一般口演
画像法と可視化 / 分子、生化学および遺伝学的手法
Imaging and Visualization / Molecular, Biochemical and Genetic Techniques
座長：船水 章大（東京大学定量生命科学研究所） |
|---|
| 2022年6月30日 16:10～16:25 沖縄コンベンションセンター 会議場B2 第5会場
1O05e1-01
CALI法を用いたシナプス分子の光不活性化技術と記憶解析
Optical inactivation of synaptic molecules using chromophore-assisted light inactivation and its application to memory analysis.
*竹本 研(1)、高橋 琢哉(2)、實木 亨(1)
1. 三重大学、2. 横浜市立大学
*Kiwamu Takemoto(1), takuya Takahashi(2), Susumu Jitsuki(1)
1. Mie university, 2. Yokohama City University
Keyword: Optical technology, CALI, synaptic plasticity, AMPA receptor
Synaptic plasticity is an important molecular mechanism for learning and memory. Elucidation of the physiological functions of AMPA receptors, which play a central role in this process, is considered to be an important step in elucidating the mechanisms of memory acquisition and maintenance. With the recent development of GFP technology, the behavior of synaptic molecules, such as AMPA receptors during learning are gradually becoming clearer, but the physiological function of these behaviors remains unclear. In order to elucidate the physiological significance of them, it is important to develop a light-induced molecular manipulation technique that can locally and arbitrarily manipulate the loss of function in the target molecules. The CALI (Chromophore-assisted light inactivation, Jay DG, Proc. Natl. Acad. Sci. U.S.A. 1988) method is a promising technique to induce the localized and acute inactivation of target molecules by using photosensitizers that generates reactive oxygen species in a light-dependent manner. We have developed elemental technologies for the CALI and established methods for molecular analysis at various levels from in vitro to in vivo (Takemoto K et al. ACS.Chem.Biol. 2011, Takemoto K et al. Sci. Rep. 2013 etc.). In addition, we have been analyzing the mechanism of learning and memory through manipulation of AMPA receptors using the CALI technology (Takemoto K et al. Nat. Biotechnol. 2017, Trusel M et al. Neuron 2019). In this presentation, we will report on the development of the CALI method for different AMPA receptor complexes, and the differences in the functions of different AMPA receptor complexes in vivo by using CALI technology. In addition, we would like to introduce a new elemental technology in the CALI method that will contribute to the development of optical manipulation techniques for other molecules related to synaptic functions. | | 2022年6月30日 16:25～16:40 沖縄コンベンションセンター 会議場B2 第5会場
1O05e1-02
斜軸回転機構によるオルガノイド多面４Dイメージング
Multi-directional 4D imaging with oblique rotational mechanisms for organoid culture
*萩原 将也(1)
1. 理化学研究所
*Masaya Hagiwara(1)
1. RIKEN
Keyword: 4D imaging, Organoid, multi-directional imaging
Reconstitution of complex 3D organoids such as mini-brain is now a global challenge. Many protocols have been established to replicate organ functions in vitro; however, the 3D imaging of whole tissue is the one of the huge obstacles. In order to capture the millimeter-sized organoid, it requires low magnification lens while it is associated with large point spread function (PSF) and it severely deteriorates the image resolution especially in z direction. Besides, the laser cannot penetrate the tissue to reach deep area. Transparency reagents can counter the laser penetration issue, but it prohibits live imaging during development which potentially provides huge amount of information for organ development. Here we have developed image enhancing device enabling multi-directional scanning to achieve large scale 4D imaging with the current microscope. We have previously developed cubic culture device, which comprises a polycarbonate frame with rigid agarose walls and an inner ECM hydrogel. The agarose wall allows nutrition from medium to reach to the cultured cells while the cube improved the handling ability significantly. Using the advantages of cube, we have developed system to continuously rotate the cube under the microscope by employing only single stepping motor. When the cube is rotated along with the diagonal axis, 3 orthogonal planes (xy, yz, zx planes) will show up in sequence so that scanning can be done from different axis. The size of the system is reasonable under microscope and it allows to setup most of prevailing microscopes to supply multi-directional imaging. In order to demonstrate the 4D imaging using the developed system, we carried out the time-lapse imaging scanned from 3 orthogonal planes. Human iPS spheroid with GFP was made and the spheroids were inserted in the cube with Matrigel. During culturing for 3 days in neuron induction medium, the cube was rotated every 4 minutes for 120 degree so that the spheroid can be scanned from another axis. Low magnification lens (10x) was used to capture the whole spheroid, but it still revealed high z area clearly thanks to the multi-directional imaging. The cube itself also applicable to the fluidics chip to generate morphogen gradient surround cells. Then, the body axis information can be applied to the stem cells. The developed system is quite simple structure and that is why it has broaden utility and has a great potential to accelerate the research in neuron and organoid technologies. | | 2022年6月30日 16:40～16:55 沖縄コンベンションセンター 会議場B2 第5会場
1O05e1-03
ゲノム編集を介した化学タグノックインによる脳内内在性タンパク質の定量的時空間プロファイリング
Quantitative, spatiotemporal profiling of endogenous proteins in mammalian brain tissues via CRISPR/Cas9-based knock-in of chemical tags
*内ヶ島 基政(1,2)、井口 理沙(1)、劉 歆儀(1)、藤井 和磨(3)、阿部 学(1)、﨑村 建司(1)、備瀬 竜馬(3)、三國 貴康(1)
1. 新潟大学脳研究所、2. 東京大学国際高等研究所ニューロインテリジェンス国際研究機構、3. 九州大学大学院システム情報科学研究院
*Motokazu Uchigashima(1,2), Risa Iguchi(1), Xinyi Liu(1), Kazuma Fujii(3), Manabu Abe(1), Kenji Sakimura(1), Ryoma Bise(3), Takayasu Mikuni(1)
1. Brain Res Inst, Niigata Univ, Niigata, Japan, 2. IRCN, Inst Adv Stu, Univ of Tokyo, Tokyo, Japan, 3. Faculty Info Sci and Elect Eng, Kyushu Univ, Fukuoka, Japan
Keyword: molecular imaging, genome editing, chemical tag, synaptic proteins
Proteins in spatially and temporally different subcellular pools can play different roles in cellular function. Thus, separate imaging of the dynamics of spatiotemporally different pools of endogenous proteins provides a good clue to understanding the cellular function at molecular levels, especially in brain cells with highly compartmentalized structures. Here, we developed a toolkit to interrogate the localization and dynamics of spatiotemporally-separable pools of endogenous proteins at single cell levels in mammalian brain tissues. By using CRISPR/Cas9-based genome editing in sparse neural cells, endogenous proteins of interest were fused with chemical tags which allow for an irreversible binding to small-molecule fluorescent ligands. We achieved a quantitative labeling of a variety of endogenous proteins tagged with chemical tags in single cells in fixed or living organotypic slice cultures and also in vivo brain tissues. Multicolor labeling of chemical tag-fused cell membrane-spanning proteins with cell membrane-permeable and -impermeable ligands visualized their intracellular and extracellular pools. Importantly, the intracellular and extracellular pools of AMPA glutamate receptor GluA2 subunit at individual spines were positively correlated with spine size. Simultaneous targeting of distinct chemical tags into different genes allowed for the detection of the extracellular pools of NMDA glutamate receptor GluN1 and GluA2 subunits at the same spine, enabling the visualization of “silent synapses” which contain NMDA but no AMPA receptors. Living cell pulse-chase labeling separated the newly-synthesized pool of calcium/calmodulin-dependent kinase II alpha (Camk2a) from the pre-existing one both in vitro and in vivo. Interestingly, the signal ratio of newly-synthesized and pre-existing pools of Camk2a was constant between different spines. Thus, our toolkit enables the quantification of spatiotemporally-different pools of endogenous proteins in single cells with synaptic resolution in fixed or living mammalian brain tissues. | | 2022年6月30日 16:55～17:10 沖縄コンベンションセンター 会議場B2 第5会場
1O05e1-04
Compact device for simultaneous cell electrophysiological signal and fluorescence imaging acquisition
*Barbara Teixeira Sais(1), Makito Haruta(1), Kuang-Chih Tso(1), Mizuki Hagita(1), Takanori Hagiwara(1), Kenji Sugie(1), Ayaka Kimura(2), Hironari Takehara(1), Hiroyuki Tashiro(1,3), Kiyotaka Sasagawa(1), Jun Ohta(1)
1. Dpto of Material Science, Nara Institute of Science and Technology, Nara, Japan, 2. Dementia Research Unit, Osaka Psychiatric Medical Center, Osaka, Japan, 3. Division of Medical Technology, Kyushu University, Fukuoka, Japan
Keyword: multielectrode array, lensless fluorescent imaging, multifunctional device, miniaturization
When observing living cells interactions, several techniques can be used, such as fluorescence microscopy and electrophysiological signal measurement. Measuring both signals is important to have a better understanding of the physiological activity, such as spikes or action potentials, of cells that have signals and dynamics such as neurons and muscle cells. However, observing those physiological traits long-term with cultured cells is difficult for the common microscope. In this study we fabricated a device capable of recording both electrophysiological and fluorescence signal from cells, in a size compact enough to fit inside any incubator. For that, we used the multi-electrode array (MEA) approach to record the in vitro electrical activity of neural cells under physiological and stimulation conditions. We fabricated our microelectrodes on a fiber optical plate (FOP) substrate by using the conventional microfabrication process, such as photolithography, metal and insulator deposition and etching. The metal we chose to fabricate the electrodes was gold, as parameters such as biocompatibility, resistance to corrosion, longevity and conductivity were of our concern. The FOP was chosen as our substrate due to its imaging properties such as conveying the incident image to the output surface. It is the FOP that promotes the device’s miniaturization by lensless fluorescent imaging. By using FOP as substrate for the cell culture chamber, we are able to measure both the fluorescent signal and electrophysiology signal in the same experiment. For the imaging module, a device composed by a commercially available CMOS image sensor, fiber-optic plate (FOP), blue LED, and optical filters was fabricated. To test our device, we used neuronal blastoma NG108-15 cells. Our device was able to satisfactorily observed fluorescent images and MEA signals in normal and under stimulation conditions. | |
|
|
