scholarly journals Deciphering dynamics of clathrin-mediated endocytosis in a living organism

2016 ◽  
Vol 214 (3) ◽  
pp. 347-358 ◽  
Author(s):  
Joshua P. Ferguson ◽  
Nathan M. Willy ◽  
Spencer P. Heidotting ◽  
Scott D. Huber ◽  
Matthew J. Webber ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Real Time ◽  
Growth Rates ◽  
Cultured Cells ◽  
Living Organism ◽  
Particle Detection ◽  
Detection And Tracking ◽  
Spatiotemporal Changes ◽  
Multicellular Organisms

Current understanding of clathrin-mediated endocytosis (CME) dynamics is based on detection and tracking of fluorescently tagged clathrin coat components within cultured cells. Because of technical limitations inherent to detection and tracking of single fluorescent particles, CME dynamics is not characterized in vivo, so the effects of mechanical cues generated during development of multicellular organisms on formation and dissolution of clathrin-coated structures (CCSs) have not been directly observed. Here, we use growth rates of fluorescence signals obtained from short CCS intensity trace fragments to assess CME dynamics. This methodology does not rely on determining the complete lifespan of individual endocytic assemblies. Therefore, it allows for real-time monitoring of spatiotemporal changes in CME dynamics and is less prone to errors associated with particle detection and tracking. We validate the applicability of this approach to in vivo systems by demonstrating the reduction of CME dynamics during dorsal closure of Drosophila melanogaster embryos.

scholarly journals Effector caspase Dcp-1 and IAP protein Bruce regulate starvation-induced autophagy during Drosophila melanogaster oogenesis

2008 ◽  
Vol 182 (6) ◽  
pp. 1127-1139 ◽  
Author(s):  
Ying-Chen Claire Hou ◽  
Suganthi Chittaranjan ◽  
Sharon González Barbosa ◽  
Kimberly McCall ◽  
Sharon M. Gorski
Keyword(s):  
Cell Death ◽  
Drosophila Melanogaster ◽  
Molecular Mechanisms ◽  
Cultured Cells ◽  
Mitogen Activated Protein Kinase ◽  
Nuclear Condensation ◽  
Effector Caspase ◽  
Apoptosis Protein ◽  
Egg Chambers

A complex relationship exists between autophagy and apoptosis, but the regulatory mechanisms underlying their interactions are largely unknown. We conducted a systematic study of Drosophila melanogaster cell death–related genes to determine their requirement in the regulation of starvation-induced autophagy. We discovered that six cell death genes—death caspase-1 (Dcp-1), hid, Bruce, Buffy, debcl, and p53—as well as Ras–Raf–mitogen activated protein kinase signaling pathway components had a role in autophagy regulation in D. melanogaster cultured cells. During D. melanogaster oogenesis, we found that autophagy is induced at two nutrient status checkpoints: germarium and mid-oogenesis. At these two stages, the effector caspase Dcp-1 and the inhibitor of apoptosis protein Bruce function to regulate both autophagy and starvation-induced cell death. Mutations in Atg1 and Atg7 resulted in reduced DNA fragmentation in degenerating midstage egg chambers but did not appear to affect nuclear condensation, which indicates that autophagy contributes in part to cell death in the ovary. Our study provides new insights into the molecular mechanisms that coordinately regulate autophagic and apoptotic events in vivo.

scholarly journals A novel mass assay to measure phosphatidylinositol-5-phosphate from cells and tissues

2019 ◽  
Vol 39 (10) ◽  
Author(s):  
Avishek Ghosh ◽  
Sanjeev Sharma ◽  
Dhananjay Shinde ◽  
Visvanathan Ramya ◽  
Padinjat Raghu
Keyword(s):  
Cultured Cells ◽  
Insulin Signalling ◽  
Acyl Chain ◽  
High Sensitivity ◽  
Tissue Extracts ◽  
Liquid Chromatography Tandem Mass ◽  
Multicellular Organisms ◽  
Drosophila Cells

Abstract Phosphatidylinositol-5-phosphate (PI5P) is a low abundance lipid proposed to have functions in cell migration, DNA damage responses, receptor trafficking and insulin signalling in metazoans. However, studies of PI5P function are limited by the lack of scalable techniques to quantify its level from cells and tissues in multicellular organisms. Currently, PI5P measurement requires the use of radionuclide labelling approaches that are not easily applicable in tissues or in vivo samples. In the present study, we describe a simple and reliable, non-radioactive mass assay to measure total PI5P levels from cells and tissues of Drosophila, a genetically tractable multicellular model. We use heavy oxygen-labelled ATP (18O-ATP) to label PI5P from tissue extracts while converting it into PI(4,5)P2 using an in vitro kinase reaction. The product of this reaction can be selectively detected and quantified with high sensitivity using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform. Further, using this method, we capture and quantify the unique acyl chain composition of PI5P from Drosophila cells and tissues. Finally, we demonstrate the use of this technique to quantify elevations in PI5P levels, from Drosophila larval tissues and cultured cells depleted of phosphatidylinositol 5 phosphate 4-kinase (PIP4K), that metabolizes PI5P into PI(4,5)P2 thus regulating its levels. Thus, we demonstrate the potential of our method to quantify PI5P levels with high sensitivity from cells and tissues of multicellular organisms thus accelerating understanding of PI5P functions in vivo.

Oligodendrocyte precursor (O-2A progenitor cell) migration; a model system for the study of cell migration in the developing central nervous system

Development ◽  
1993 ◽  
Vol 119 (Supplement) ◽  
pp. 219-225 ◽  
Author(s):  
B. W. Kiernan ◽  
Charles ffrench-Constant
Keyword(s):  
Nervous System ◽  
Cell Migration ◽  
Progenitor Cell ◽  
Molecular Mechanisms ◽  
Cultured Cells ◽  
Large Numbers ◽  
Multicellular Organisms ◽  
Developing Nervous System

Cell migration plays an important role in the development of complex multicellular organisms. The molecular mechanisms that regulate this migration arc therefore of great interest. Unfortunately, however, analysis of cell migration in vertebrates is hampered by the inaccessability of the cells and the difficulty of manipulating their environment within the embryo. This review focusses on one particular migratory cell population, the oligodendrocyte p1ecursor cell or O-2A progenitor cell, that gives rise to the myelin-forming oligodendrocytes within the CNS. These cells mi grate extensively during normal development. They can be purified and grown in large numbers in cell culture, so allowing the use of reductionist approaches using cell and molecular biology techniques. Moreover, cultured cells will migrate within the CNS following transplantation. As a result, the migration of these cells in vivo can be analysed following manipulation in vitro. Taken together, we believe that the different properties of these cells makes them excellent candidates for studies addressing the control of cell migration in the developing nervous system.

scholarly journals Real-Time Monitoring of Nuclear Factor κB Activity in Cultured Cells and in Animal Models

2009 ◽  
Vol 8 (5) ◽  
pp. 7290.2009.00026 ◽  
Author(s):  
Christian E. Badr ◽  
Johanna M. Niers ◽  
Lee-Ann Tjon-Kon-Fat ◽  
David P. Noske ◽  
Thomas Wurdinger ◽  
...  
Keyword(s):  
Real Time ◽  
Cultured Cells ◽  
Tumor Development ◽  
Nuclear Factor Κb ◽  
Tumor Formation ◽  
Nuclear Factor ◽  
Reporter System ◽  
Human Disorders

Nuclear factor κB (NF-κB) is a transcription factor that plays a major role in many human disorders, including immune diseases and cancer. We designed a reporter system based on NF-κB responsive promoter elements driving expression of the secreted Gaussia princeps luciferase (Gluc). We show that this bioluminescent reporter is a highly sensitive tool for noninvasive monitoring of the kinetics of NF-κB activation and inhibition over time, both in conditioned medium of cultured cells and in the blood and urine of animals. NF-κB activation was successfully monitored in real time in endothelial cells in response to tumor angiogenic signaling, as well as in monocytes in response to inflammation. Further, we demonstrated dual blood monitoring of both NF-κB activation during tumor development as correlated to tumor formation using the NF-κB Gluc reporter, as well as the secreted alkaline phosphatase reporter. This NF-κB reporter system provides a powerful tool for monitoring NF-κB activity in real time in vitro and in vivo.

scholarly journals Clasp-mediated microtubule bundling regulates persistent motility and contact repulsion in Drosophila macrophages in vivo

2010 ◽  
Vol 189 (4) ◽  
pp. 681-689 ◽  
Author(s):  
Brian Stramer ◽  
Severina Moreira ◽  
Tom Millard ◽  
Iwan Evans ◽  
Chieh-Yin Huang ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
High Resolution ◽  
Fluorescent Probes ◽  
In Vivo Imaging ◽  
Living Organism ◽  
Developmental Guidance ◽  
Cytoskeletal Networks ◽  
Migratory Cells ◽  
Dynamic Interplay

Drosophila melanogaster macrophages are highly migratory cells that lend themselves beautifully to high resolution in vivo imaging experiments. By expressing fluorescent probes to reveal actin and microtubules, we can observe the dynamic interplay of these two cytoskeletal networks as macrophages migrate and interact with one another within a living organism. We show that before an episode of persistent motility, whether responding to developmental guidance or wound cues, macrophages assemble a polarized array of microtubules that bundle into a compass-like arm that appears to anticipate the direction of migration. Whenever cells collide with one another, their microtubule arms transiently align just before cell–cell repulsion, and we show that forcing depolymerization of microtubules by expression of Spastin leads to their defective polarity and failure to contact inhibit from one another. The same is true in orbit/clasp mutants, indicating a pivotal role for this microtubule-binding protein in the assembly and/or functioning of the microtubule arm during polarized migration and contact repulsion.

scholarly journals TRF4 Is Involved in Polyadenylation of snRNAs in Drosophila melanogaster

2008 ◽  
Vol 28 (21) ◽  
pp. 6620-6631 ◽  
Author(s):  
Ryoichi Nakamura ◽  
Ryo Takeuchi ◽  
Kei-ichi Takata ◽  
Kaori Shimanouchi ◽  
Yoko Abe ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Cellular Localization ◽  
Polymerase Activity ◽  
Rna Quality Control ◽  
Localization Analysis ◽  
Nuclear Exosome ◽  
Quality Control Mechanism ◽  
Multicellular Organisms

ABSTRACT The Saccharomyces cerevisiae poly(A) polymerases Trf4 and Trf5 are involved in an RNA quality control mechanism, where polyadenylated RNAs are degraded by the nuclear exosome. Although Trf4/5 homologue genes are distributed throughout multicellular organisms, their biological roles remain to be elucidated. We isolated here the two homologues of Trf4/5 in Drosophila melanogaster, named DmTRF4-1 and DmTRF4-2, and investigated their biological function. DmTRF4-1 displayed poly(A) polymerase activity in vitro, whereas DmTRF4-2 did not. Gene knockdown of DmTRF4-1 by RNA interference is lethal in flies, as is the case for the trf4 trf5 double mutants. In contrast, disruption of DmTRF4-2 results in viable flies. Cellular localization analysis suggested that DmTRF4-1 localizes in the nucleolus. Abnormal polyadenylation of snRNAs was observed in transgenic flies overexpressing DmTRF4-1 and was slightly increased by the suppression of DmRrp6, the 3′-5′ exonuclease of the nuclear exosome. These results suggest that DmTRF4-1 and DmRrp6 are involved in the polyadenylation-mediated degradation of snRNAs in vivo.

scholarly journals Analysis of the H-Ras mobility pattern in vivo shows cellular heterogeneity inside epidermal tissue

2021 ◽  
Author(s):  
Radoslaw J. Gora ◽  
Babette de Jong ◽  
Patrick van Hage ◽  
Mary Ann Rhiemus ◽  
Fjodor van Steenis ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Cultured Cells ◽  
Living Organism ◽  
Cellular Heterogeneity ◽  
Mobility Patterns ◽  
Protein Mobility ◽  
Protein Activation ◽  
Membrane Anchoring

Developments in single-molecule microscopy (SMM) have enabled imaging individual proteins in biological systems, focusing on the analysis of protein mobility patterns inside cultured cells. In the present study, SMM was applied in vivo, using the zebrafish embryo model. We studied dynamics of the membrane protein H-Ras, its membrane-anchoring domain, C10H-Ras, and mutants, using total internal reflection fluorescence microscopy (TIRFM). Our results consistently confirm the presence of fast- and slow-diffusing subpopulations of molecules, which confine to microdomains within the plasma membrane. The active mutant H-RasV12 exhibits higher diffusion rates and is confined to larger domains than the wild-type H-Ras and its inactive mutant H-RasN17. Subsequently, we demonstrate that the structure and composition of the plasma membrane have an imperative role in modulating H-Ras mobility patterns. Ultimately, we establish that differences between cells within the same embryo largely contribute to the overall data variability. Our findings agree with a model where the cell architecture and the protein activation state determine protein mobility, underlining the importance of SMM imaging to study factors influencing protein dynamics in an intact living organism.

Real time micro/nano particle detection and tracking with nanosecond resolution

2009 ◽  
Author(s):  
Feng Qian ◽  
Qi Song ◽  
En-kuang Tien ◽  
Ozdal Boyraz
Keyword(s):  
Real Time ◽  
Nano Particle ◽  
Particle Detection ◽  
Detection And Tracking

scholarly journals Drosophila melanogaster γ-TuRC is dispensable for targeting γ-tubulin to the centrosome and microtubule nucleation

2006 ◽  
Vol 172 (4) ◽  
pp. 517-528 ◽  
Author(s):  
Christel Vérollet ◽  
Nathalie Colombié ◽  
Thomas Daubon ◽  
Henri-Marc Bourbon ◽  
Michel Wright ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Cultured Cells ◽  
Microtubule Assembly ◽  
Microtubule Nucleation ◽  
Mitotic Delay ◽  
Ring Complexes ◽  
Higher Eukaryotes ◽  
First Time

In metazoans, γ-tubulin acts within two main complexes, γ-tubulin small complexes (γ-TuSCs) and γ-tubulin ring complexes (γ-TuRCs). In higher eukaryotes, it is assumed that microtubule nucleation at the centrosome depends on γ-TuRCs, but the role of γ-TuRC components remains undefined. For the first time, we analyzed the function of all four γ-TuRC–specific subunits in Drosophila melanogaster: Dgrip75, Dgrip128, Dgrip163, and Dgp71WD. Grip-motif proteins, but not Dgp71WD, appear to be required for γ-TuRC assembly. Individual depletion of γ-TuRC components, in cultured cells and in vivo, induces mitotic delay and abnormal spindles. Surprisingly, γ-TuSCs are recruited to the centrosomes. These defects are less severe than those resulting from the inhibition of γ-TuSC components and do not appear critical for viability. Simultaneous cosilencing of all γ-TuRC proteins leads to stronger phenotypes and partial recruitment of γ-TuSC. In conclusion, γ-TuRCs are required for assembly of fully functional spindles, but we suggest that γ-TuSC could be targeted to the centrosomes, which is where basic microtubule assembly activities are maintained.

Abstract 12840: In vivo Visualization of ATP Dynamics Under Hypoxia Reveals That G0/G1 Switch Gene 2 Provides Ischemic Tolerance Through the Increase of ATP Production

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Hidetaka Kioka ◽  
Hisakazu Kato ◽  
Yoshihiro Asano ◽  
Yasushi Sakata ◽  
Masafumi Kitakaze ◽  
...  
Keyword(s):  
Energy Metabolism ◽  
Cardiac Function ◽  
Real Time ◽  
Cultured Cells ◽  
Imaging Technique ◽  
Heart Diseases ◽  
Transgenic Zebrafish ◽  
Hypoxic Stress ◽  
Zebrafish Heart

Introduction: We have recently established the method for the selective measurement of intra-mitochondrial ATP levels ([ATP]mito) and have identified the hypoxia-inducible protein G0/G1 switch gene 2 (G0s2) as a positive regulator of mitochondrial oxidative phosphorylation by cultured cardiomyocyte-based experiments. However, the energy metabolism in cultured cells may be much different from that in the living tissue. In this study, we examined the in vivo role of G0s2 under hypoxia by using a novel real-time in vivo ATP imaging technique in zebrafish heart. Methods and Results: We first established the in vivo ATP imaging technique by introducing a FRET-based ATP biosensor named Mit-ATeam into zebrafish heart (Mit-ATeam zebrafish). This system also allows us the simultaneous evaluation of cardiac function. Using Mit-ATeam zebrafish, we successfully observed a decline in [ATP]mito and cardiac function under hypoxic stress and the recovery of these parameters by sequential re-oxygenation. Cardiac specific G0s2 transgenic zebrafish had significantly stronger tolerance against hypoxic stress than wild type zebrafish, while mutant G0s2 transgenic zebrafish did not. In addition, we generated a chimeric zebrafish model in which G0s2 was regionally overexpressed to examine whether focal overexpression of G0s2 could preserve regional cardiac function under hypoxia. Only G0s2-overexpressing cardiomyocyte populations showed enhanced contractility with increased [ATP]mito in hypoxia compared to the control region (i.e. non-G0s2-overexpressing cardiomyocyte populations). Furthermore, we detected enhanced protein expression of G0s2 protein in the risk area in canine ischemic preconditioned heart, suggesting the possible involvement of G0s2 in ischemic preconditioning mediated cardioprotection. Conclusions: These results suggest that G0s2 functions as a guardian of ischemic myocardium and could become a therapeutic target for ischemic heart diseases. Additionally, this study is the first to connect cardiac energy metabolism and function in living animal in real-time.

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