Probing Multicellular Dynamics in Xenopus Laevis Embryonic Development Using a Mechanical Engineering Based Microfluidic Feedback Approach

2010 ◽  
Author(s):  
YongTae Kim ◽  
Sagar D. Joshi ◽  
Philip R. LeDuc ◽  
Lance A. Davidson ◽  
William C. Messner
Keyword(s):  
Embryonic Development ◽  
Xenopus Laevis ◽  
High Speed ◽  
Mechanical Engineering ◽  
Fluorescent Protein ◽  
Microfluidic Channel ◽  
Temporal Regulation ◽  
Chemical Conditions ◽  
Green Fluorescent

Spatial and temporal regulation of chemical environments in and around cells or tissues for long time periods is important to understand multicellular signaling since the responses to chemical factors control the resulting coordinated events in development. Although progress has been made in command of single cell environments, both long-term and high-speed control of multicellular chemical environments in development is still challenging. We have developed a mechanical engineering based microfluidic feedback approach that allows long-term and high-speed manipulation of a laminar flow interface in a microfluidic channel. This approach enabled long-term spatiotemporal control of chemical conditions over Animal Cap (AC) explants during the gastrulation stage in Xenopus laevis embryonic development. We present the responses of the explants to periodic stimulation of steroid hormone dexamethasone (DEX) by tracking a hormone-activated nuclear-localizing green fluorescent protein tagged glucocorticoid receptor (nuc-GR-GFP). We believe that our approach will be useful in diverse areas including dynamic system and control in microfluidics, embryonic development, and spatiotemporally integrated biological responses.

scholarly journals Multiciliated cell basal bodies align in stereotypical patterns coordinated by the apical cytoskeleton

2016 ◽  
Vol 214 (5) ◽  
pp. 571-586 ◽  
Author(s):  
Elisa Herawati ◽  
Daisuke Taniguchi ◽  
Hatsuho Kanoh ◽  
Kazuhiro Tateishi ◽  
Shuji Ishihara ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Imaging System ◽  
Basal Bodies ◽  
Large Numbers ◽  
Multiciliated Cells ◽  
Alignment Process ◽  
Flow Through ◽  
Linear Alignment ◽  
Green Fluorescent

Multiciliated cells (MCCs) promote fluid flow through coordinated ciliary beating, which requires properly organized basal bodies (BBs). Airway MCCs have large numbers of BBs, which are uniformly oriented and, as we show here, align linearly. The mechanism for BB alignment is unexplored. To study this mechanism, we developed a long-term and high-resolution live-imaging system and used it to observe green fluorescent protein–centrin2–labeled BBs in cultured mouse tracheal MCCs. During MCC differentiation, the BB array adopted four stereotypical patterns, from a clustering “floret” pattern to the linear “alignment.” This alignment process was correlated with BB orientations, revealed by double immunostaining for BBs and their asymmetrically associated basal feet (BF). The BB alignment was disrupted by disturbing apical microtubules with nocodazole and by a BF-depleting Odf2 mutation. We constructed a theoretical model, which indicated that the apical cytoskeleton, acting like a viscoelastic fluid, provides a self-organizing mechanism in tracheal MCCs to align BBs linearly for mucociliary transport.

scholarly journals GluA1 trafficking and metabotropic NMDA: addressing results from other laboratories inconsistent with ours

2014 ◽  
Vol 369 (1633) ◽  
pp. 20130145 ◽  
Author(s):  
Sadegh Nabavi ◽  
Rocky Fox ◽  
Stephanie Alfonso ◽  
Jonathan Aow ◽  
Roberto Malinow
Keyword(s):  
Fluorescent Protein ◽  
Long Term Potentiation ◽  
Long Term Depression ◽  
Mk 801 ◽  
Over Expression ◽  
Term Potentiation ◽  
The Difference ◽  
Green Fluorescent ◽  
Gfp Tag

We have previously shown that when over-expressed in neurons, green fluorescent protein (GFP) tagged GluA1 (GluA1-GFP) delivery into synapses is dependent on plasticity. A recent study suggests that GluA1 over-expression leads to its incorporation into the synapse, in the absence of additional long-term potentiation-like manipulations. It is possible that a GFP tag was responsible for the difference. Using rectification index as a measure of synaptic delivery of GluA1, we found no difference in the synaptic delivery of GluA1-GFP versus untagged GluA1. We recently published a study showing that while D-APV blocks NMDAr-dependent long-term depression (LTD), MK-801 and 7-chloro kynurenate (7CK) fail to block LTD. We propose a metabotropic function for the NMDA receptor in LTD induction. In contrast to our observations, recent unpublished data suggest that the above antagonists are equally effective in blocking LTD. We noticed different methodology in their study. Here, we show that their methodology has complex effects on synaptic transmission. Therefore, it is not possible to conclude that 7CK is effective in blocking LTD from their type of experiment.

scholarly journals Tools to Study Plant Organelle Biogenesis. Point Mutation Lines with Disrupted Vacuoles and High-Speed Confocal Screening of Green Fluorescent Protein-Tagged Organelles

2003 ◽  
Vol 133 (4) ◽  
pp. 1673-1676 ◽  
Author(s):  
Emily L. Avila ◽  
Jan Zouhar ◽  
April E. Agee ◽  
David G. Carter ◽  
S. Narasimha Chary ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Point Mutation ◽  
High Speed ◽  
Fluorescent Protein ◽  
Organelle Biogenesis ◽  
Green Fluorescent

scholarly journals EXPRESSION OF THE GREEN FLUORESCENT PROTEIN DERIVATIVE S65T IN XENOPUS LAEVIS OOCYTES

1996 ◽  
Vol 17 (3) ◽  
pp. 221-225 ◽  
Author(s):  
JULIE M. MATHESON ◽  
ATSUSHI MIYAWAKI ◽  
AKIRA MUTO ◽  
TAKAFUMI INOUE ◽  
KATSUHIKO MIKOSHIBA
Keyword(s):  
Green Fluorescent Protein ◽  
Xenopus Laevis ◽  
Fluorescent Protein ◽  
Green Fluorescent

scholarly journals A high-speed, bright, red fluorescent voltage sensor to detect neural activity

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Connor Beck ◽  
Yiyang Gong
Keyword(s):  
Blue Light ◽  
Neural Activity ◽  
High Speed ◽  
Fluorescent Protein ◽  
Resonance Energy ◽  
Fluorescent Sensors ◽  
Fast Kinetics ◽  
Spectral Separation ◽  
Green Fluorescent

Abstract Genetically encoded voltage indicators (GEVIs) have emerged as a technology to optically record neural activity with genetic specificity and millisecond-scale temporal resolution using fluorescence microscopy. GEVIs have demonstrated ultra-fast kinetics and high spike detection fidelity in vivo, but existing red-fluorescent voltage indicators fall short of the response and brightness achieved by green fluorescent protein-based sensors. Furthermore, red-fluorescent GEVIs suffer from incomplete spectral separation from green sensors and blue-light-activated optogenetic actuators. We have developed Ace-mScarlet, a red fluorescent GEVI that fuses Ace2N, a voltage-sensitive inhibitory rhodopsin, with mScarlet, a bright red fluorescent protein (FP). Through fluorescence resonance energy transfer (FRET), our sensor detects changes in membrane voltage with high sensitivity and brightness and has kinetics comparable to the fastest green fluorescent sensors. Ace-mScarlet’s red-shifted absorption and emission spectra facilitate virtually complete spectral separation when used in combination with green-fluorescent sensors or with blue-light-sensitive sensors and rhodopsins. This spectral separation enables both simultaneous imaging in two separate wavelength channels and high-fidelity voltage recordings during simultaneous optogenetic perturbation.

scholarly journals Retrograde Transport of Transcription Factor NF-κB in Living Neurons

2000 ◽  
Vol 276 (15) ◽  
pp. 11821-11829 ◽  
Author(s):  
Henning Wellmann ◽  
Barbara Kaltschmidt ◽  
Christian Kaltschmidt
Keyword(s):  
Transcription Factor ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Retrograde Transport ◽  
Receptor Activation ◽  
Transcriptional Changes ◽  
Localization Signal ◽  
Green Fluorescent ◽  
Living Neurons

The mechanism by which signals such as those produced by glutamate are transferred to the nucleus may involve direct transport of an activated transcription factor to trigger long-term transcriptional changes. Ionotropic glutamate receptor activation or depolarization activates transcription factor NF-κB and leads to translocation of NF-κB from the cytoplasm to the nucleus. We investigated the dynamics of NF-κB translocation in living neurons by tracing the NF-κB subunit RelA (p65) with jellyfish green fluorescent protein. We found that green fluorescent protein-RelA was located in either the nucleus or cytoplasm and neurites, depending on the coexpression of the cognate inhibitor of NF-κB, IκB-α. Stimulation with glutamate, kainate, or potassium chloride resulted in a redistribution of NF-κB from neurites to the nucleus. This transport depended on an intact nuclear localization signal on RelA. Thus, in addition to its role as a transcription factor, NF-κB may be a signal transducer, transmitting transient glutamatergic signals from distant sites to the nucleus.

Persistence of eGFP Marked Bone Marrow Cells in Long-Term Hematopoiesis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2111-2111
Author(s):  
Ingo H. Pilz ◽  
Manfred Schmidt ◽  
Claudia Ball ◽  
Hanno Glimm ◽  
Fritz von Weizsäcker ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Alkylating Agent ◽  
Fluorescent Protein ◽  
Bone Marrow Cells ◽  
Egfp Expression ◽  
Hind Limbs ◽  
Long Term Follow Up ◽  
Green Fluorescent

Abstract To study transplanted unperturbed and mobilized long-term hematopoiesis after selection with an alkylating agent, bone marrow (BM) from 5 C57BL/6J mice was pooled, repeatedly transduced with retroviruses encoding the alkylating agent resistance protein O6-Methylguanine-DNA and enhanced green fluorescent protein (eGFP) as an easily traceable marker. Between 1 to 9x105 transfected BM cells were transplanted into 15 myeloablatively irradiated sex-mismatched C57BL/6J mice. Subsequently, 3 to 4 selection rounds with BCNU/O6-BG were carried out, enriching eGFP marked hematopoiesis in these mice up to 70–90%. Between 1 and 7x107BM cells of different mice were transplanted according to marrow location into groups of 5 sex-matched Bri44[1] mice. Two mice each received BM from the hind limbs, two from the pelvis and one received cells from the spleen, only, respectively. Altogether the study comprised 15 groups divided into 6 female and 9 male groups. Of these, 4 male and 3 female groups received 3 HSC-mobilization courses with G-CSF at intervals of 2 months starting 3 month after transplantation. Hematopoiesis in the other fraction remained unperturbed. During the observation period of 11–14 months in these tertiary recipients, repeated FACS analyses as well as linear amplification mediated (LAM) PCRs were carried out to track the clonal contributions. A decrease in the percentage of eGFP expressing marked hematopoiesis was observed in most cases. However, eGFP expression never disappeared altogether and could still be detected in the different hematopoietic lineages and successfully sorted for further analyses by MoFlo (Dako-Cytomation). Assessment of the clonal status of the Bri44 by LAM-PCR displayed interesting results. In some mice a decline in clone numbers was observed, whereas clone numbers remained stable in others. Tertiary transplantation with long-term follow-up indicates that this observation may be related to the transplantation of limited long-term repopulating clone numbers and progenitor cell exhaustion over time.

scholarly journals Large and Small Dendritic Spines Serve Different Interacting Functions in Hippocampal Synaptic Plasticity and Homeostasis

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Joshua J. W. Paulin ◽  
Peter Haslehurst ◽  
Alexander D. Fellows ◽  
Wenfei Liu ◽  
Joshua D. Jackson ◽  
...  
Keyword(s):  
Dendritic Spines ◽  
Fluorescent Protein ◽  
Long Term Potentiation ◽  
Functional Differentiation ◽  
Strong Stimulus ◽  
Term Potentiation ◽  
Original Size ◽  
Green Fluorescent ◽  
Homeostatic Response

The laying down of memory requires strong stimulation resulting in specific changes in synaptic strength and corresponding changes in size of dendritic spines. Strong stimuli can also be pathological, causing a homeostatic response, depressing and shrinking the synapse to prevent damage from too much Ca2+influx. But do all types of dendritic spines serve both of these apparently opposite functions? Using confocal microscopy in organotypic slices from mice expressing green fluorescent protein in hippocampal neurones, the size of individual spines along sections of dendrite has been tracked in response to application of tetraethylammonium. This strong stimulus would be expected to cause both a protective homeostatic response and long-term potentiation. We report separation of these functions, with spines of different sizes reacting differently to the same strong stimulus. The immediate shrinkage of large spines suggests a homeostatic protective response during the period of potential danger. In CA1, long-lasting growth of small spines subsequently occurs consolidating long-term potentiation but only after the large spines return to their original size. In contrast, small spines do not change in dentate gyrus where potentiation does not occur. The separation in time of these changes allows clear functional differentiation of spines of different sizes.

scholarly journals Characterization of Leptin-Responsive Neurons in the Caudal Brainstem

Endocrinology ◽  
2006 ◽  
Vol 147 (7) ◽  
pp. 3190-3195 ◽  
Author(s):  
Kate L. J. Ellacott ◽  
Ilia G. Halatchev ◽  
Roger D. Cone
Keyword(s):  
Green Fluorescent Protein ◽  
Energy Homeostasis ◽  
Leptin Receptor ◽  
Fluorescent Protein ◽  
Physiological Role ◽  
Cell Group ◽  
Melanocortin System ◽  
Peptide Precursor ◽  
Green Fluorescent

The central melanocortin system plays a key role in the regulation of energy homeostasis. Neurons containing the peptide precursor proopiomelanocortin (POMC) are found at two sites in the brain, the arcuate nucleus of the hypothalamus (ARC) and the caudal region of the nucleus of the solitary tract (NTS). ARC POMC neurons, which also express cocaine- and amphetamine-regulated transcript (CART), are known to mediate part of the response to factors regulating energy homeostasis, such as leptin and ghrelin. In contrast, the physiological role(s) of the POMC neurons in the caudal brainstem are not well characterized. However, development of a transgenic mouse expressing green fluorescent protein under the control of the POMC promoter [POMC-enhanced green fluorescent protein (EGFP) mouse] has aided the study of these neurons. Indeed, recent studies have shown significant activation of NTS POMC-EGFP cells by the gut released satiety factor cholecystokinin (CCK). Here we show that peripheral leptin administration induces the expression of phospho-signal transducer and activator of transcription 3 immunoreactivity (pSTAT3-IR), a marker of leptin receptor signaling, in more than 50% of NTS POMC-EGFP neurons. Furthermore, these POMC-EGFP neurons comprise 30% of all pSTAT3-IR cells in the NTS. Additionally, we also show that in contrast to the ARC population, NTS POMC-EGFP neurons do not coexpress CART immunoreactivity. These data suggest that NTS POMC neurons may participate with ARC POMC cells in mediating some of the effects of leptin and thus comprise a novel cell group regulated by both long-term adipostatic signals and satiety factors such as CCK.

scholarly journals Identification of an Outer Segment Targeting Signal in the Cooh Terminus of Rhodopsin Using Transgenic Xenopus laevis

2000 ◽  
Vol 151 (7) ◽  
pp. 1369-1380 ◽  
Author(s):  
Beatrice M. Tam ◽  
Orson L. Moritz ◽  
Lawrence B. Hurd ◽  
David S. Papermaster
Keyword(s):  
Amino Acids ◽  
Xenopus Laevis ◽  
Fusion Proteins ◽  
Outer Segment ◽  
Fluorescent Protein ◽  
Localization Signal ◽  
Targeting Signal ◽  
Retinal Degenerative Diseases ◽  
Membrane Spanning ◽  
Green Fluorescent

Mislocalization of the photopigment rhodopsin may be involved in the pathology of certain inherited retinal degenerative diseases. Here, we have elucidated rhodopsin's targeting signal which is responsible for its polarized distribution to the rod outer segment (ROS). Various green fluorescent protein (GFP)/rhodopsin COOH-terminal fusion proteins were expressed specifically in the major red rod photoreceptors of transgenic Xenopus laevis under the control of the Xenopus opsin promoter. The fusion proteins were targeted to membranes via lipid modifications (palmitoylation and myristoylation) as opposed to membrane spanning domains. Membrane association was found to be necessary but not sufficient for efficient ROS localization. A GFP fusion protein containing only the cytoplasmic COOH-terminal 44 amino acids of Xenopus rhodopsin localized exclusively to ROS membranes. Chimeras between rhodopsin and α adrenergic receptor COOH-terminal sequences further refined rhodopsin's ROS localization signal to its distal eight amino acids. Mutations/deletions of this region resulted in partial delocalization of the fusion proteins to rod inner segment (RIS) membranes. The targeting and transport of endogenous wild-type rhodopsin was unaffected by the presence of mislocalized GFP fusion proteins.

Sign in / Sign up

Export Citation Format

Share Document