公募シンポジウム８：超解像度顕微鏡から見た新しい神経系の分子像
Symposium8 : New Vistas using superresolusion microscopy in neurobiology |
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| 2020/9/11 10:55～11:12 Zoom A
SY8-01
超解像顕微鏡による成長円錐と神経細胞の観察
Observation of growth cones and neuronal cells with super resolution microscopy
*加藤 薫1、田中 みなみ1、波平 昌一1、光山 光山1
1. 産業技術総合研究所 バイオメディカル研究部門
*Kaoru Katoh1, Minami Tanaka1, Masakazu Namihira1, Toutai Mitsuyama1
1. Biomedical Research Institute, AIST
Growth cones are enlargement of thin cytoplasm at the tips of growing neurites. The growth cones crawl about and find the path of neuronal elongation. Their movements depend on the dynamics of actin filaments, which are too small to observe with conventional optical microscopes. We, therefore, applied polarized light microscope (Polscope), apodised phase contrast microscope, and recently super resolution microscopes (SIM, STED and Spinning disk) to visualize actin cytoskeleton and other structures.
In this presentation, we will summarize the images of fine structures of the cell, recorded with conventional (polarized light and apodised phase contrast) and super resolution microscopy. Polscope revealed radially aligned actin bundles in the growth cones of living unstained cells. Apodised phase contrast revealed intra nuclear structures and intracellular organelle (mitochondria, ER etc.). Super resolution microscopes revealed fine structures of actin meshwork. SIM (Structure Illumination Microscope ) visualized actin meshwork in live cells with resolution of 110 nm as time lapse movie and STED (Stimulated emission depletion) microscope visualized them in the fixed cells with higher resolution (30-50 nm) and with thin optical section (140 nm thick in each optical slice). Two camera SIM showed co-localization of actin and actin bundling protein (fascin) in live cells with high resolution and dissociation of fascin from actin bundles as time lapse records. Spinning disk type super resolution revealed fine structures in the small fish.
Based on the summarized image data, we will discuss future of the imaging with optical microscopes. This will include big image data (of several TB size (1 million frame images) ), AI and robots in biomedical imaging with optical microscopy.
| | 2020/9/11 11:12～11:29 Zoom A
SY8-02
超解像観察により見出された新たな成長円錐構造と動態
Super-resolution imaging unveils the novel structures in growth cones
*野住 素広1、五十嵐 道弘1
1. 新潟大学大学院医歯学総合研究科 分子細胞機能学
*Motohiro Nozumi1, Michihiro Igarashi1
1. Div Mol Cell Biol, Niigata Univ Grad Sch Med Dent Sci
The growth cone is a highly motile structure in the tips of growing axons. From the proteomic findings, the growth cone is thought to be equipped with minimum molecular requirements for 1) motility as a driving force to axonal growth, 2) signal pathways in response to axon guidance cues, 3) synapse formation after the target recognition (PNAS, 2009). The complex protein interactions underlying such complicated functions are densely packed in the narrow area of each growth cone, which prevented us from understanding the molecular basis of the growth cone. Super-resolution (SR) microscopy is available to observe the fluorescence-labeled molecules by overcoming the diffraction limit of optical microscopy. 3D-structured illumination microscopy (SIM), one of the SR microscopy devices, is utilized to analyze the dynamics of the molecules for live-cell imaging. We have applied it to the proteins involved in the growth cone behaviors as follows. Namely, in the peripheral (P-) domain of the growth cone, highly concentrated F-actin elongates toward the leading edge, and they then push the plasma membrane to advance. Although the P-domain was thought to be a flat structure F-actin filled simply, we found the F-actin were distributed along the non-adhesion surface in the area (Neurochem Int, 2018; J Neurosci, 2018). A part of the F-actin formed non-adhesion filopodia, which protrude from the surface of growth cones. An axon guidance receptor, neuropilin-1, was accumulated in the plasma membrane of the elongating filopodia in the z-axis direction, in a lipid-raft-dependent fashion. Some components of clathrin-independent endocytosis, i.e., synaptophysin, endophilin-A3, and dynamin-1, also were localized in such z-axis filopodia. Our results based on the 3D-SIM observation suggest that there is a new actin-dependent mechanism for the accumulation and retrieval of axon guidance receptors on/from the surface of the growth cone. We also will talk about an original method using SR microscopy, to find the unidentified interactions between organelles within the growth cone.
| | 2020/9/11 11:25～11:46 Zoom A
SY8-03
神経伝達物質放出を制御する超分子ナノ構造体
Supramolecular nanostructures that regulate neurotransmitter release
*廣瀬 謙造1
1. 東京大学
*Kenzo Hirose1
1. The University of Tokyo
More than sixty years have passed since the quantal model of synaptic transmission was proposed, in which three parameters, N, P, and Q, determine the size of transmission. Q is a postsynaptic response upon release of single vesicular neurotransmitter release. N denotes the number of release sites where a synaptic vesicle undergoes exocytosis. P is the release probability at the release site. However molecular correlates for these synaptic parameters had remained largely unknown. We have approached the problems by combining two imaging techniques: the glutamate imaging technique using a fluorescent glutamate probe EOS, and superresolution microscopy of presynaptic proteins. Using the glutamate imaging, we successfully estimated synaptic parameters for each single synapse and found that synapses are heterogeneous in N and P values. Interestingly, these two parameters showed no correlation. This finding indicates that there are independent molecular mechanisms to determine N and P values. We used 3D-STORM to obtain superresolution images of proteins at synapses and found that Munc13-1 molecules forms supramolecular assemblies with a nanoscale diameter (~ 50 nm). There exist multiple Munc13-1 assemblies, and the number of the assemblies and the N value showed close match for each synapse. Reduction of Munc13-1 by knockdown decreased both the numbers of the Munc13-1 assemblies and the N values. These results indicate that the molecular entity of the release site is the Munc13-1 assembly. We also found that syntaxin-1, an essential protein machinery for vesicular release was associated with Munc13-1 assemblies. Furthermore, we analyzed supreresolution images of other presynaptic proteins (RIM, Cav2.1, CAST, Neurexin-1), and found their unique distributions. Therefore, supramolecular nanostructures formed by presynaptic proteins might play important roles in regulating synaptic transmission.
| | 2020/9/11 11:46～12:03 Zoom A
SY8-04
Photo-convertible fluorescent proteins for CLEM microscopy
*Xu Pingyong1
1. Institute of Biophysics, Chinese Academy of Sciences
*Pingyong Xu1
1. Institute of Biophysics, Chinese Academy of Sciences
Superresolution correlative light and electron microscopy (SR-CLEM) is a powerful tool for localizing a specific molecular context under electron microscopy (EM) beyond the diffraction limit of light microscopy (LM). The sampling method and labelling fluorophore are critical to maintain both bright fluorescent signals for LM and ultrastructure of the EM images. Epon epoxy resin is superior to other resins for conventional EM chemical fixation because it preserves the cellular ultrastructure and has better sectioning properties. However, no fluorescent protein (FP) was reported to survive Epon embedding after osmium tetroxide (OsO4) fixation for use in SR-CLEM. Here, we have developed an OsO4 fixation-resistant photoconvertible FP (PCFP), PCEM, which not only is much brighter than mEos4b after OsO4 treatment but also retains its fluorescence and photoswitching property after Epon embedding. PCEM and Epon-embedding-based SR-CLEM microscopy (ESR-CLEM) enable the visualization of biomolecules and their cellular context in the same section.
| | 2020/9/11 12:03～12:20 Zoom A
SY8-05
Molecular tools for super-resolution imaging of sub-organelle structures
*多喜 正泰1
1. 名古屋大学 トランスフォーマティブ生命分子研究所
*Masayasu Taki1
1. Institute of Transformative Bio-Molecules, Nagoya University
The new technologies in fluorescence microscopy have been contributing much to various areas of life sciences. Especially, stimulation emission depletion (STED) microscopy enables ultrastructural imaging of organelle dynamics with a high spatiotemporal resolution in living cells. However, owing to the very intense laser irradiation used for excitation and depletion in STED microscopy, most fluorescent dyes suffered from rapid photobleaching during the repeated recording of images. Therefore, to visualize biological phenomena in detail for a long period of time under microscopic conditions, fluorophores with high photostability are strongly desired. Herein, I'd like to demonstrate new chemical tools for visualizing the organelle membranes and their applications to STED imaging.
MitoPB Yellow is a super-photostable probe to selectively capture the ultra-structures of the mitochondrial cristae with a resolution of ~60 nm when depleted at 660 nm. This chemical tool provides morphological information of the cristae, which has so far only been observed in fixed cells using electron microscopy. Moreover, this method gives information about the dynamic ultrastructures such as the inter-membrane fusion in different mitochondria as well as the inter-cristae mergence in a single mitochondrion during the apoptosis-like mitochondrial swelling process.
More recently, we have developed a lysosomal membrane marker LysoPB Yellow, where lysosome directing groups were introduced into the same fluorophore as MitoPB Yellow. Using LysoPB Yellow, donut-shape structures corresponding to the lysosomal membrane can be clearly visualized in STED microscopy.
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