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 Photoactive Split Green Fluorescent Protein: Engineering a New Optogenetic and Imaging System

2018 ◽  
Vol 114 (3) ◽  
pp. 177a-178a
Author(s):  
Matthew G. Romei ◽  
Chelsea K. Longwell ◽  
Jennifer R. Cochran ◽  
Steven G. Boxer
Keyword(s):  
Green Fluorescent Protein ◽  
Protein Engineering ◽  
Fluorescent Protein ◽  
Imaging System ◽  
Green Fluorescent

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 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.

scholarly journals Longitudinal evaluation of expression of virally delivered transgenes in gerbil cone photoreceptors

2008 ◽  
Vol 25 (3) ◽  
pp. 273-282 ◽  
Author(s):  
MATTHEW C. MAUCK ◽  
KATHERINE MANCUSO ◽  
JAMES A. KUCHENBECKER ◽  
THOMAS B. CONNOR ◽  
WILLIAM W. HAUSWIRTH ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Imaging System ◽  
Regulatory Elements ◽  
Meriones Unguiculatus ◽  
Opsin Gene ◽  
Post Injection ◽  
Cone Photoreceptors ◽  
Long Wavelength ◽  
Green Fluorescent

Delivery of foreign opsin genes to cone photoreceptors using recombinant adeno-associated virus (rAAV) is a potential tool for studying the basic mechanisms underlying cone based vision and for treating vision disorders. We used an in vivo retinal imaging system to monitor, over time, expression of virally-delivered genes targeted to cone photoreceptors in the Mongolian gerbil (Meriones unguiculatus). Gerbils have a well-developed photopic visual system, with 11–14% of their photoreceptors being cones. We used replication deficient serotype 5 rAAV to deliver a gene for green fluorescent protein (GFP). In an effort to direct expression of the gene specifically to either S or M cones, the transgene was under the control of either the human X-chromosome opsin gene regulatory elements, i.e., an enhancer termed the locus control region (LCR) and L promoter, or the human S-opsin promoter. Longitudinal fluorescence images reveal that gene expression is first detectable about 14 days post-injection, reaches a peak after about 3 months, and is observed more than a year post-injection if the initial viral concentration is sufficiently high. The regulatory elements are able to direct expression to a subpopulation of cones while excluding expression in rods and non-photoreceptor retinal cells. When the same viral constructs are used to deliver a human long-wavelength opsin gene to gerbil cones, stimulation of the introduced human photopigment with long-wavelength light produces robust cone responses.

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 Time-Lapse Microscopy of Streptomyces coelicolor Growth and Sporulation

2008 ◽  
Vol 74 (21) ◽  
pp. 6774-6781 ◽  
Author(s):  
Vinod Jyothikumar ◽  
Emma J. Tilley ◽  
Rashmi Wali ◽  
Paul R. Herron
Keyword(s):  
Protein Trafficking ◽  
Growth And Development ◽  
Fluorescent Protein ◽  
Imaging System ◽  
Streptomyces Coelicolor ◽  
Time Lapse ◽  
Time Lapse Microscopy ◽  
Use Of Time ◽  
Aerial Hyphae ◽  
Green Fluorescent

ABSTRACT Bacteria from the genus Streptomyces are among the most complex of all prokaryotes; not only do they grow as a complex mycelium, they also differentiate to form aerial hyphae before developing further to form spore chains. This developmental heterogeneity of streptomycete microcolonies makes studying the dynamic processes that contribute to growth and development a challenging procedure. As a result, in order to study the mechanisms that underpin streptomycete growth, we have developed a system for studying hyphal extension, protein trafficking, and sporulation by time-lapse microscopy. Through the use of time-lapse microscopy we have demonstrated that Streptomyces coelicolor germ tubes undergo a temporary arrest in their growth when in close proximity to sibling extension sites. Following germination, in this system, hyphae extended at a rate of ∼20 μm h−1, which was not significantly different from the rate at which the apical ring of the cytokinetic protein FtsZ progressed along extending hyphae through a spiraling movement. Although we were able to generate movies for streptomycete sporulation, we were unable to do so for either the erection of aerial hyphae or the early stages of sporulation. Despite this, it was possible to demonstrate an arrest of aerial hyphal development that we suggest is through the depolymerization of FtsZ-enhanced green fluorescent protein (GFP). Consequently, the imaging system reported here provides a system that allows the dynamic movement of GFP-tagged proteins involved in growth and development of S. coelicolor to be tracked and their role in cytokinesis to be characterized during the streptomycete life cycle.

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