scholarly journals Fluorescent and bioluminescent calcium indicators with tuneable colors and affinities

2021 ◽  
Author(s):  
Nicole Mertes ◽  
Marvin Busch ◽  
Magnus-Carsten Huppertz ◽  
Christina Nicole Hacker ◽  
Clara-Marie Guerth ◽  
...  
Keyword(s):  
Action Potentials ◽  
Hippocampal Neurons ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Ease Of Use ◽  
Fluorescent Indicators ◽  
Biological Assays ◽  
Single Action ◽  
Calcium Indicators ◽  
Cellular Compartments

We introduce a family of bright, rhodamine-based calcium indicators with tuneable affinities and colors. The indicators can be specifically localized to different cellular compartments and are compatible with both fluorescence and bioluminescence readouts through conjugation to HaloTag fusion proteins. Importantly, their increase in fluorescence upon localization enables no-wash live-cell imaging, which greatly facilitates their use in biological assays. Applications as fluorescent indicators in rat hippocampal neurons include the detection of single action potentials and of calcium fluxes in the endoplasmic reticulum (ER). Applications as bioluminescent indicators include the recording of the pharmacological modulation of nuclear calcium in high-throughput-compatible assays. The versatility and remarkable ease of use of these indicators make them powerful tools for bioimaging and bioassays.

Activity-dependent release of tissue plasminogen activator from the dendritic spines of hippocampal neurons revealed by live-cell imaging

2006 ◽  
Vol 66 (6) ◽  
pp. 564-577 ◽  
Author(s):  
Janis E. Lochner ◽  
Leah S. Honigman ◽  
Wilmon F. Grant ◽  
Sarah K. Gessford ◽  
Alexis B. Hansen ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Dendritic Spines ◽  
Hippocampal Neurons ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Tissue Plasminogen ◽  
Activity Dependent

The Fate of Nascent APP in Hippocampal Neurons: A Live Cell Imaging Study

2018 ◽  
Vol 9 (9) ◽  
pp. 2225-2232
Author(s):  
Claire E. DelBove ◽  
Xian-zhen Deng ◽  
Qi Zhang
Keyword(s):  
Hippocampal Neurons ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Imaging Study ◽  
Live Cell

scholarly journals PI(3,5)P2 biosynthesis regulates oligodendrocyte differentiation by intrinsic and extrinsic mechanisms

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Yevgeniya A Mironova ◽  
Guy M Lenk ◽  
Jing-Ping Lin ◽  
Seung Joon Lee ◽  
Jeffery L Twiss ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Action Potentials ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Primary Cells ◽  
Oligodendrocyte Differentiation ◽  
Compound Action Potentials ◽  
Myelin Associated Glycoprotein ◽  
Cns Myelination ◽  
Compound Action

Proper development of the CNS axon-glia unit requires bi-directional communication between axons and oligodendrocytes (OLs). We show that the signaling lipid phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2] is required in neurons and in OLs for normal CNS myelination. In mice, mutations of Fig4, Pikfyve or Vac14, encoding key components of the PI(3,5)P2 biosynthetic complex, each lead to impaired OL maturation, severe CNS hypomyelination and delayed propagation of compound action potentials. Primary OLs deficient in Fig4 accumulate large LAMP1+ and Rab7+ vesicular structures and exhibit reduced membrane sheet expansion. PI(3,5)P2 deficiency leads to accumulation of myelin-associated glycoprotein (MAG) in LAMP1+perinuclear vesicles that fail to migrate to the nascent myelin sheet. Live-cell imaging of OLs after genetic or pharmacological inhibition of PI(3,5)P2 synthesis revealed impaired trafficking of plasma membrane-derived MAG through the endolysosomal system in primary cells and brain tissue. Collectively, our studies identify PI(3,5)P2 as a key regulator of myelin membrane trafficking and myelinogenesis.

scholarly journals A novel epitope tagging system to visualize and monitor antigens in live cells with chromobodies

2020 ◽  
Author(s):  
Bjoern Traenkle ◽  
Sören Segan ◽  
Philipp D. Kaiser ◽  
Ulrich Rothbauer
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Drug Action ◽  
Live Cells ◽  
Antigen Concentration ◽  
Epitope Tagging ◽  
Dynamic Regulation ◽  
Short Peptide ◽  
Cellular Compartments ◽  
Tagging System

SummaryEpitope tagging is a versatile approach to study different proteins using a well-defined and established methodology. To date, most epitope tags such as myc, HA, V5 and FLAG tags are recognized by antibodies which limits their application to fixed cells, tissues or protein samples. Here we introduce a broadly applicable tagging strategy utilizing a short peptide tag/chromobody (PepTag/PepCB) system. The addition of the small PepTag does not interfere with the examined structures in different cellular compartments and its detection with the fluorescently labeled Pep-chromobody (PepCB) enables optical antigen tracing in real time. By employing the phenomenon of antigen-mediated chromobody stabilization (AMCBS) using a turnover-accelerated PepCB we demonstrated that the system is suitable to visualize and quantify changes in Pep-tagged antigen concentration by quantitative live-cell imaging. We expect that this novel tagging strategy offers new opportunities to study the dynamic regulation of proteins, e.g. during cellular signaling, cell differentiation, or upon drug action.

scholarly journals ADAM22 and ADAM23 modulate the targeting of the Kv1 channel-associated protein LGI1 to the axon initial segment

2018 ◽  
Author(s):  
Bruno Hivert ◽  
Laurène Marien ◽  
Komlan Nassirou Agbam ◽  
Catherine Faivre-Sarrailh
Keyword(s):  
Hippocampal Neurons ◽  
Live Cell Imaging ◽  
Initial Segment ◽  
Cell Imaging ◽  
Autosomal Dominant ◽  
Neurological Diseases ◽  
Axon Initial Segment ◽  
Intrinsic Excitability ◽  
Missense Mutant ◽  
Combinatorial Expression

AbstractThe distribution of voltage-gated potassium channels Kv1 at the axon initial segment (AIS), along the axon and at presynaptic terminals influences intrinsic excitability and transmitter release. Kv1.1/1.2 subunits are associated with cell adhesion molecules (CAMs), including Caspr2 and LGI1 that are implicated in autoimmune and genetic neurological diseases with seizures. In particular, mutations in the LGI1 gene cause autosomal dominant lateral temporal lobe epilepsy (ADTLE). In the present study, we used rat hippocampal neurons in culture to assess whether interplay between distinct Kv1-associated CAMs contributes to targeting at the AIS. Strikingly, LGI1 was highly restricted to the AIS surface when transfected alone, whereas the missense mutant LGI1S473L associated with ADLTE was not. Next, we showed that ADAM22 and ADAM23 acted as chaperones to promote axonal vesicular transport of LGI1 reducing its density at the AIS. Moreover, live-cell imaging of fluorescently labelled CAMs indicated that LGI1 was co-transported in axonal vesicles with ADAM22 or ADAM23. Finally, we showed that ADAM22 and ADAM23 also associate with Caspr2 and TAG-1 to be selectively targeted within different axonal sub-regions. The combinatorial expression of Kv1-associated CAMs may be critical to tune intrinsic excitability in a physiological or an epileptogenic context.

scholarly journals Bright and tunable far-red chemigenetic indicators

2020 ◽  
Author(s):  
Claire Deo ◽  
Ahmed S. Abdelfattah ◽  
Hersh K. Bhargava ◽  
Adam J. Berro ◽  
Natalie Falco ◽  
...  
Keyword(s):  
Functional Imaging ◽  
Action Potentials ◽  
Chemical Properties ◽  
The Self ◽  
Red Emission ◽  
Fluorescent Indicators ◽  
Single Action ◽  
Voltage Sensors ◽  
Environmentally Sensitive ◽  
And Chemical Properties

AbstractFunctional imaging using fluorescent indicators has revolutionized biology but additional sensor scaffolds are needed to access properties such as bright, far-red emission. We introduce a new platform for ‘chemigenetic’ fluorescent indicators, utilizing the self-labeling HaloTag protein conjugated to environmentally sensitive synthetic fluorophores. This approach affords bright, far-red calcium and voltage sensors with highly tunable photophysical and chemical properties, which can reliably detect single action potentials in neurons.

scholarly journals Local ERM activation and dynamic growth cones at Schwann cell tips implicated in efficient formation of nodes of Ranvier

2003 ◽  
Vol 162 (3) ◽  
pp. 489-498 ◽  
Author(s):  
Cheryl L. Gatto ◽  
Barbara J. Walker ◽  
Stephen Lambert
Keyword(s):  
Schwann Cell ◽  
Action Potentials ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Explant Culture ◽  
Nodes Of Ranvier ◽  
Sodium Hydrogen ◽  
Sodium Hydrogen Exchanger ◽  
Dynamic Growth ◽  
Node Formation

Nodes of Ranvier are specialized, highly polarized axonal domains crucial to the propagation of saltatory action potentials. In the peripheral nervous system, axo–glial cell contacts have been implicated in Schwann cell (SC) differentiation and formation of the nodes of Ranvier. SC microvilli establish axonal contact at mature nodes, and their components have been observed to localize early to sites of developing nodes. However, a role for these contacts in node formation remains controversial. Using a myelinating explant culture system, we have observed that SCs reorganize and polarize microvillar components, such as the ezrin-binding phosphoprotein 50 kD/regulatory cofactor of the sodium-hydrogen exchanger isoform 3 (NHERF-1), actin, and the activated ezrin, radixin, and moesin family proteins before myelination in response to inductive signals. These components are targeted to the SC distal tips where live cell imaging reveals novel, dynamic growth cone–like behavior. Furthermore, localized activation of the Rho signaling pathway at SC tips gives rise to these microvillar component–enriched “caps” and influences the efficiency of node formation.

scholarly journals Super-resolved live-cell imaging using Random Illumination Microscopy

2020 ◽  
Author(s):  
Thomas Mangeat ◽  
Simon Labouesse ◽  
Marc Allain ◽  
Emmanuel Martin ◽  
Renaud Poincloux ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
New Technology ◽  
Super Resolution ◽  
Ease Of Use ◽  
Live Cell ◽  
Three Dimensions ◽  
Tissue Imaging ◽  
Wide Range ◽  
Random Illumination

SummarySuper-resolution fluorescence microscopy has been instrumental to progress in biology. Yet, the photo-induced toxicity, the loss of resolution into scattering samples or the complexity of the experimental setups curtail its general use for functional cell imaging. Here, we describe a new technology for tissue imaging reaching a 114nm/8Hz resolution at 30 µm depth. Random Illumination Microscopy (RIM) consists in shining the sample with uncontrolled speckles and extracting a high-fidelity super-resolved image from the variance of the data using a reconstruction scheme accounting for the spatial correlation of the illuminations. Super-resolution unaffected by optical aberrations, undetectable phototoxicity, fast image acquisition rate and ease of use, altogether, make RIM ideally suited for functional live cell imaging in situ. RIM ability to image molecular and cellular processes in three dimensions and at high resolution is demonstrated in a wide range of biological situations such as the motion of Myosin II minifilaments in Drosophila.

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