An ultrasensitive biosensor for high-resolution kinase activity imaging in awake mice

Zhang, Jinfan; Liu, B.; Hong, Ingie; Mo, Albert; Roth, Richard H.; Tenner, Brian; Lin, Wei; Zhang, Jason Z.; Molina, Rosana S.; Drobizhev, Mikhail; Hughes, Thomas E.; Tian, Lin; Huganir, Richard L.; Mehta, Sohum; Zhang, Jin

doi:10.1038/s41589-020-00660-y

Cited by 64 publications

(74 citation statements)

References 59 publications

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“…The recent extensive effort to develop multiplexed imaging platforms has enabled a better understanding of complex biological processes. For instance, complex signaling networks in intact cells, such as a change in kinase activity and second messenger transient responses to stimulations, were elegantly dissected by multiplexed imaging [32,33]. Moreover, emerging NIR fluorescent proteins, combined with newly developed NIR-FRET technologies, have opened a new window to understanding how complex biological processes, including calcium dynamics, signaling pathways, and/or proteolysis, are spatiotemporally regulated in cells [19,20,[34][35][36][37].…”

Section: Discussionmentioning

confidence: 99%

Limited Substrate Specificity of PS/γ-Secretase Is Supported by Novel Multiplexed FRET Analysis in Live Cells

et al. 2021

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Presenilin (PS)/γ-secretase is an aspartyl protease that processes a wide range of transmembrane proteins such as the amyloid precursor protein (APP) and Notch1, playing essential roles in normal biological events and diseases. However, whether there is a substrate preference for PS/γ-secretase processing in cells is not fully understood. Structural studies of PS/γ-secretase enfolding a fragment of APP or Notch1 showed that the two substrates engage the protease in broadly similar ways, suggesting the limited substrate specificity of PS/γ-secretase. In the present study, we developed a new multiplexed imaging platform that, for the first time, allowed us to quantitatively monitor how PS/γ-secretase processes two different substrates (e.g., APP vs. Notch1) in the same cell. In this assay, we utilized the recently reported, spectrally compatible visible and near-infrared (NIR)-range Förster resonance energy transfer (FRET) biosensors that permit quantitative recording of PS/γ-secretase activity in live cells. Here, we show that, overall, PS/γ-secretase similarly cleaves Notch1 N100, wild-type APP C99, and familial Alzheimer’s disease (FAD)-linked APP C99 mutants in Chinese hamster ovary (CHO) cells, which further supports the limited PS/γ-secretase substrate specificity. On the other hand, a cell-by-cell basis analysis demonstrates a certain degree of variability in substrate recognition and processing by PS/γ-secretase among different cells. Our new multiplexed FRET assay could be a useful tool to better understand how PS/γ-secretase processes its multiple substrates in normal and disease conditions in live, intact cells.

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Section: Discussionmentioning

confidence: 99%

Limited Substrate Specificity of PS/γ-Secretase Is Supported by Novel Multiplexed FRET Analysis in Live Cells

et al. 2021

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“…Especially in the case of our in vivo fiber photometry measurements, we may have missed changes in cAMP in response to unexpected delivery of palatable food or tail shocks. We overcame this limitation via additional experiments using a fluorescent biosensor of PKA activity (Zhang et al, 2021a), which had improved signal-to-noise in response to unexpected tail shocks and revealed clear depression with repeated salient events. An additional limitation is that our sensor measurements were not targeted specifically to dendritic compartments where cAMP increases may be concentrated, and thus our bulk measurements across somatic and dendritic compartments may have limited our sensitivity further.…”

Section: Potential Limitations Imposed By the Sensitivity Of The Camp Sensormentioning

confidence: 99%

Dopamine-dependent cAMP dynamics in basal amygdala glutamatergic neurons

Lutas

Fernando

Zhang

et al. 2021

Preprint

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Dopaminergic inputs to basal amygdala (BA) instruct learning of motivational salience. Here, we investigated the dynamics of dopamine release and downstream signaling during multiple salient events occurring within tens of seconds. We established in vitro and in vivo real-time tracking and manipulation of cAMP - a key intracellular plasticity signal downstream of dopamine receptor activation. Optogenetically-evoked release of dopamine drove proportional increases in cAMP in almost all BA glutamatergic neurons, suggesting widespread actions of dopamine across neurons preferring positive or negative valence. These cAMP responses decayed more slowly than dopamine release, potentially extending the window of plasticity. cAMP levels accumulated following direct photostimulation of cAMP but not repeated stimulation of dopamine axons, due to potent depression of dopamine release. cAMP and protein kinase A (PKA) responses to repeated appetitive or aversive stimuli also exhibited pronounced depression. Thus, history-dependent dynamics of dopamine and cAMP may regulate learning of temporally clustered, salient stimuli.

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“…In the past two decades, masses of genetically encoded fluorescent reporters capable of continuously monitoring kinase activities in live cells and even in vivo were designed by fusing fluorescent proteins (FPs) or luciferase with substrate sequence and phosphoamino acid binding domain [11–14] . Further efforts were made on upgrading kinase activity reporters in the following years in terms of elevating the sensitivity and expanding the kinase tracking channels [15, 16] . However, the expression of fused FPs‐based biosensors requires the expertise in life science, and sophisticated library screening was included in the procedure to attain high‐sensitivity biosensors [16] .…”

Section: Introductionmentioning

confidence: 99%

Nanomediator–Effector Cascade Systems for Amplified Protein Kinase Activity Imaging and Phosphorylation‐Induced Drug Release In Vivo

et al. 2021

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Protein kinases constitute a rich pool of biomarkers and therapeutic targets of tremendous diseases including cancer. However, sensing kinase activity in vivo while implementing treatments according to kinase hyperactivation remains challenging. Herein, we present a nanomediator–effector cascade system that can in situ magnify the subtle events of kinase‐catalyzed phosphorylation via DNA amplification machinery to achieve kinase activity imaging and kinase‐responsive drug release in vivo. In this cascade, the phosphorylation‐mediated disassembly of DNA/peptide complex on the nanomediators initiated the detachment of fluorescent hairpin DNAs from the nanoeffectors via hybridization chain reaction (HCR), leading to fluorescence recovery and therapeutic cargo release. We demonstrated that this nanosystem simultaneously enabled trace protein kinase A (PKA) activity imaging and on‐demand drug delivery for inhibition of tumor cell growth both in vitro and in vivo, affording a kinase‐specific sense‐and‐treat paradigm for cancer theranostics.

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An ultrasensitive biosensor for high-resolution kinase activity imaging in awake mice

Cited by 64 publications

References 59 publications

Limited Substrate Specificity of PS/γ-Secretase Is Supported by Novel Multiplexed FRET Analysis in Live Cells

Limited Substrate Specificity of PS/γ-Secretase Is Supported by Novel Multiplexed FRET Analysis in Live Cells

Dopamine-dependent cAMP dynamics in basal amygdala glutamatergic neurons

Nanomediator–Effector Cascade Systems for Amplified Protein Kinase Activity Imaging and Phosphorylation‐Induced Drug Release In Vivo

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