  
Julien Ratelade ◽  
Alan S. Verkman
Keyword(s):  
Plasma Membrane ◽  
Protein Complexes ◽  
Water Channel ◽  
Aquaporin 4 ◽  
Cellular Functions ◽  
Aggregation State ◽  
Functional Roles ◽  
State Dependent ◽  
And Function

The astrocyte water channel aquaporin-4 (AQP4) is expressed as heterotetramers of M1 and M23 isoforms in which the presence of M23–AQP4 promotes formation of large macromolecular aggregates termed orthogonal arrays. Here, we demonstrate that the AQP4 aggregation state determines its subcellular localization and cellular functions. Individually expressed M1–AQP4 was freely mobile in the plasma membrane and could diffuse into rapidly extending lamellipodial regions to support cell migration. In contrast, M23–AQP4 formed large arrays that did not diffuse rapidly enough to enter lamellipodia and instead stably bound adhesion complexes and polarized to astrocyte end-feet in vivo. Co-expressed M1– and M23–AQP4 formed aggregates of variable size that segregated due to diffusional sieving of small, mobile M1–AQP4-enriched arrays into lamellipodia and preferential interaction of large, M23–AQP4-enriched arrays with the extracellular matrix. Our results therefore demonstrate an aggregation state–dependent mechanism for segregation of plasma membrane protein complexes that confers specific functional roles to M1– and M23–AQP4.

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 AP-1B facilitates endocytosis during cell migration

2019 ◽  
Author(s):  
Margaret Johnson Kell ◽  
Su Fen Ang ◽  
Lucy Pigati ◽  
Abby Halpern ◽  
Heike Fölsch
Keyword(s):  
Plasma Membrane ◽  
Cell Migration ◽  
Epithelial Cell ◽  
Metastatic Cancer ◽  
Basolateral Membrane ◽  
Focal Adhesions ◽  
Pcr Analysis ◽  
Collective Cell Migration ◽  
Coated Pits ◽  
Cell Protrusion

ABSTRACTThe epithelial cell-specific clathrin adaptor AP-1B has a well-established role in polarized sorting of cargos to the basolateral membrane. Here we demonstrate a novel function for AP-1B during collective cell migration of epithelial sheets. We show that AP-1B colocalized with β1 integrin in focal adhesions during cell migration using confocal microscopy and total internal reflection fluorescence (TIRF) microscopy on fixed specimens. Further, AP-1B labeling in cell protrusion was distinct from labeling for the canonical endocytic adaptor complex AP-2. Using stochastic optical reconstruction microscopy (STORM) and live TIRF imaging we identified numerous AP-1B-coated structures at or close to the plasma membrane in cell protrusions. Importantly, immuno-electron microscopy (EM) showed AP-1B in clathrin-coated pits and budding vesicles at the plasma membrane during cell migration. Our data therefore established a novel function for AP-1B in endocytosis. We further show that β1 integrin was dependent on AP-1B and its co-adaptor, autosomal recessive hypercholesterolemia protein (ARH), for sorting to the basolateral membrane. Notably, we found that expression of AP-1B (and ARH) slowed epithelial-cell migration, and qRT-PCR analysis of human epithelial-derived cell lines revealed a loss of AP-1B expression in highly metastatic cancer cells indicating that AP-1B-facilitated endocytosis during cell migration might be an anti-cancer mechanism.

scholarly journals Cellular transduction of mechanical oscillations in plants by the plasma-membrane mechanosensitive channel MSL10

2020 ◽  
Vol 118 (1) ◽  
pp. e1919402118
Author(s):  
Daniel Tran ◽  
Tiffanie Girault ◽  
Marjorie Guichard ◽  
Sébastien Thomine ◽  
Nathalie Leblanc-Fournier ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Complexes ◽  
Electrical Signal ◽  
Efficient System ◽  
Mechanical Oscillations ◽  
Multimeric Protein ◽  
Membrane Stretching ◽  
Static Conditions ◽  
Open Question ◽  
Rapid Activation

Plants spend most of their life oscillating around 1–3 Hz due to the effect of the wind. Therefore, stems and foliage experience repetitive mechanical stresses through these passive movements. However, the mechanism of the cellular perception and transduction of such recurring mechanical signals remains an open question. Multimeric protein complexes forming mechanosensitive (MS) channels embedded in the membrane provide an efficient system to rapidly convert mechanical tension into an electrical signal. So far, studies have mostly focused on nonoscillatory stretching of these channels. Here, we show that the plasma-membrane MS channel MscS-LIKE 10 (MSL10) from the model plant Arabidopsis thaliana responds to pulsed membrane stretching with rapid activation and relaxation kinetics in the range of 1 s. Under sinusoidal membrane stretching MSL10 presents a greater activity than under static stimulation. We observed this amplification mostly in the range of 0.3–3 Hz. Above these frequencies the channel activity is very close to that under static conditions. With a localization in aerial organs naturally submitted to wind-driven oscillations, our results suggest that the MS channel MSL10, and by extension MS channels sharing similar properties, represents a molecular component allowing the perception of oscillatory mechanical stimulations by plants.

scholarly journals Focal adhesion-generated cues in extracellular matrix regulate cell migration by local induction of clathrin-coated plaques

2018 ◽  
Author(s):  
Delia Bucher ◽  
Markus Mukenhirn ◽  
Kem A. Sochacki ◽  
Veronika Saharuka ◽  
Christian Huck ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Cell Migration ◽  
Focal Adhesions ◽  
Plaque Formation ◽  
Cell Contact ◽  
Coated Pits ◽  
Independent Manner ◽  
A Cell ◽  
And Function ◽  
Topographical Cues

AbstractClathrin is a unique scaffold protein, which forms polyhedral lattices with flat and curved morphology. The function of curved clathrin-coated pits in forming endocytic structures is well studied. On the contrary, the role of large flat clathrin arrays, called clathrin-coated plaques, remains ambiguous. Previous studies suggested an involvement of plaques in cell adhesion. However, the molecular origin leading to their formation and their precise functions remain to be determined. Here, we study the origin and function of clathrin-coated plaques during cell migration. We revealed that plaque formation is intimately linked to extracellular matrix (ECM) modification by focal adhesions (FAs). We show that in migrating cells, FAs digest the ECM creating extracellular topographical cues that dictate the future location of clathrin-coated plaques. We identify Eps15 and Eps15R as key regulators for the formation of clathrin-coated plaques at locally remodelled ECM sites. Using a genetic silencing approach to abrogate plaque formation and 3D-micropatterns to spatially control the location of clathrin-coated plaques, we could directly correlate cell migration directionality with the formation of clathrin-coated plaques and their ability to recognize extracellular topographical cues. We here define the molecular mechanism regulating the functional interplay between FAs and plaques and propose that clathrin-coated plaques act as regulators of cell migration promoting contact guidance-mediated collective migration in a cell-to-cell contact independent manner.

scholarly journals Transduction of mechanical cellular oscillation by the plasma-membrane mechanosensitive channel MSL10

2019 ◽  
Author(s):  
Daniel Tran ◽  
Tiffanie Girault ◽  
Marjorie Guichard ◽  
Sébastien Thomine ◽  
Nathalie Leblanc-Fournier ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Complexes ◽  
Electrical Signal ◽  
Mechanosensitive Channel ◽  
Efficient System ◽  
Universal System ◽  
Thermal Exchange ◽  
Model Plant Arabidopsis Thaliana ◽  
Membrane Stretching ◽  
Open Question

AbstractThroughout their life, plants are submitted to recurrent cyclic mechanical loading due to wind. The resulting passive oscillation movements of stem and foliage is an important phenomenon for biological and ecological issues such as photosynthesis optimization 1–3 and thermal exchange 4. The induced motions at plant scale are well described and analyzed, with oscillations at typically 1 to 3 Hz in trees 5–10. However, the cellular perception and transduction of such recurring mechanical signals remains an open question. Multimeric protein complexes forming mechanosensitive (MS) channels embedded in the membrane provide an efficient system to rapidly convert mechanical tension into electrical signal 11. Here we show that the plasma membrane mechanosensitive channel MscS-LIKE 10 (MSL10) from the model plant Arabidopsis thaliana responds to pulsed membrane stretching with rapid activation and relaxation kinetics in the range of one second. Under sinusoidal membrane stretching MSL10 presents a greater activity than under static stimulation and behaves as a large bandpass oscillation “follower” without filtering the signal in the range of 0.3 to 3 Hz. With a localization in aerial organs naturally submitted to oscillations, our results suggest that the mechanosensitive channel MSL10 represents a molecular component of a universal system of oscillatory perception in plants.

scholarly journals Three-dimensional reconstruction of the membrane skeleton at the plasma membrane interface by electron tomography

2006 ◽  
Vol 174 (6) ◽  
pp. 851-862 ◽  
Author(s):  
Nobuhiro Morone ◽  
Takahiro Fujiwara ◽  
Kotono Murase ◽  
Rinshi S. Kasai ◽  
Hiroshi Ike ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Filaments ◽  
High Speed ◽  
Plasma Membranes ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Membrane Skeleton ◽  
Single Particle Tracking ◽  
Coated Pits ◽  
Cytoplasmic Surface

Three-dimensional images of the undercoat structure on the cytoplasmic surface of the upper cell membrane of normal rat kidney fibroblast (NRK) cells and fetal rat skin keratinocytes were reconstructed by electron tomography, with 0.85-nm–thick consecutive sections made ∼100 nm from the cytoplasmic surface using rapidly frozen, deeply etched, platinum-replicated plasma membranes. The membrane skeleton (MSK) primarily consists of actin filaments and associated proteins. The MSK covers the entire cytoplasmic surface and is closely linked to clathrin-coated pits and caveolae. The actin filaments that are closely apposed to the cytoplasmic surface of the plasma membrane (within 10.2 nm) are likely to form the boundaries of the membrane compartments responsible for the temporary confinement of membrane molecules, thus partitioning the plasma membrane with regard to their lateral diffusion. The distribution of the MSK mesh size as determined by electron tomography and that of the compartment size as determined from high speed single-particle tracking of phospholipid diffusion agree well in both cell types, supporting the MSK fence and MSK-anchored protein picket models.

Conditional fast expression and function of multimeric TRPV5 channels using Shield-1

2009 ◽  
Vol 296 (1) ◽  
pp. F204-F211 ◽  
Author(s):  
Joost P. H. Schoeber ◽  
Stan F. J. van de Graaf ◽  
Kyu Pil Lee ◽  
Hanneke G. M. Wittgen ◽  
Joost G. J. Hoenderop ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Protein Complexes ◽  
Fk506 Binding Protein ◽  
Synthetic Cell ◽  
Conditional Expression ◽  
Model Protein ◽  
Dose Dependent Fashion ◽  
Regulate Protein ◽  
And Function

A recently described novel controllable method to regulate protein expression is based on a mutated FK506-binding protein-12 (mtFKBP) that is unstable and rapidly degraded in mammalian cells. This instability can be conferred to other proteins directly fused to mtFKBP. Binding of a synthetic cell-permeant ligand (Shield-1) to mtFKBP reverses the instability, allowing conditional expression of mtFKBP-fused proteins. We adapted this strategy to study multimeric plasma membrane proteins using the ion channel TRPV5 as model protein. mtFKBP-TRPV5 forms functional ion channels and its expression can be controlled in a time- and dose-dependent fashion using Shield-1. Moreover, in the presence of Shield-1, mtFKBP-TRPV5 formed heteromultimeric channels with untagged TRPV5, which were codegraded upon washout of Shield-1, providing a strategy to study multimeric plasma membrane protein complexes without the need to destabilize all individual subunits.

scholarly journals An evidence based hypothesis on the existence of two pathways of mitochondrial crista formation

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Max E Harner ◽  
Ann-Katrin Unger ◽  
Willie JC Geerts ◽  
Muriel Mari ◽  
Toshiaki Izawa ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Structure And Function ◽  
Evidence Based ◽  
Molecular Pathways ◽  
Metabolic Function ◽  
F1fo Atp Synthase ◽  
Higher Eukaryotes ◽  
And Function ◽  
Open Question ◽  
Boundary Membrane

Metabolic function and architecture of mitochondria are intimately linked. More than 60 years ago, cristae were discovered as characteristic elements of mitochondria that harbor the protein complexes of oxidative phosphorylation, but how cristae are formed, remained an open question. Here we present experimental results obtained with yeast that support a novel hypothesis on the existence of two molecular pathways that lead to the generation of lamellar and tubular cristae. Formation of lamellar cristae depends on the mitochondrial fusion machinery through a pathway that is required also for homeostasis of mitochondria and mitochondrial DNA. Tubular cristae are formed via invaginations of the inner boundary membrane by a pathway independent of the fusion machinery. Dimerization of the F1FO-ATP synthase and the presence of the MICOS complex are necessary for both pathways. The proposed hypothesis is suggested to apply also to higher eukaryotes, since the key components are conserved in structure and function throughout evolution.

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