scholarly journals Gland cell responses to feeding in Drosera capensis, a carnivorous plant

PROTOPLASMA ◽  
2021 ◽  
Author(s):  
Irene Lichtscheidl ◽  
Sue Lancelle ◽  
Marieluise Weidinger ◽  
Wolfram Adlassnig ◽  
Marianne Koller-Peroutka ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Gland Cell ◽  
Cell Types ◽  
Carnivorous Plant ◽  
Dynamic Processes ◽  
Cell Responses ◽  
Excellent Preservation ◽  
Parenchymal Cells

AbstractGlands of Drosera absorb and transport nutrients from captured prey, but the mechanism and dynamics remain unclear. In this study, we offered animal proteins in the form of fluorescent albumin (FITC-BSA) and observed the reactions of the glands by live cell imaging and fluorescence microscopy. The ultrastructure of these highly dynamic processes was also assessed in high-pressure frozen and freeze substituted (HPF-FS) cells. HPF-FS yielded excellent preservation of the cytoplasm of all cell types, although the cytosol looked different in gland cells as compared to endodermoid and stalk cells. Especially prominent were the ER and its contacts with the plasma membrane, plasmodesmata, and other organelles as well as continuities between organelles. Also distinct were actin microfilaments in association with ER and organelles. Application of FITC-BSA to glands caused the formation of fluorescent endosomes that pinched off the plasma membrane. Endosomes fused to larger aggregates, and accumulated in the bulk cytoplasm around the nucleus. They did not fuse with the cell sap vacuole but remained for at least three days; in addition, fluorescent vesicles also proceeded through endodermoid and transfer cells to the epidermal and parenchymal cells of the tentacle stalk.

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.

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.

scholarly journals A Simple and Efficient CRISPR Technique for Protein Tagging

Cells ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 2618
Author(s):  
Fanning Zeng ◽  
Valerie Beck ◽  
Sven Schuierer ◽  
Isabelle Garnier ◽  
Carole Manneville ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Cell Types ◽  
Luciferase Assay ◽  
Primary Cells ◽  
Homology Directed Repair ◽  
Independent Gene ◽  
Protein Tagging ◽  
Selection Of

Genetic knock-in using homology-directed repair is an inefficient process, requiring the selection of few modified cells and hindering its application to primary cells. Here, we describe Homology independent gene Tagging (HiTag), a method to tag a protein of interest by CRISPR in up to 66% of transfected cells with one single electroporation. The technique has proven effective in various cell types and can be used to knock in a fluorescent protein for live cell imaging, to modify the cellular location of a target protein and to monitor the levels of a protein of interest by a luciferase assay in primary cells.

Live Cell Imaging of Actin Dynamics in the Filamentous Fungus Aspergillus nidulans

2016 ◽  
Vol 22 (2) ◽  
pp. 264-274 ◽  
Author(s):  
Zachary Schultzhaus ◽  
Laura Quintanilla ◽  
Angelyn Hilton ◽  
Brian D. Shaw
Keyword(s):  
Aspergillus Nidulans ◽  
Filamentous Fungus ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Tip Growth ◽  
Cell Types ◽  
Actin Dynamics ◽  
Growth Site ◽  
The Face ◽  
Over Time

AbstractHyphal cells of filamentous fungi grow at their tips in a method analogous to pollen tube and root hair elongation. This process, generally referred to as tip growth, requires precise regulation of the actin cytoskeleton, and characterizing the various actin structures in these cell types is currently an active area of research. Here, the actin marker Lifeact was used to document actin dynamics in the filamentous fungus Aspergillus nidulans. Contractile double rings were observed at septa, and annular clusters of puncta were seen subtending growing hyphal tips, corresponding to the well-characterized subapical endocytic collar. However, Lifeact also revealed two additional structures. One, an apical array, was dynamic on the face opposite the tip, while a subapical web was dynamic on the apical face and was located several microns behind the growth site. Each was observed turning into the other over time, implying that they could represent different localizations of the same structure, although hyphae with a subapical web grew faster than those exhibiting an apical array. The subapical web has not been documented in any filamentous fungus to date, and is separate from the networks of F-actin seen in other tip-growing organisms surrounding septa or stationary along the plasmalemma.

scholarly journals Keeping Life in Focus New Systems Prevent Z-axis Drift in Time Lapse Studies

2006 ◽  
Vol 14 (4) ◽  
pp. 42-46 ◽  
Author(s):  
Edward Lachica
Keyword(s):  
Fluorescent Probes ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Time Lapse ◽  
Living Cells ◽  
Dynamic Processes ◽  
Biological Structure ◽  
Camera Lucida ◽  
Developmental Anatomy ◽  
Organ Level

Just two decades ago, life scientists studied biological structure, developmental anatomy and intracellular processes by describing individual snapshots of kinetic events. Today, with so much bioscience research focusing on dynamic processes that occur on the molecular, cellular and whole organ level, it is important to record events as they happen, over seconds, minutes or hours, in living cells. Photographs and camera lucida drawings of fixed, stained cells have given way to live cell imaging using fluorescent probes, warming trays to promote cell viability and cinemicrography as a method of recording events.

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.

Staining of Mitochondria with Cy5-Labeled Oligonucleotides for Long-Term Microscopy Studies

2011 ◽  
Vol 17 (3) ◽  
pp. 440-445 ◽  
Author(s):  
Steffen Lorenz ◽  
Stephanie Tomcin ◽  
Volker Mailänder
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Cell Types ◽  
Low Ph ◽  
Target Sequence ◽  
Sensitive Cell ◽  
Cellular Compartment ◽  
Short Oligonucleotide ◽  
Fluorescently Labeled Oligonucleotides

AbstractLabeling of organelles for microscopy is achieved generally by specific dyes that either accumulate in a cellular compartment such as cyanine dyes in mitochondria or are only fluorescent under specific conditions such as the low pH in the lysosome. Here we demonstrate that Cy5—a fluorescent molecule that does not enter cells by itself—can be loaded into cells by attaching a short oligonucleotide. This very inexpensive labeling procedure can be done in the presence of serum. Therefore, very sensitive cell types should also be amenable to this procedure, and longer observations can be achieved compared to other commercially available dyes as the labeling reagent does not need to be washed out. This also points to the pitfall of using fluorescently labeled oligonucleotides for live cell imaging where the oligonucleotide is supposed to detect a specific target sequence in its subcellular distribution.

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 A novel live-cell imaging assay reveals regulation of endosome maturation

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Maria Podinovskaia ◽  
Cristina Prescianotto-Baschong ◽  
Dominik P Buser ◽  
Anne Spang
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Cell Communication ◽  
Cell Types ◽  
Cellular Systems ◽  
Live Cell ◽  
Recycling Endosome ◽  
Endosomal Acidification ◽  
Turn Over ◽  
Endosome Maturation

Cell-cell communication is an essential process in life, with endosomes acting as key organelles for regulating uptake and secretion of signaling molecules. Endocytosed material is accepted by the sorting endosome where it either is sorted for recycling or remains in the endosome as it matures to be degraded in the lysosome. Investigation of the endosome maturation process has been hampered by the small size and rapid movement of endosomes in most cellular systems. Here, we report an easy versatile live-cell imaging assay to monitor endosome maturation kinetics, which can be applied to a variety of mammalian cell types. Acute ionophore treatment led to enlarged early endosomal compartments that matured into late endosomes and fused with lysosomes to form endolysosomes. Rab5-to-Rab7 conversion and PI(3)P formation and turn over were recapitulated with this assay and could be observed with a standard widefield microscope. We used this approach to show that Snx1 and Rab11-positive recycling endosome recruitment occurred throughout endosome maturation and was uncoupled from Rab conversion. In contrast, efficient endosomal acidification was dependent on Rab conversion. The assay provides a powerful tool to further unravel various aspects of endosome maturation.

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