Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons

10.3791/60287 ◽  
2019 ◽  
Author(s):  
Chengyu Sheng ◽  
Uzma Javed ◽  
Justin Rosenthal ◽  
Jun Yin ◽  
Bo Qin ◽  
...  
Keyword(s):  
Time Lapse ◽  
Live Imaging ◽  
Dendritic Branch

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 LIVE IMAGING OF ERK ACTIVITY STEPWISE PATTERNING DURING SOMITOGENESIS

2015 ◽  
Vol 2 (1) ◽  
pp. 363
Author(s):  
Dini Wahyu Kartika Sari ◽  
Ryutaro Akiyama ◽  
Takaaki Matsui ◽  
Yasumasa Bessho
Keyword(s):  
Clock Genes ◽  
Time Lapse ◽  
Live Imaging ◽  
Molecular Signature ◽  
Vertebrate Development ◽  
Segmentation Clock ◽  
Somite Formation ◽  
Repetitive Structure ◽  
Time Lapse Imaging ◽  
Erk Activity

<p>Periodical segmentation of the anterior extremity of the presomitic mesoderm (PSM) generates metameric structure of somites during vertebrate development. During somite segmentation in zebrafish, msep determines a future somite boundary at position B-2 within the PSM. However, heat shock experiments suggest that an earlier future of somite boundary exists at B-5, but the molecular signature of this boundary remains unidentified. Our recent study demonstrated that fibroblast growth factor (FGF) gradient is converted into an ON-OFF boundary of downstream Erk activity, which corresponds to the future B-5 somite boundary. Moreover, we also revealed that the segmentation clock is required for a stepwise posterior shift of the Erk activity boundary during each segmentation. To clarify this evidence, here we perform time-lapse imaging of Erk activity in living embryos using a FRET biosensor. We focused on FRET signals within the PSM to observe spatial and temporal changes of Erk activity. Consistent with the data from fixed embryos, we observed ON-OFF boundary of Erk activity within the PSM and the position stepwisely shifted to posterior during somite formation. In order to test the contribution of the segmentation clock to the stepwise movements of the ON-OFF boundary of Erk activity, we disrupted the segmentation clock in zebrafish embryos by knocking down two segmentation clock genes, her1 and her7. Double knockdown of her1 and her7 resulted in the failure of the shift of the Erk ON-OFF boundary during somite segmentation, leading to segmentation defects of somites. These results strongly suggest that the clock-dependent stepwise movement of Erk activity is a key mechanism to generate the perfect repetitive structure of somites.</p><p><br /><strong>Keywords</strong>: Live imaging, Erk, stepwise, somitogenesis</p>

scholarly journals Live Imaging of muscle histolysis inDrosophilametamorphosis

2016 ◽  
Author(s):  
Yadav Kuleesha ◽  
Wee Choo Puah ◽  
Martin Wasser
Keyword(s):  
Cell Death ◽  
Molecular Mechanisms ◽  
Cysteine Proteinase ◽  
Cathepsin L ◽  
Time Lapse ◽  
Live Imaging ◽  
Dominant Negative ◽  
Muscle Size ◽  
Genes Encoding

Background:The contribution of programmed cell death (PCD) to muscle wasting disorders remains a matter of debate.Drosophila melanogastermetamorphosis offers the opportunity to study muscle cell death in the context of development. Using live cell imaging of the abdomen, two groups of larval muscles can be observed, doomed muscles that undergo histolysis and persistent muscles that are remodelled and survive into adulthood.Method:To identify and characterize genes that control the decision between survival and cell death of muscles, we developed a method comprisingin vivoimaging, targeted gene perturbation and time-lapse image analysis. Our approach enabled us to study the cytological and temporal aspects of abnormal cell death phenotypes.Results:In a previous genetic screen for genes controlling muscle size and cell death in metamorphosis, we identified gene perturbations that induced cell death of persistent or inhibit histolysis of doomed larval muscles. RNA interference (RNAi) of the genes encoding the helicase Rm62 and the lysosomal Cathepsin-L homolog Cysteine proteinase 1 (Cp1) caused premature cell death of persistent muscle in early and mid-pupation, respectively. Silencing of the transcriptional co-repressorAtrophininhibited histolysis of doomed muscles. Overexpression of dominant-negative Target of Rapamycin (TOR) delayed the histolysis of a subset of doomed and induced ablation of all persistent muscles. RNAi ofAMPKα,which encodes a subunit of the AMPK protein complex that senses AMP and promotes ATP formation, led to loss of attachment and a spherical morphology. None of the perturbations affected the survival of newly formed adult muscles, suggesting that the method is useful to find genes that are crucial for the survival of metabolically challenged muscles, like those undergoing atrophy. The ablation of persistent muscles did not affect eclosion of adult flies.Conclusions:Live imaging is a versatile approach to uncover gene functions that are required for the survival of muscle undergoing remodelling, yet are dispensable for other adult muscles. Our approach promises to identify molecular mechanisms that can explain the resilience of muscles to PCD.

scholarly journals Long-term time-lapse live imaging reveals extensive cell migration during annelid regeneration

2016 ◽  
Vol 16 (1) ◽  
Author(s):  
Eduardo E. Zattara ◽  
Kate W. Turlington ◽  
Alexandra E. Bely
Keyword(s):  
Cell Migration ◽  
Time Lapse ◽  
Live Imaging ◽  
Extensive Cell

scholarly journals Long-Term Potentiation Induces Extrasynaptic Exocytosis of Glun2A-containing NMDA Receptors that is Mainly Controlled by SNAP23

2019 ◽  
Author(s):  
Xiaojun Yu ◽  
Wei Li ◽  
Tong Wang
Keyword(s):  
Synaptic Plasticity ◽  
Nmda Receptors ◽  
Postsynaptic Density ◽  
Super Resolution ◽  
Time Lapse ◽  
Long Term Potentiation ◽  
Live Imaging ◽  
Term Potentiation ◽  
Specific Regulation

AbstractNMDA receptors (NMDAR) are key players in the initiation of synaptic plasticity that underlies learning and memory. Long-term potentiation (LTP) of synapses require an increased calcium current via NMDA channels to trigger modifications in postsynaptic density (PSD). It is generally believed that the amount of NMDARs on the postsynaptic surface remains stationary, whereas their subunit composition is dynamically fluctuated during this plasticity process. However, the molecular machinery underlying this subunit-specific regulation remains largely elusive. Here, by detecting the time-lapse changes of surface GluN2A and GluN2B subunit levels using biochemical approaches, surface immunostaining, live-imaging and super-resolution microscopy, we uncovered a transient increase of surface GluN2A-type NMDARs shortly after the induction of chemical long term potentiation (cLTP). These augmented sub-diffraction-limited GluN2A clusters predominantly exist in extrasynaptic domains. We also showed that the spine-enriched SNARE associated protein SNAP-23, and to a minor extent its homologue SNAP-25, control both the basal and regulated surface level of GluN2A receptors. Using a total internal reflection fluorescence microscopy (TIRFM) based live-imaging assay, we resolved and analyzed individual exocytic events of NMDARs in live neurons and found that cLTP raised the frequency of NMDAR exocytosis at extrasynaptic regions, without altering the duration or the package size of these events. Our study thereby provides direct evidence that synaptic plasticity controls the postsynaptic exocytosis machinery, which induces the insertion of more GluN2A receptors into the extrasynaptic area.Significance StatementMemory formation involves the long-term modification of synapses, which is called synaptic plasticity. In the postsynaptic density (PSD) of excited neurons, this modification process occurs on a minute timescale, initiated by the opening of NMDARs that trigger downstream cascades to fix the potentiation (LTP) at specific synapses for longer timescales. Here, using a novel live-imaging assay we resolved the dynamic delivery of NMDARs to the cell surface, and found that only the insertion frequency, not the duration of individual insertion or number of GluN2A subunits each of these NMDAR vesicles contains, was altered during the synaptic potentiation process. We also identified SNAP-23 as the key molecule mediating this activity dependent NMDAR surface delivery. This study provides a novel mechanism of how NMDARs are regulated in the short window to initiate the long-lasting synaptic modifications.

scholarly journals The Tip Growth Apparatus of Aspergillus nidulans

2008 ◽  
Vol 19 (4) ◽  
pp. 1439-1449 ◽  
Author(s):  
Naimeh Taheri-Talesh ◽  
Tetsuya Horio ◽  
Lidia Araujo-Bazán ◽  
Xiaowei Dou ◽  
Eduardo A. Espeso ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Aspergillus Nidulans ◽  
Fusion Proteins ◽  
Tip Growth ◽  
Time Lapse ◽  
Live Imaging ◽  
Medical Importance ◽  
Cytoskeletal Elements ◽  
Time Lapse Imaging ◽  
Hyphal Tip

Hyphal tip growth in fungi is important because of the economic and medical importance of fungi, and because it may be a useful model for polarized growth in other organisms. We have investigated the central questions of the roles of cytoskeletal elements and of the precise sites of exocytosis and endocytosis at the growing hyphal tip by using the model fungus Aspergillus nidulans. Time-lapse imaging of fluorescent fusion proteins reveals a remarkably dynamic, but highly structured, tip growth apparatus. Live imaging of SYNA, a synaptobrevin homologue, and SECC, an exocyst component, reveals that vesicles accumulate in the Spitzenkörper (apical body) and fuse with the plasma membrane at the extreme apex of the hypha. SYNA is recycled from the plasma membrane by endocytosis at a collar of endocytic patches, 1–2 μm behind the apex of the hypha, that moves forward as the tip grows. Exocytosis and endocytosis are thus spatially coupled. Inhibitor studies, in combination with observations of fluorescent fusion proteins, reveal that actin functions in exocytosis and endocytosis at the tip and in holding the tip growth apparatus together. Microtubules are important for delivering vesicles to the tip area and for holding the tip growth apparatus in position.

Ultrastructure of melanocyte-keratinocyte interactions and pigment transfer in vitro

1978 ◽  
Vol 36 (2) ◽  
pp. 650-651
Author(s):  
Raul I. Garcia ◽  
Evelyn A. Flynn ◽  
George Szabo
Keyword(s):  
Direct Injection ◽  
Skin Pigmentation ◽  
Freeze Fracture ◽  
Pigment Granule ◽  
Time Lapse ◽  
Ultrastructural Level ◽  
Lanthanum Tracer ◽  
A Cell

Skin pigmentation in mammals involves the interaction of epidermal melanocytes and keratinocytes in the structural and functional unit known as the Epidermal Melanin Unit. Melanocytes(M) synthesize melanin within specialized membrane-bound organelles, the melanosome or pigment granule. These are subsequently transferred by way of M dendrites to keratinocytes(K) by a mechanism still to be clearly defined. Three different, though not necessarily mutually exclusive, mechanisms of melanosome transfer have been proposed: cytophagocytosis by K of M dendrite tips containing melanosomes, direct injection of melanosomes into the K cytoplasm through a cell-to-cell pore or communicating channel formed by localized fusion of M and K cell membranes, release of melanosomes into the extracellular space(ECS) by exocytosis followed by K uptake using conventional phagocytosis. Variability in methods of transfer has been noted both in vivo and in vitro and there is evidence in support of each transfer mechanism. We Have previously studied M-K interactions in vitro using time-lapse cinemicrography and in vivo at the ultrastructural level using lanthanum tracer and freeze-fracture.

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