Imaging the Insertion of Superecliptic pHluorin‐Labeled Dopamine D2 Receptor Using Total Internal Reflection Fluorescence Microscopy

Daly, Kathryn M.; Li, Yun; Lin, Da-Ting

doi:10.1002/0471142301.ns0531s70

Cited by 3 publications

(3 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As the above experiments used non-neuronal cells (HEK293) to examine D2R internalization, we wished to examine this response within a neuronal context. Thus, we performed studies to examine D2R internalization and recycling in striatal neurons from E15-E18 mouse embryos using both immunocytochemistry (ICC) and total internal refection fluorescent (TIRF) microscopy (Li et al, 2012a , b ; Daly et al, 2015 ). Figure 2A shows quantification of ICC assays following treatment of the transfected cells with either 10 μM DA, 30 μM MLS1547, 30 μM of the D2R antagonist sulpiride, or vehicle (control).…”

Section: Resultsmentioning

confidence: 99%

“…For TIRF microscopy studies, the neurons were transfected with a D2R construct tagged with a pH-dependent GFP (pHluorin) that loses fluorescence in the acidic environment of internalized vesicles (Li et al, 2012a , b ; Daly et al, 2015 ). This enables the visualization of vesicle exocytosis or “reinsertion,” as pHluorin fluorescence is regained the moment the vesicle fuses with the cell membrane.…”

Section: Resultsmentioning

confidence: 99%

“…Isolation and culturing of cells was conducted as described previously (Li et al, 2012b ; Daly et al, 2015 ) using C57BL6/J mice. All experiments using laboratory animals were conducted with approved protocols from the NIH/NIDA Animal Care and Use Committee.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Structure-Activity Investigation of a G Protein-Biased Agonist Reveals Molecular Determinants for Biased Signaling of the D2 Dopamine Receptor

Chun

Vekariya

Free

et al. 2018

Front. Synaptic Neurosci.

Self Cite

View full text Add to dashboard Cite

The dopamine D2 receptor (D2R) is known to elicit effects through activating two major signaling pathways mediated by either G proteins (Gi/o) or β-arrestins. However, the specific role of each pathway in physiological or therapeutic activities is not known with certainty. One approach to the dissection of these pathways is through the use of drugs that can selectively modulate one pathway vs. the other through a mechanism known as functional selectivity or biased signaling. Our laboratory has previously described a G protein signaling-biased agonist, MLS1547, for the D2R using a variety of in vitro functional assays. To further evaluate the biased signaling activity of this compound, we investigated its ability to promote D2R internalization, a process known to be mediated by β-arrestin. Using multiple cellular systems and techniques, we found that MLS1547 promotes little D2R internalization, which is consistent with its inability to recruit β-arrestin. Importantly, we validated these results in primary striatal neurons where the D2R is most highly expressed suggesting that MLS1547 will exhibit biased signaling activity in vivo. In an effort to optimize and further explore structure-activity relationships (SAR) for this scaffold, we conducted an iterative chemistry campaign to synthesize and characterize novel analogs of MLS1547. The resulting analysis confirmed previously described SAR requirements for G protein-biased agonist activity and, importantly, elucidated new structural features that are critical for agonist efficacy and signaling bias of the MLS1547 scaffold. One of the most important determinants for G protein-biased signaling is the interaction of a hydrophobic moiety of the compound with a defined pocket formed by residues within transmembrane five and extracellular loop two of the D2R. These results shed new light on the mechanism of biased signaling of the D2R and may lead to improved functionally-selective molecules.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Structure-Activity Investigation of a G Protein-Biased Agonist Reveals Molecular Determinants for Biased Signaling of the D2 Dopamine Receptor

Chun

Vekariya

Free

et al. 2018

Front. Synaptic Neurosci.

Self Cite

View full text Add to dashboard Cite

show abstract

High‐content cell death imaging using quantum dot‐based TIRF microscopy for the determination of anticancer activity against breast cancer stem cell

Shim

Song

2016

Journal of Biophotonics

View full text Add to dashboard Cite

We report a two color monitoring of drug-induced cell deaths using total internal reflection fluorescence (TIRF) as a novel method to determine anticancer activity. Instead of cancer cells, breast cancer stem cells (CSCs) were directly tested in the present assay to determine the effective concentration (EC ) values of camptothecin and cisplatin. Phosphatidylserine and HMGB1 protein were concurrently detected to observe apoptotic and necrotic cell death induced by anticancer drugs using quantum dot (Qdot)-antibody conjugates. Only 50-to-100 breast CSCs were consumed at each cell chamber due to the high sensitivity of Qdot-based TIRF. The high sensitivity of Qdot-based TIRF, that enables the consumption of a small number of cells, is advantageous for cost-effective large-scale drug screening. In addition, unlike MTT assay, this approach can provide a more uniform range of EC values because the average values of single breast CSCs fluorescence intensities are observed to acquire EC values as a function of dose. This research successfully demonstrated the possibility that Qdot-based TIRF can be widely used as an improved alternative to MTT assay for the determination of anticancer drug efficacies.

show abstract

BDNF rescues prefrontal dysfunction elicited by pyramidal neuron-specific DTNBP1 deletion in vivo

Zhang

Daly

Liang

et al. 2016

Journal of Molecular Cell Biology

View full text Add to dashboard Cite

Dystrobrevin-binding protein 1 (Dtnbp1) is one of the earliest identified schizophrenia susceptibility genes. Reduced expression of DTNBP1 is commonly found in brain areas of schizophrenic patients. Dtnbp1-null mutant mice exhibit abnormalities in behaviors and impairments in neuronal activities. However, how diminished DTNBP1 expression contributes to clinical relevant features of schizophrenia remains to be illustrated. Here, using a conditional Dtnbp1 knockout mouse line, we identified an in vivo schizophrenia-relevant function of DTNBP1 in pyramidal neurons of the medial prefrontal cortex (mPFC). We demonstrated that DTNBP1 elimination specifically in pyramidal neurons of the mPFC impaired mouse pre-pulse inhibition (PPI) behavior and reduced perisomatic GABAergic synapses. We further revealed that loss of DTNBP1 in pyramidal neurons diminished activity-dependent secretion of brain-derived neurotrophic factor (BDNF). Finally, we showed that chronic BDNF infusion in the mPFC fully rescued both GABAergic synaptic dysfunction and PPI behavioral deficit induced by DTNBP1 elimination from pyramidal neurons. Our findings highlight brain region- and cell type-specific functions of DTNBP1 in the pathogenesis of schizophrenia, and underscore BDNF restoration as a potential therapeutic strategy for schizophrenia.

show abstract

Imaging the Insertion of Superecliptic pHluorin‐Labeled Dopamine D2 Receptor Using Total Internal Reflection Fluorescence Microscopy

Cited by 3 publications

References 29 publications

Structure-Activity Investigation of a G Protein-Biased Agonist Reveals Molecular Determinants for Biased Signaling of the D2 Dopamine Receptor

Structure-Activity Investigation of a G Protein-Biased Agonist Reveals Molecular Determinants for Biased Signaling of the D2 Dopamine Receptor

High‐content cell death imaging using quantum dot‐based TIRF microscopy for the determination of anticancer activity against breast cancer stem cell

BDNF rescues prefrontal dysfunction elicited by pyramidal neuron-specific DTNBP1 deletion in vivo

Contact Info

Product

Resources

About