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 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.

Lipid order and molecular assemblies in the plasma membrane of eukaryotic cells

2009 ◽  
Vol 37 (5) ◽  
pp. 1056-1060 ◽  
Author(s):  
Marek Cebecauer ◽  
Dylan M. Owen ◽  
Anna Markiewicz ◽  
Anthony I. Magee
Keyword(s):  
Plasma Membrane ◽  
Physical Properties ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Eukaryotic Cells ◽  
Dynamic Nature ◽  
Membrane Components ◽  
Technological Improvements

Multimolecular assemblies on the plasma membrane exhibit dynamic nature and are often generated during the activation of eukaryotic cells. The role of lipids and their physical properties in helping to control the existence of these structures is discussed. Technological improvements for live cell imaging of membrane components are also reviewed.

scholarly journals PGE2 upregulates the Na+/K+ ATPase in HepG2 cells via EP4 receptors and intracellular calcium

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0245400
Author(s):  
Rawad Hodeify ◽  
Mohamed Chakkour ◽  
Reem Rida ◽  
Sawsan Kreydiyyeh
Keyword(s):  
Intracellular Calcium ◽  
Hepg2 Cells ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Time Lapse ◽  
Live Cells ◽  
Ionic Homeostasis ◽  
Significant Stimulation ◽  
Time Lapse Imaging ◽  
Stimulation Of

The Na+/K+ ATPase is a key regulator of the hepatocytes ionic homeostasis, which when altered may lead to many liver disorders. We demonstrated recently, a significant stimulation of the Na+/K+ ATPase in HepG2 cells treated with the S1P analogue FTY 720P, that was mediated through PGE2. The mechanism by which the prostaglandin exerts its effect was not investigated, and is the focus of this work. The type of receptors involved was determined using pharmacological inhibitors, while western blot analysis, fluorescence imaging of GFP-tagged Na+/K+ ATPase, and time-lapse imaging on live cells were used to detect changes in membrane abundance of the Na+/K+ ATPase. The activity of the ATPase was assayed by measuring the amount of inorganic phosphate liberated in the presence and absence of ouabain. The enhanced activity of the ATPase was not observed when EP4 receptors were blocked but still appeared in presence inhibitors of EP1, EP2 and EP3 receptors. The involvement of EP4 was confirmed by the stimulation observed with EP4 agonist. The stimulatory effect of PGE2 did not appear in presence of Rp-cAMP, an inhibitor of PKA, and was imitated by db-cAMP, a PKA activator. Chelating intracellular calcium with BAPTA-AM abrogated the effect of db-cAMP as well as that of PGE2, but PGE2 treatment in a calcium-free PBS medium did not, suggesting an involvement of intracellular calcium, that was confirmed by the results obtained with 2-APB treatment. Live cell imaging showed movement of GFP–Na+/K+ ATPase-positive vesicles to the membrane and increased abundance of the ATPase at the membrane after PGE2 treatment. It was concluded that PGE2 acts via EP4, PKA, and intracellular calcium.

scholarly journals Role of turgor pressure in endocytosis in fission yeast

2014 ◽  
Vol 25 (5) ◽  
pp. 679-687 ◽  
Author(s):  
Roshni Basu ◽  
Emilia Laura Munteanu ◽  
Fred Chang
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Fission Yeast ◽  
Turgor Pressure ◽  
Time Lapse ◽  
Actin Assembly ◽  
Latrunculin A ◽  
The Media ◽  
Time Lapse Imaging

Yeast and other walled cells possess high internal turgor pressure that allows them to grow and survive in the environment. This turgor pressure, however, may oppose the invagination of the plasma membrane needed for endocytosis. Here we study the effects of turgor pressure on endocytosis in the fission yeast Schizosaccharomyces pombe by time-lapse imaging of individual endocytic sites. Decreasing effective turgor pressure by addition of sorbitol to the media significantly accelerates early steps in the endocytic process before actin assembly and membrane ingression but does not affect the velocity or depth of ingression of the endocytic pit in wild-type cells. Sorbitol also rescues endocytic ingression defects of certain endocytic mutants and of cells treated with a low dose of the actin inhibitor latrunculin A. Endocytosis proceeds after removal of the cell wall, suggesting that the cell wall does not contribute mechanically to this process. These studies suggest that endocytosis is governed by a mechanical balance between local actin-dependent inward forces and opposing forces from high internal turgor pressure on the plasma membrane.

Nir1 constitutively localizes at ER-PM junctions and promotes Nir2 recruitment for PIP2 homeostasis

2022 ◽  
Author(s):  
Carlo Giovanni Quintanilla ◽  
Wan-Ru Lee ◽  
Jen Liou
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Live Cell Imaging ◽  
Biochemical Analysis ◽  
Cell Imaging ◽  
Lipid Transfer Protein ◽  
Homeostatic Regulation ◽  
Lipid Transfer ◽  
Transfer Protein ◽  
The Family

Homeostatic regulation of plasma membrane (PM) phosphatidylinositol 4,5-bisphosphate (PIP2) in receptor-stimulated cells is mediated by the lipid transfer protein Nir2. Nir2 is dynamically recruited to endoplasmic reticulum-plasma membrane (ER-PM) junctions to facilitate replenishment of PM PIP2 hydrolyzed during receptor-mediated signaling. However, our knowledge regarding the activation and sustainment of Nir2-mediated replenishment of PM PIP2 is limited. Here, we describe the functions of Nir1 as a positive regulator of Nir2 and PIP2 homeostasis. In contrast to the family proteins Nir2 and Nir3, Nir1 constitutively localizes at ER-PM junctions. Nir1 potentiates Nir2 targeting to ER-PM junctions during receptor-mediated signaling and is required for efficient PM PIP2 replenishment. Live-cell imaging and biochemical analysis reveal that Nir1 interacts with Nir2 via a region between the FFAT motif and the DDHD domain. Combined, results from this study identify Nir1 as an ER-PM junction localized protein that promotes Nir2 recruitment for PIP2 homeostasis.

scholarly journals Time-Lapse, Live-Cell Imaging of Potassium Efflux in Cell Exposed to Nanosecond Pulsed Electric Fields

2021 ◽  
Vol 120 (3) ◽  
pp. 223a
Author(s):  
Flavia Mazzarda ◽  
Esin B. Sozer ◽  
Julia L. Pittaluga ◽  
Claudia Muratori ◽  
P. Thomas Vernier
Keyword(s):  
Electric Fields ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Pulsed Electric Fields ◽  
Time Lapse ◽  
Live Cell ◽  
Potassium Efflux ◽  
Nanosecond Pulsed Electric Fields

Dye selection for live cell imaging of intact siRNA

2012 ◽  
Vol 393 (1-2) ◽  
pp. 23-35 ◽  
Author(s):  
Markus Hirsch ◽  
Dennis Strand ◽  
Mark Helm
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Dyes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Lapse ◽  
Live Cell ◽  
High Yield ◽  
Ph Variation ◽  
Rnai Efficiency

Abstract Investigations into the fate of small interfering RNA (siRNA) after transfection may unravel new ways to improve RNA interference (RNAi) efficiency. Because intracellular degradation of RNA may prevent reliable observation of fluorescence-labeled siRNA, new tools for fluorescence microscopy are warranted to cover the considerable duration of the RNAi effect. Here, the characterization and application of new fluorescence resonance energy transfer (FRET) dye pairs for sensing the integrity of duplex siRNA is reported, which allows an assessment of the degradation status of an siRNA cell population by live cell imaging. A panel of high-yield fluorescent dyes has been investigated for their suitability as FRET pairs for the investigation of RNA inside the cell. Nine dyes in 13 FRET pairs were evaluated based on the performance in assays of photostability, cross-excitation, bleed-through, as well as on quantified changes of fluorescence as a consequence of, e.g., RNA strand hybridization and pH variation. The Atto488/Atto590 FRET pair has been applied to live cell imaging, and has revealed first aspects of unusual trafficking of intact siRNA. A time-lapse study showed highly dynamic movement of siRNA in large perinuclear structures. These and the resulting optimized FRET labeled siRNA are expected to have significant impact on future observations of labeled RNAs in living cells.

scholarly journals LRRK2 mediates tubulation and vesicle sorting from membrane damaged lysosomes

2020 ◽  
Author(s):  
Luis Bonet-Ponce ◽  
Alexandra Beilina ◽  
Chad D. Williamson ◽  
Eric Lindberg ◽  
Jillian H. Kluss ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Super Resolution ◽  
Adaptor Protein ◽  
Dependent Manner ◽  
Leucine Rich Repeat ◽  
Biological Functions ◽  
New Process ◽  
Sporadic Parkinson’S Disease

ABSTRACTMutations in the leucine rich repeat kinase 2 (LRRK2) gene are a cause of familial and sporadic Parkinson’s disease (PD). Nonetheless, the biological functions of LRRK2 remain incompletely understood. Here, we observed that LRRK2 is recruited to lysosomes that have a ruptured membrane. Using unbiased proteomics, we observed that LRRK2 is able to recruit the motor adaptor protein JIP4 to permeabilized lysosomes in a kinase-dependent manner through the phosphorylation of RAB35 and RAB10. Super-resolution live cell imaging microscopy and FIB-SEM revealed that once at the lysosomal membrane, JIP4 promotes the formation of LAMP1-negative lysosomal tubules that release membranous content from ruptured lysosomes. Released vesicular structures are able to interact with other lysosomes. Thus, we described a new process that uses lysosomal tubulation to release vesicular structures from permeabilized lysosomes. LRRK2 orchestrates this process that we name LYTL (LYsosomal Tubulation/sorting driven by LRRK2) that, given the central role of the lysosome in PD, is likely to be disease relevant.

scholarly journals Conformational specificity of opioid receptors is determined by subcellular location irrespective of agonist

2021 ◽  
Author(s):  
Stephanie E. Crilly ◽  
Wooree Ko ◽  
Zara Y. Weinberg ◽  
Manojkumar A. Puthenveedu
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Opioid Receptors ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Subcellular Location ◽  
Unanswered Question ◽  
Receptor Coupling ◽  
Δ Opioid Receptor ◽  
G Protein Coupled

AbstractThe prevailing model for the variety in drug responses is that they stabilize distinct active states of their G protein-coupled receptor (GPCR) targets, allowing coupling to different effectors. However, whether the same ligand can produce different GPCR active states based on the environment of receptors in cells is a fundamental unanswered question. Here we address this question using live cell imaging of conformational biosensors that read out distinct active conformations of the δ-opioid receptor (DOR), a physiologically relevant GPCR localized to Golgi and the surface in neurons. We show that, although Golgi and surface pools of DOR regulated cAMP, the two pools engaged distinct conformational biosensors in response to the same ligand. Further, DOR recruited arrestin on the plasma membrane but not the Golgi. Our results suggest that the same agonist drives different conformations of a GPCR at different locations, allowing receptor coupling to distinct effectors at different locations.

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