Browsing by Subject "Luminescent Proteins"

Now showing 1 - 7 of 7
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    Arc/Arg3.1 translation is controlled by convergent N-methyl-D-aspartate and Gs-coupled receptor signaling pathways.
    (The Journal of biological chemistry, 2008-01) Bloomer, Wendy AC; VanDongen, Hendrika MA; VanDongen, Antonius MJ
    Arc/Arg3.1 is an immediate early gene whose expression is necessary for the late-phase of long-term potentiation (LTP) and memory consolidation. Whereas pathways regulating Arc transcription have been extensively investigated, less is known about the role of post-transcriptional mechanisms in Arc expression. Fluorescence microscopy experiments in cultured hippocampal neurons revealed that Arc protein level was dramatically increased by activation of the cAMP-dependent protein kinase (PKA) pathway, which is implicated in long-term memory. A PKA-dependent increase in Arc protein level was observed after pharmacological or synaptic activation of N-methyl-D-aspartate (NMDA) receptors, which play a critical role in both LTP induction and learning. Arc protein was also up-regulated by activation of PKA through G(s)-coupled dopamine and beta-adrenergic receptors, which regulate the late-phase of LTP and memory. When agonists for the NMDA and G(s)-coupled receptors were co-applied, they had an additive effect on Arc protein expression. Interestingly, G(s)-coupled receptor stimulation was ineffective in the presence of an NMDA receptor antagonist, suggesting calcium influx through the NMDA receptor plays a gating role in this pathway. Stimulation of the cAMP/PKA pathway did not affect Arc mRNA level or protein stability, identifying translational efficacy as the main determinant of Arc protein expression level. It is concluded that efficient Arc translation requires NMDA receptor activity, whereas a further enhancement can be achieved with activation of G(s)-coupled receptors. These experiments have, therefore, revealed remarkable similarities in the signaling pathways that control Arc expression and those that regulate LTP, learning, and memory.
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    beta-Arrestin-mediated PDE4 cAMP phosphodiesterase recruitment regulates beta-adrenoceptor switching from Gs to Gi.
    (Proc Natl Acad Sci U S A, 2003-02-04) Baillie, George S; Sood, Arvind; McPhee, Ian; Gall, Irene; Perry, Stephen J; Lefkowitz, Robert J; Houslay, Miles D
    Phosphorylation of the beta(2) adrenoreceptor (beta(2)AR) by cAMP-activated protein kinase A (PKA) switches its predominant coupling from stimulatory guanine nucleotide regulatory protein (G(s)) to inhibitory guanine nucleotide regulatory protein (G(i)). beta-Arrestins recruit the cAMP-degrading PDE4 phosphodiesterases to the beta(2)AR, thus controlling PKA activity at the membrane. Here we investigate a role for PDE4 recruitment in regulating G protein switching by the beta(2)AR. In human embryonic kidney 293 cells overexpressing a recombinant beta(2)AR, stimulation with isoprenaline recruits beta-arrestins 1 and 2 as well as both PDE4D3 and PDE4D5 to the receptor and stimulates receptor phosphorylation by PKA. The PKA phosphorylation status of the beta(2)AR is enhanced markedly when cells are treated with the selective PDE4-inhibitor rolipram or when they are transfected with a catalytically inactive PDE4D mutant (PDE4D5-D556A) that competitively inhibits isoprenaline-stimulated recruitment of native PDE4 to the beta(2)AR. Rolipram and PDE4D5-D556A also enhance beta(2)AR-mediated activation of extracellular signal-regulated kinases ERK12. This is consistent with a switch in coupling of the receptor from G(s) to G(i), because the ERK12 activation is sensitive to both inhibitors of PKA (H89) and G(i) (pertussis toxin). In cardiac myocytes, the beta(2)AR also switches from G(s) to G(i) coupling. Treating primary cardiac myocytes with isoprenaline induces recruitment of PDE4D3 and PDE4D5 to membranes and activates ERK12. Rolipram robustly enhances this activation in a manner sensitive to both pertussis toxin and H89. Adenovirus-mediated expression of PDE4D5-D556A also potentiates ERK12 activation. Thus, receptor-stimulated beta-arrestin-mediated recruitment of PDE4 plays a central role in the regulation of G protein switching by the beta(2)AR in a physiological system, the cardiac myocyte.
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    How the kinetochore couples microtubule force and centromere stretch to move chromosomes.
    (Nature cell biology, 2016-04) Suzuki, Aussie; Badger, Benjamin L; Haase, Julian; Ohashi, Tomoo; Erickson, Harold P; Salmon, Edward D; Bloom, Kerry
    The Ndc80 complex (Ndc80, Nuf2, Spc24 and Spc25) is a highly conserved kinetochore protein essential for end-on anchorage to spindle microtubule plus ends and for force generation coupled to plus-end polymerization and depolymerization. Spc24/Spc25 at one end of the Ndc80 complex binds the kinetochore. The N-terminal tail and CH domains of Ndc80 bind microtubules, and an internal domain binds microtubule-associated proteins (MAPs) such as the Dam1 complex. To determine how the microtubule- and MAP-binding domains of Ndc80 contribute to force production at the kinetochore in budding yeast, we have inserted a FRET tension sensor into the Ndc80 protein about halfway between its microtubule-binding and internal loop domains. The data support a mechanical model of force generation at metaphase where the position of the kinetochore relative to the microtubule plus end reflects the relative strengths of microtubule depolymerization, centromere stretch and microtubule-binding interactions with the Ndc80 and Dam1 complexes.
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    Intercellular Protein Transfer from Thymocytes to Thymic Epithelial Cells.
    (PLoS One, 2016) Wang, Hong-Xia; Qiu, Yu-Rong; Zhong, Xiao-Ping
    Promiscuous expression of tissue restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs) is crucial for negative selection of self-reactive T cells to establish central tolerance. Intercellular transfer of self-peptide-MHC complexes from mTECs to thymic dendritic cells (DCs) allows DCs to acquire TRAs, which in turn contributes to negative selection and regulatory T cell generation. However, mTECs are unlikely to express all TRAs, such as immunoglobulins generated only in B cells after somatic recombination, hyper-mutation, or class-switches. We report here that both mTECs and cortical TECs can efficiently acquire not only cell surface but also intracellular proteins from thymocytes. This reveals a previously unappreciated intercellular sharing of molecules from thymocytes to TECs, which may broaden the TRA inventory in mTECs for establishing a full spectrum of central tolerance.
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    Liposome division by a simple bacterial division machinery.
    (Proceedings of the National Academy of Sciences of the United States of America, 2013-07) Osawa, Masaki; Erickson, Harold P
    We previously reconstituted Z rings in tubular multilamellar liposomes with FtsZ-YFP-mts, where mts is a membrane-targeting amphiphilic helix. These reconstituted Z rings generated a constriction force but did not divide the thick-walled liposomes. Here we developed a unique system to observe Z rings in unilamellar liposomes. FtsZ-YFP-mts incorporated inside large, unilamellar liposomes formed patches that produced concave distortions when viewed at the equator of the liposome. When viewed en face at the top of the liposome, many of the patches were seen to be small Z rings, which still maintained the concave depressions. We also succeeded in reconstituting the more natural, two-protein system, with FtsA and FtsZ-YFP (having the FtsA-binding peptide instead of the mts). Unilamellar liposomes incorporating FtsA and FtsZ-YFP showed a variety of distributions, including foci and linear arrays. A small fraction of liposomes had obvious Z rings. These Z rings could constrict the liposomes and in some cases appeared to complete the division, leaving a clear septum between the two daughter liposomes. Because complete liposome divisions were not seen with FtsZ-mts, FtsA may be critical for the final membrane scission event. We demonstrate that reconstituted cell division machinery apparently divides the liposome in vitro.
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    Presynaptic Inputs to Any CNS Projection Neuron Identified by Dual Recombinant Virus Infection.
    (PLoS One, 2015) Bráz, João M; Wang, Fan; Basbaum, Allan I
    Although neuroanatomical tracing studies have defined the origin and targets of major projection neurons (PN) of the central nervous system (CNS), there is much less information about the circuits that influence these neurons. Recently, genetic approaches that use Cre recombinase-dependent viral vectors have greatly facilitated such circuit analysis, but these tracing approaches are limited by the availability of Cre-expressing mouse lines and the difficulty in restricting Cre expression to discrete regions of the CNS. Here, we illustrate an alternative approach to drive Cre expression specifically in defined subsets of CNS projection neurons, so as to map both direct and indirect presynaptic inputs to these cells. The method involves a combination of Cre-dependent transneuronal viral tracers that can be used in the adult and that does not require genetically modified mice. To trigger Cre-expression we inject a Cre-expressing adenovirus that is retrogradely transported to the projection neurons of interest. The region containing the retrogradely labeled projection neurons is next injected with Cre-dependent pseudorabies or rabies vectors, which results in labeling of poly- and monosynaptic neuronal inputs, respectively. In proof-of-concept experiments, we used this novel tracing system to study the circuits that engage projection neurons of the superficial dorsal horn of the spinal cord and trigeminal nucleus caudalis, neurons of the parabrachial nucleus of the dorsolateral pons that project to the amygdala and cortically-projecting neurons of the lateral geniculate nucleus. Importantly, because this dual viral tracing method does not require genetically derived Cre-expressing mouse lines, inputs to almost any projection system can be studied and the analysis can be performed in larger animals, such as the rat.
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    The evolving capabilities of rhodopsin-based genetically encoded voltage indicators.
    (Curr Opin Chem Biol, 2015-08) Gong, Yiyang
    Protein engineering over the past four years has made rhodopsin-based genetically encoded voltage indicators a leading candidate to achieve the task of reporting action potentials from a population of genetically targeted neurons in vivo. Rational design and large-scale screening efforts have steadily improved the dynamic range and kinetics of the rhodopsin voltage-sensing domain, and coupling these rhodopsins to bright fluorescent proteins has supported bright fluorescence readout of the large and rapid rhodopsin voltage response. The rhodopsin-fluorescent protein fusions have the highest achieved signal-to-noise ratios for detecting action potentials in neuronal cultures to date, and have successfully reported single spike events in vivo. Given the rapid pace of current development, the genetically encoded voltage indicator class is nearing the goal of robust spike imaging during live-animal behavioral experiments.