Biology subjects

Chien, C.-T.

Publications and source records attributed to Chien, C.-T..

5 recordsLinked to original sources

bioRxiv2025

Molecular mechanism of exchange coupling in CLC chloride/proton antiporters

The ubiquitous CLC membrane transporters are unique in their ability to exchange anions for cations. Despite extensive study, there is no mechanistic model that fully explains their 2:1 Cl-/H+ stoichiometric exchange mechanism. Here, we provide such a model. Using differential hydrogen-deuterium exchange mass spectrometry, cryo-EM structure determination, and molecular dynamics simulations, we uncovered conformational dynamics in CLC-ec1, a bacterial CLC homolog that has served as a paradigm for this family of transporters. Simulations based on a cryo-EM structure at pH 3 revealed critical steps in the transport mechanism, including release of Cl- ions to the extracellular side, opening of the inner gate, and water wires that facilitate H+ transport. Surprisingly, these water wires occurred independently of Cl-binding, prompting us to reassess the relationship between Cl- binding and Cl-/H+ coupling. Using isothermal titration calorimetry and quantitative flux assays on mutants with reduced Cl- binding affinity, we conclude that, while Cl- binding is necessary for coupling, even weak binding can support Cl-/H+ coupling. By integrating our findings with existing literature, we establish a complete and efficient CLC 2:1 Cl-/H+ exchange mechanism.

biophysics↗

bioRxiv2022

Enhanced expression of 4-Cl-IAA and 6-Cl-IAA by touch stimulus for rapid and differential growths of the Madeira vine

Madeira vine (MV) grows 30 times faster after encountering a support. In vivo x-ray diffraction made on live MV stems revel the appearance of crystallized IAA (C10H9NO2), 4-Cl-IAA (C10H8ClNO2) and 6-Cl-IAA (C10H8ClNO2) in the stems. Small angle neutron scattering spectra of the IAA extracted from MVs stem reveal a progressive increase in the size of crystallized IAA transported downward from the apex of the shoots. High resolution X-ray diffractions made on the extracted IAA reveal significantly larger amounts of 4-Cl-IAA and 6-Cl-IAA in the climbing MVs than in the swaying around MVs. The gas chromatography-mass spectrometry spectra reveal the production of 9% more IAA and 90% more 4-Cl-IAA+6-Cl-IAA at the apexes of climbing MVs than swaying MVs. More 4-Cl-IAA+6-Cl-IAA were transported to the contact-free side than to the contact side of the vine. In vivo neutron tomography of naturally climbing MVs reveals a substantially higher H+ concentration in the contact-free parts than in the contact parts. The absorption spectra also reveal more expansin in the contact-free parts than in the contact parts. These results provide a view, at the molecular level, of what triggers the faster and differential growths in MVs in response to touching a support.

plant biology↗

bioRxiv2021

Gastrodia elata Blume prevents dopaminergic neuron degeneration via glial Nrf2 signaling in Lrrk2-G2019S Parkinson's disease models

The most frequent missense mutations in familial Parkinsons disease (PD) occur in the highly conserved LRRK2/PARK8 gene with G2019S mutation. We previously established a fly model of PD carrying the LRRK2-G2019S mutation that exhibited the parkinsonism-like phenotypes. An herbal medicine--Gastrodia elata Blume (GE), has been reported to have neuroprotective effects in toxin- induced PD models. However, the underpinning molecular mechanisms of GE beneficiary to G2019S- induced PD remain unclear. Here, we show that these G2019S flies treated with water extracts of GE (WGE) and its bioactive compounds, gastrodin and 4-HBA, displayed locomotion improvement and dopaminergic neuron protection. WGE suppressed the accumulation and hyperactivation of G2019S proteins in dopaminergic neurons, and activated the antioxidation and detoxification factor Nrf2 mostly in the astrocyte-like and ensheathing glia. Glial activation of Nrf2 antagonizes G2019S-induced Mad/Smad signaling. Moreover, we treated LRRK2-G2019S transgenic mice with WGE and found the locomotion declines, the loss of dopaminergic neurons, and the number of hyperactive microglia were restored. WGE also suppressed the hyperactivation of G2019S proteins and regulated the Smad2/3 pathways in the mice brains. We conclude that WGE prevents locomotion defects and the neuronal loss induced by G2019S mutation via glial Nrf2/Mad signaling, unveiling a potential therapeutic avenue for PD.

neuroscience↗

bioRxiv2019

Glia-derived exosomal miR-274 targets Sprouty in trachea and synaptic boutons to modulate growth and responses to hypoxia

Secreted exosomal miRNAs mediate inter-organ/tissue communication by downregulating gene expression, thereby modulating developmental and physiological functions. However, the source, route, and function have not been formally established for specific miRNAs. Here, we show that glial miR-274 non-cell autonomously modulates the growth of synaptic boutons and tracheal branches. Whereas precursor miR-274 was expressed in glia, mature miR-274 was secreted. miR-274 secretion to circulating hemolymph was detected in exosomes, a process requiring ESCRT components in exosome biogenesis and Rab11 and Syx1A in exosome release. miR-274 downregulated Sprouty to activate MAPK in synaptic boutons and tracheal branches, thereby promoting their growth. Expression of miR-274 solely in glia of a mir-274 null mutant reset normal levels of Sprouty and MAPK, and hemolymphatic exosomal miR-274. mir-274 mutant larvae were hypersensitive to hypoxia, which was suppressed by increasing tracheal branches. Thus, glia-derived miR-274 coordinates growth of synaptic boutons and tracheal branches to modulate larval hypoxia responses.

developmental biology↗

bioRxiv2019

Glial response to hypoxia in trachealess mutants induces synapse remodeling.

Synaptic structure and activity are sensitive to environmental alterations. Modulation of synaptic morphology and function is often induced by signals from glia. However, the process by which glia mediate synaptic responses to environmental perturbations such as hypoxia remains unknown. Here, we report that, in the Drosophila trachealess (trh) mutant, smaller synaptic boutons form clusters named bunch boutons appear at larval neuromuscular junctions (NMJs), which is induced by the reduction of internal oxygen levels due to defective tracheal branches. Thus, the bunch bouton phenotype in the trh mutant is suppressed by hyperoxia, and recapitulated in wild-type larvae raised under hypoxia. We further show that hypoxia-inducible factor (HIF)-1/Similar (Sima) is critical in mediating hypoxia-induced bunch bouton formation. Sima upregulates the level of the Wnt/Wingless (Wg) signal in glia, leading to reorganized microtubule structures within presynaptic sites. Finally, hypoxia-induced bunch boutons maintain normal synaptic transmission at the NMJs, which is crucial for coordinated larval locomotion. Author summaryOxygen is essential for animals to maintain their life such as growth, metabolism, responsiveness, and movement. It is therefore important to understand how animal cells trigger hypoxia response and adapt to hypoxia thereafter. Both mammalian vascular and insect tracheal branches are induced to enhance the oxygen delivery. However, the study of hypoxia response in the nervous system remains limited. In this study, we assess the morphology of Drosophila neuromuscular junctions (NMJs), a model system to study development and function of synapses, in two hypoxia conditions, one with raising wild-type larvae in hypoxia, and the other in the trachealess (trh) mutant in which the trachea is defective, causing insufficient oxygen supply. Interestingly, glia, normally wrapping the axons of NMJs, invade into synapse and trigger Wg signals to reconstitute the synaptic structure under hypoxia. This synaptic remodeling maintains the synaptic transmission of synapse, which associate the locomotor behavior of larvae.

genetics↗