scholarly journals A nanobody-based toolset to monitor and modify the mitochondrial GTPase Miro1

2021 ◽  
Author(s):  
Funmilayo O Fagbadebo ◽  
Philipp D Kaiser ◽  
Katharina Zittlau ◽  
Natascha Bartlick ◽  
Teresa R Wagner ◽  
...  
Keyword(s):  
Time Lapse ◽  
Model Systems ◽  
Live Cells ◽  
Mitochondrial Outer Membrane ◽  
Potential Biomarker ◽  
Time Lapse Imaging ◽  
Lateral Sclerosis ◽  
Neuronal Disorders ◽  
Research Tools

The mitochondrial outer membrane (MOM)-anchored GTPase Miro1, is a central player in mitochondrial transport and homeostasis. The dysregulation of Miro1 in amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD) suggests that Miro1 may be a potential biomarker or drug target in neuronal disorders. However, the molecular functionality of Miro1 under (patho-) physiological conditions is poorly known. For a more comprehensive understanding of the molecular functions of Miro1, we have developed Miro1-specific nanobodies (Nbs) as novel research tools. We identified seven Nbs that bind either the N- or C-terminal GTPase domain of Miro1 and demonstrate their application as research tools for proteomic and imaging approaches. To visualize the dynamics of Miro1 in real time, we selected intracellularly functional Nbs, which we reformatted into chromobodies (Cbs) for time-lapse imaging of Miro1. By genetic fusion to an Fbox domain, these Nbs were further converted into Miro1-specific degrons and applied for targeted degradation of Miro1 in live cells. In summary, this study presents a collection of novel Nbs that serve as a toolkit for advanced biochemical and intracellular studies and modulations of Miro1, thereby contributing to the understanding of the functional role of Miro1 in disease-derived model systems.

scholarly journals Assemblies of DivIVA Mark Sites for Hyphal Branching and Can Establish New Zones of Cell Wall Growth in Streptomyces coelicolor

2008 ◽  
Vol 190 (22) ◽  
pp. 7579-7583 ◽  
Author(s):  
Antje Marie Hempel ◽  
Sheng-bing Wang ◽  
Michal Letek ◽  
José A. Gil ◽  
Klas Flärdh
Keyword(s):  
Cell Wall ◽  
Streptomyces Coelicolor ◽  
Time Lapse ◽  
Cell Wall Assembly ◽  
Peptidoglycan Synthesis ◽  
Wall Growth ◽  
Lateral Branches ◽  
Wall Assembly ◽  
Time Lapse Imaging

ABSTRACT Time-lapse imaging of Streptomyces hyphae revealed foci of the essential protein DivIVA at sites where lateral branches will emerge. Overexpression experiments showed that DivIVA foci can trigger establishment of new zones of cell wall assembly, suggesting a key role of DivIVA in directing peptidoglycan synthesis and cell shape in Streptomyces.

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 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 High-throughput live-imaging of embryos in microwell arrays using a modular, inexpensive specimen mounting system

2017 ◽  
Author(s):  
Seth Donoughe ◽  
Chiyoung Kim ◽  
Cassandra G. Extavour
Keyword(s):  
High Throughput ◽  
Fruit Fly ◽  
Time Lapse ◽  
Live Imaging ◽  
Model Systems ◽  
Wide Range ◽  
Amphipod Crustacean ◽  
Live Image ◽  
Time Lapse Imaging ◽  
Annelid Worm

AbstractLive-imaging embryos in a high-throughput manner is essential for shedding light on a wide range of questions in developmental biology, but it is difficult and costly to mount and image embryos in consistent conditions. Here, we present OMMAwell, a simple, reusable device that makes it easy to mount up to hundreds of embryos in arrays of agarose microwells with customizable dimensions and spacing. OMMAwell can be configured to mount specimens for upright or inverted microscopes, and includes a reservoir to hold live-imaging medium to maintain constant moisture and osmolarity of specimens during time-lapse imaging. All device components can be cut from a sheet of acrylic using a laser cutter. Even a novice user will be able to cut the pieces and assemble the device in less than an hour. At the time of writing, the total materials cost is less than five US dollars. We include all device design files in a commonly used format, as well as complete instructions for its fabrication and use. We demonstrate a detailed workflow for designing a custom mold and employing it to simultaneously live-image dozens of embryos at a time for more than five days, using embryos of the cricket Gryllus bimaculatus as an example. Further, we include descriptions, schematics, and design files for molds that can be used with 14 additional vertebrate and invertebrate species, including most major traditional laboratory models and a number of emerging model systems. Molds have been user-tested for embryos including zebrafish (Danio rerio), fruit fly (Drosophila melanogaster), coqui frog (Eleutherodactylus coqui), annelid worm (Capitella teleta), amphipod crustacean (Parhyale hawaiensis), red flour beetle (Tribolium castaneum), and three-banded panther worm (Hofstenia miamia), as well mouse organoids (Mus musculus). Finally, we provide instructions for researchers to customize OMMAwell inserts for embryos or tissues not described herein.Summary StatementThis Techniques and Resources article describes an inexpensive, customizable device for mounting and live-imaging a wide range of tissues and species; complete design files and instructions for assembly are included.

scholarly journals Identification of astroglia-like cardiac nexus glia that are critical regulators of cardiac development and function

PLoS Biology ◽  
2021 ◽  
Vol 19 (11) ◽  
pp. e3001444
Author(s):  
Nina L. Kikel-Coury ◽  
Jacob P. Brandt ◽  
Isabel A. Correia ◽  
Michael R. O’Dea ◽  
Dana F. DeSantis ◽  
...  
Keyword(s):  
Heart Rate ◽  
Nervous System ◽  
Cardiac Development ◽  
Time Lapse ◽  
Zebrafish Model ◽  
Single Cell Sequencing ◽  
Parasympathetic System ◽  
And Function ◽  
Time Lapse Imaging

Glial cells are essential for functionality of the nervous system. Growing evidence underscores the importance of astrocytes; however, analogous astroglia in peripheral organs are poorly understood. Using confocal time-lapse imaging, fate mapping, and mutant genesis in a zebrafish model, we identify a neural crest–derived glial cell, termed nexus glia, which utilizes Meteorin signaling via Jak/Stat3 to drive differentiation and regulate heart rate and rhythm. Nexus glia are labeled with gfap, glast, and glutamine synthetase, markers that typically denote astroglia cells. Further, analysis of single-cell sequencing datasets of human and murine hearts across ages reveals astrocyte-like cells, which we confirm through a multispecies approach. We show that cardiac nexus glia at the outflow tract are critical regulators of both the sympathetic and parasympathetic system. These data establish the crucial role of glia on cardiac homeostasis and provide a description of nexus glia in the PNS.

scholarly journals JMJD6 Dysfunction Due to Iron Deficiency in Preeclampsia Disrupts Fibronectin Homeostasis Resulting in Diminished Trophoblast Migration

2021 ◽  
Vol 9 ◽  
Author(s):  
Sruthi Alahari ◽  
Abby Farrell ◽  
Leonardo Ermini ◽  
Chanho Park ◽  
Julien Sallais ◽  
...  
Keyword(s):  
Time Lapse ◽  
Lysosomal Degradation ◽  
Related Disorder ◽  
Novel Target ◽  
And Function ◽  
Time Lapse Imaging ◽  
Fibronectin Assembly ◽  
Degradation Time ◽  
Trophoblast Migration

The mechanisms contributing to excessive fibronectin in preeclampsia, a pregnancy-related disorder, remain unknown. Herein, we investigated the role of JMJD6, an O2- and Fe2+-dependent enzyme, in mediating placental fibronectin processing and function. MALDI-TOF identified fibronectin as a novel target of JMJD6-mediated lysyl hydroxylation, preceding fibronectin glycosylation, deposition, and degradation. In preeclamptic placentae, fibronectin accumulated primarily in lysosomes of the mesenchyme. Using primary placental mesenchymal cells (pMSCs), we found that fibronectin fibril formation and turnover were markedly impeded in preeclamptic pMSCs, partly due to impaired lysosomal degradation. JMJD6 knockdown in control pMSCs recapitulated the preeclamptic FN phenotype. Importantly, preeclamptic pMSCs had less total and labile Fe2+ and Hinokitiol treatment rescued fibronectin assembly and promoted lysosomal degradation. Time-lapse imaging demonstrated that defective ECM deposition by preeclamptic pMSCs impeded HTR-8/SVneo cell migration, which was rescued upon Hinokitiol exposure. Our findings reveal new Fe2+-dependent mechanisms controlling fibronectin homeostasis/function in the placenta that go awry in preeclampsia.

scholarly journals Identification of multiple kinetic populations of DNA-binding proteins in live cells

2019 ◽  
Author(s):  
Han N. Ho ◽  
Daniel Zalami ◽  
Jürgen Köhler ◽  
Antoine M. van Oijen ◽  
Harshad Ghodke
Keyword(s):  
Dna Binding ◽  
Fluorescence Imaging ◽  
Single Molecule ◽  
Binding Proteins ◽  
Dynamic Range ◽  
Time Lapse ◽  
Dna Binding Proteins ◽  
Live Cells ◽  
Single Molecule Fluorescence ◽  
Time Lapse Imaging

ABSTRACTUnderstanding how multi-protein complexes function in cells requires detailed quantitative understanding of their association and dissociation kinetics. Analysis of the heterogeneity of binding lifetimes enables interrogation of the various intermediate states formed during the reaction. Single-molecule fluorescence imaging permits the measurement of reaction kinetics inside living organisms with minimal perturbation. However, poor photo-physical properties of fluorescent probes limit the dynamic range and accuracy of measurements of off rates in live cells. Time-lapse single-molecule fluorescence imaging can partially overcome the limits of photobleaching, however, limitations of this technique remain uncharacterized. Here, we present a structured analysis of which timescales are most accessible using the time-lapse imaging approach and explore uncertainties in determining kinetic sub-populations. We demonstrate the effect of shot noise on the precision of the measurements, as well as the resolution and dynamic range limits that are inherent to the method. Our work provides a convenient implementation to determine theoretical errors from measurements and to support interpretation of experimental data.STATEMENT OF SIGNIFICANCEMeasuring lifetimes of interactions between DNA-binding proteins and their substrates is important for understanding how they function in cells. In principle, time-lapse imaging of fluorescently-tagged proteins using single-molecule methods can be used to identify multiple sub-populations of DNA-binding proteins and determine binding lifetimes lasting for several tens of minutes. Despite this potential, currently available guidelines for the selection of binding models are unreliable, and the practical implementation of this approach is limited. Here, using experimental and simulated data we identify the minimum size of the dataset required to resolve multiple populations reliably and measure binding lifetimes with desired accuracy. This work serves to provide a guide to data collection, and measurement of DNA-binding lifetimes from single-molecule time-lapse imaging data.

Abstract 599: Lighting Up P2Y12 Oligomers: Insights into the Role of Oligomerization in P2Y12 Function

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Aasma Khan ◽  
Jeffrey L Caplan ◽  
Donna Woulfe
Keyword(s):  
Flow Cytometry ◽  
Confocal Microscopy ◽  
G Protein ◽  
Time Lapse ◽  
Bimolecular Fluorescence Complementation ◽  
P2y12 Receptor ◽  
Live Cells ◽  
Bleeding Disorders ◽  
Mutant Forms

Introduction: Little is known about the role of P2Y12 oligomerization in receptor function and whether P2Y12 receptor mutations associated with human bleeding disorders may be explained by alterations in oligomerization. Objectives: 1) To determine whether P2Y12 homo- and hetero-oligomers are constitutive or dynamically regulated. 2) To explore whether P2Y12 mutants R256Q and R265W (previously detected in patients with abnormal bleeding, but with unaltered ADP binding) have different oligomerization affinities or kinetics and determine whether differences in P2Y12 oligomerization explain the functional defects. Methods: We employed a Venus-based Bimolecular Fluorescence Complementation (BiFC) approach in HEK293T cells transiently co-expressing P2Y12 or its mutant forms (R256Q or R265W) tagged with either the N-terminal (P2Y12-VN) or C terminal fragment (P2Y12-VC) of Venus, to characterize their homomeric interactions, in live cells using confocal microscopy and quantitative flow cytometry assays. Results: Agonist-independent formation of P2Y12 receptor homo-oligomers were detected on cell membranes. Time lapse imaging showed movement of P2Y12 receptor pairs from the endoplamic reticulum and Golgi network to the plasma membrane, suggesting that they are constitutive and required for export. Co-expression of P2Y12-VN with increasing amounts of P2Y12-VC demonstrated a dose-dependent increase in the fluorescence intensity of Venus, and reached saturation at a ratio of 1:3. Interestingly, the fluorescence intensities of homomeric P2Y12-R256Q-VN and R256Q-VC and, separately, P2Y12-R265W-VN and P2Y12R265W-VC were almost 4 times stronger than that of the wild type receptor as quantified in flow cytometry-based BiFC. Similar results were obtained in confocal microscopy. This suggests that these P2Y12 mutants form an increased number of dimers or oligomers with increased self-affinities. Conclusion: We demonstrate that P2Y12 forms constitutive homo-oligomers. Two mutations associated with bleeding disorders in patients have altered receptor-receptor interactions. Future investigation will explore the effect of mutations and receptor oligomers on G protein coupling and receptor: G protein stoichiometry.

scholarly journals Nuclear Aggresomes Form by Fusion of PML-associated Aggregates

2005 ◽  
Vol 16 (10) ◽  
pp. 4905-4917 ◽  
Author(s):  
Lianwu Fu ◽  
Ya-sheng Gao ◽  
Albert Tousson ◽  
Anish Shah ◽  
Tung-Ling L. Chen ◽  
...  
Keyword(s):  
Neurodegenerative Diseases ◽  
Fluorescence Recovery After Photobleaching ◽  
Nuclear Architecture ◽  
Time Lapse ◽  
Promyelocytic Leukemia ◽  
Live Cells ◽  
Aggregate Formation ◽  
Pml Bodies ◽  
Nuclear Domains ◽  
Time Lapse Imaging

Nuclear aggregates formed by proteins containing expanded poly-glutamine (poly-Q) tracts have been linked to the pathogenesis of poly-Q neurodegenerative diseases. Here, we show that a protein (GFP170*) lacking poly-Q tracts forms nuclear aggregates that share characteristics of poly-Q aggregates. GFP170*aggregates recruit cellular chaperones and proteasomes, and alter the organization of nuclear domains containing the promyelocytic leukemia (PML) protein. These results suggest that the formation of nuclear aggregates and their effects on nuclear architecture are not specific to poly-Q proteins. Using GFP170*as a model substrate, we explored the mechanistic details of nuclear aggregate formation. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching analyses show that GFP170*molecules exchange rapidly between aggregates and a soluble pool of GFP170*, indicating that the aggregates are dynamic accumulations of GFP170*. The formation of cytoplasmic and nuclear GFP170*aggregates is microtubule-dependent. We show that within the nucleus, GFP170*initially deposits in small aggregates at or adjacent to PML bodies. Time-lapse imaging of live cells shows that small aggregates move toward each other and fuse to form larger aggregates. The coalescence of the aggregates is accompanied by spatial rearrangements of the PML bodies. Significantly, we find that the larger nuclear aggregates have complex internal substructures that reposition extensively during fusion of the aggregates. These studies suggest that nuclear aggregates may be viewed as dynamic multidomain inclusions that continuously remodel their components.

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