Browsing by Author "Huang, Yung-An"

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Actin waves transport RanGTP to the neurite tip to regulate non-centrosomal microtubules in neurons
(2020-05-01)
Huang, Yung-An
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Hsu, Chih-Hsuan
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Chiu, Ho-Chieh
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Hsi, Pei-Yu
;
Ho, Chris T.
Microtubules (MTs) are the most abundant cytoskeleton in neurons, and control multiple facets of their development. While the MT-organizing center (MTOC) in mitotic cells is typically located at the centrosome, the MTOC in neurons switches to non-centrosomal sites. A handful of cellular components have been shown to promote non-centrosomal MT (ncMT) formation in neurons, yet the regulation mechanism remains unknown. Here, we demonstrate that the small GTPase Ran is a key regulator of ncMTs in neurons. Using an optogenetic tool that enables light-induced local production of RanGTP, we demonstrate that RanGTP promotes ncMT plus-end growth along the neurite. Additionally, we discovered that actin waves drive the anterograde transport of RanGTP. Pharmacological disruption of actin waves abolishes the enrichment of RanGTP and reduces growing ncMT plus-ends at the neurite tip. These observations identify a novel regulation mechanism for ncMTs and pinpoint an indirect connection between the actin and MT cytoskeletons in neurons.
An inverted method for culturing dissociated mouse hippocampal neurons
(2011-05-01)
Chen, Wen-Shin
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Yueh, Chao-Yen
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Huang, Yung-An
;
Hwang, Eric
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生物科技學系
Dissociated hippocampal neuron culture has long been the model system of choice for many neuroscientists. The ability to culture dissociated hippocampal neurons from genetically modified mice provides an invaluable tool for studying many neuronal processes. In this study, we established a novel method to culture dissociated hippocampal neurons from embryonic and neonatal mice. Dissociated neurons were cultured in a microchamber between the glass coverslip and the plastic cell container without the use of glial feeder cells. Our method significantly simplifies the preparation while produces healthy and long-lived neuronal cultures that are difficult to achieve without the use of feeder cells. (C) 2011 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
Application of aurintricarboxylic acid for the adherence of mouse P19 neurons and primary hippocampal neurons to noncoated surface in serum-free culture
(2012-11-01)
Lee, Tsung-Yih
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Chen, Wen-Shin
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Huang, Yung-An
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Liu, Ting-Wei
;
Hwang, Eric
Dissociated primary neuron culture has been the most widely used model systems for neuroscience research. Most of these primary neurons are cultured on adhesion matrix-coated surface to provide a proper environment for cell anchorage under serum-free conditions. In this study, we provide an alternative technique to promote the adhesions of these neurons using aurintricarboxylic acid (ATA), a nonpeptide compound, without surface manipulations. We first demonstrated that ATA could promote Chinese hamster ovary cell attachment and proliferation in serum-free medium in a dosage-dependent manner. We later showed that ATA significantly enhanced the attachment of the retinoic acid differentiated P19 mouse embryonal carcinoma (P19) neurons, with an optimal concentration around 30 mu g/mL. A similar result was seen in primary hippocampal neurons, with an optimal ATA concentration around 15 mu g/mL. Further morphological assessments revealed that the average neurite length and neuronal polarization were almost identical to that obtained using a conventional method with poly-L-lysine surface. The advantages of using the ATA treatment technique for immunochemical analysis are discussed. (C) 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012
Biocompatible evaluation of fluorescent nanodiamonds in neurons in vitro
(2014-04-01)
Kao, Jun-Wei
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Huang, Yung-An
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Chiang, Ming-Han
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Huang, Hou-Syun
;
Liu, Kuang-Kai
Examining the function of TPX2 during neurite morphogenesis
(2014-04-01)
Hwang, Eric
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Chen, Wen-Shin
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Chen, Yi-Ru
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Huang, Yung-An
;
交大名義發表
Microtubule-Associated Type II Protein Kinase A Is Important for Neurite Elongation
(2013-08-13)
Huang, Yung-An
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Kao, Jun-Wei
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Tseng, Dion Tzu-Huan
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Chen, Wen-Shin
;
Chiang, Ming-Han
Neuritogenesis is a process through which neurons generate their widespread axon and dendrites. The microtubule cytoskeleton plays crucial roles throughout neuritogenesis. Our previous study indicated that the amount of type II protein kinase A (PKA) on microtubules significantly increased upon neuronal differentiation and neuritogenesis. While the overall pool of PKA has been shown to participate in various neuronal processes, the function of microtubule-associated PKA during neuritogenesis remains largely unknown. First, we showed that PKA localized to microtubule-based region in different neurons. Since PKA is essential for various cellular functions, globally inhibiting PKA activity will causes a wide variety of phenotypes in neurons. To examine the function of microtubule-associated PKA without changing the total PKA level, we utilized the neuron-specific PKA anchoring protein MAP2. Overexpressing the dominant negative MAP2 construct that binds to type II PKA but cannot bind to the microtubule cytoskeleton in dissociated hippocampal neurons removed PKA from microtubules and resulted in compromised neurite elongation. In addition, we demonstrated that the association of PKA with microtubules can also enhance cell protrusion using the non-neuronal P19 cells. Overexpressing a MAP2 deletion construct which does not target PKA to the microtubule cytoskeleton caused non-neuronal cells to generate shorter cell protrusions than control cells overexpressing wild-type MAP2 that anchors PKA to microtubules. Finally, we demonstrated that the ability of microtubule-associated PKA to promote protrusion elongation was independent of MAP2 phosphorylation. This suggests other proteins in close proximity to the microtubule cytoskeleton are involved in this process.
Photocontrollable Probe Spatiotemporally Induces Neurotoxic Fibrillar Aggregates and Impairs Nucleocytoplasmic Trafficking
(2017-07-01)
He, Ruei-Yu
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Chao, Shu-Han
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Tsai, Yu-Ju
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Lee, Chi-Chang
;
Yu, Chu-Yi
The abnormal assembly of misfolded proteins into neurotoxic aggregates is the hallmark associated with neurodegenerative diseases. Herein, we establish a photocontrollable platform to trigger amyloidogenesis to recapitulate the pathogenesis of amyotrophic lateral sclerosis (ALS) by applying a chemically engineered probe as a "switch" in live cells. This probe is composed of an amyloidogenic peptide from TDP-43, a photolabile linker, a polycationic sequence both to mask amyloidogenicity and for cell penetration, and a fluorophore for visualization. The photocontrollable probe can self-assemble into a spherical vesicle but rapidly develops massive nanofibrils with amyloid properties upon photo activation. The photoinduced in vitro fibrillization process is characterized by biophysical techniques. In cellular experiments, this cell-penetrable vesicle was retained in the cytoplasm, seeded the mislocalized endogenous TDP-43 into aggregates upon irradiation, and consequently initiated apoptosis. In addition, this photocontrollable vesicle interfered with nucleocytoplasmic protein transport and triggered cortical neuron degeneration. Our developed strategy provides in vitro and in vivo spatiotemporal control of neurotoxic fibrillar aggregate formation, which can be readily applied in the studies of protein misfolding, aggregation-induced protein mislocalization, and amyloid-induced pathogenesis in different diseases.
Ran-dependent TPX2 activation promotes acentrosomal microtubule nucleation in neurons
(2017-02-13)
Chen, Wen-Shin
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Chen, Yi-Ju
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Huang, Yung-An
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Hsieh, Bing-Yuan
;
Chiu, Ho-Chieh
The microtubule (MT) cytoskeleton is essential for the formation of morphologically appropriate neurons. The existence of the acentrosomal MT organizing center in neurons has been proposed but its identity remained elusive. Here we provide evidence showing that TPX2 is an important component of this acentrosomal MT organizing center. First, neurite elongation is compromised in TPX2-depleted neurons. In addition, TPX2 localizes to the centrosome and along the neurite shaft bound to MTs. Depleting TPX2 decreases MT formation frequency specifically at the tip and the base of the neurite, and these correlate precisely with the regions where active GTP-bound Ran proteins are enriched. Furthermore, overexpressing the downstream effector of Ran, importin, compromises MT formation and neuronal morphogenesis. Finally, applying a Ran-importin signaling interfering compound phenocopies the effect of TPX2 depletion on MT dynamics. Together, these data suggest a model in which Ran-dependent TPX2 activation promotes acentrosomal MT nucleation in neurons.
The effect of fluorescent nanodiamonds on neuronal survival and morphogenesis
(2014-11-05)
Huang, Yung-An
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Kao, Chun-Wei
;
Liu, Kuang-Kai
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Huang, Hou-Syun
;
Chiang, Ming-Han
Nanodiamond (ND) has emerged as a promising carbon nanomaterial for therapeutic applications. In previous studies, ND has been reported to have outstanding biocompatibility and high uptake rate in various cell types. ND containing nitrogen-vacancy centers exhibit fluorescence property is called fluorescent nanodiamond (FND), and has been applied for bio-labeling agent. However, the influence and application of FND on the nervous system remain elusive. In order to study the compatibility of FND on the nervous system, neurons treated with FNDs in vitro and in vivo were examined. FND did not induce cytotoxicity in primary neurons from either central (CNS) or peripheral nervous system (PNS); neither did intracranial injection of FND affect animal behavior. The neuronal uptake of FNDs was confirmed using flow cytometry and confocal microscopy. However, FND caused a concentration-dependent decrease in neurite length in both CNS and PNS neurons. Time-lapse live cell imaging showed that the reduction of neurite length was due to the spatial hindrance of FND on advancing axonal growth cone. These findings demonstrate that FNDs exhibit low neuronal toxicity but interfere with neuronal morphogenesis, and should be taken into consideration when applications involve actively growing neurites (e.g. nerve regeneration).