{"created":"2023-06-20T13:20:56.489331+00:00","id":1009,"links":{},"metadata":{"_buckets":{"deposit":"ce7c843d-8cd7-43f0-be5d-e09461617bd1"},"_deposit":{"created_by":1,"id":"1009","owners":[1],"pid":{"revision_id":0,"type":"depid","value":"1009"},"status":"published"},"_oai":{"id":"oai:ir.soken.ac.jp:00001009","sets":["2:430:20"]},"author_link":["0","0","0"],"item_1_creator_2":{"attribute_name":"著者名","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"勝木, 健雄"}],"nameIdentifiers":[{}]}]},"item_1_creator_3":{"attribute_name":"フリガナ","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"カツキ, タケオ"}],"nameIdentifiers":[{}]}]},"item_1_date_granted_11":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"2005-03-24"}]},"item_1_degree_grantor_5":{"attribute_name":"学位授与機関","attribute_value_mlt":[{"subitem_degreegrantor":[{"subitem_degreegrantor_name":"総合研究大学院大学"}]}]},"item_1_degree_name_6":{"attribute_name":"学位名","attribute_value_mlt":[{"subitem_degreename":"博士（理学）"}]},"item_1_description_12":{"attribute_name":"要旨","attribute_value_mlt":[{"subitem_description":"　During the development of the nervous system, neurons extend their axons over a long distance to their targets with the assistance of guidance cues and guidance receptors. Although we have detailed knowledge about the variety of these guidance molecules, little is known about how the distributions of the guidance molecules are controlled in space and time. Immunohistochemical studies have revealed that the spatial distribution of axon guidance receptors is often regulated at sub-cellular levels, or rather at sub-axonal levels. For example, in the Drosophila ventral nerve cords, a repulsive guidance receptor Roundabout and its family members (Robo2 and Robo3) are specifically expressed in the longitudinal axon tracts, which indicates that Robo receptors are localized to the distal segment of the axons but excluded from the proximal segment of the same axons. This restricted expression of Robo within axons is largely conserved from fruit flies to mammals. Moreover, recent findings that receptors can influence the distribution of guidance cues underscore the importance of the regulated expression of guidance receptors. <br />　A fundamental question about such sub-axonal localization of molecules is whether the localization is dependent on extrinsic signals such as cell-cell contacts, or is due to intrinsic properties of neurons. Although it has long been known that neurons possess an intrinsic ability to acquire axonal and somatodendritic domains, the possibility that neurons have an intrinsic ability to generate sub-axonal domains remains to be explored. Another question is how the localization of transmembrane molecules in certain region of the axonal membrane is established and maintained. Molecules should first be asymmetrically delivered to the axonal membrane, and then the asymmetric distribution of the molecules should be retained despite the fact that the axonal membrane is continuous and fluid.<br />　To distinguish between the contributions of extrinsic factors and intrinsic properties on the development of the sub-axonal localization of molecules, I used a low-density primary culture system of Drosophila neurons where neurons extend their axons without cell-cell contacts. I reasoned that if neurons have an intrinsic property to create sub-axonal localization of molecules, localization of Robo receptors could be seen in the cultured neurons. When cultured neurons were immunostained for Robo proteins, cultured neurons exhibited uniform distribution of Robo along their axons, suggesting that Robo may require extrinsic cues for its localization. In contrast, Robo2 and Robo3 were localized to the distal segment of the axons even in cultured neurons. This result indicates that neurons possess an intrinsic property to generate the localization of Robo2 and Robo3.<br />　I next asked how the localization of Robo3 in the distal axon segment is established during the course of axonal elongation. One possibility is that molecules are delivered only to the growing tip of the axon, and the localization pattern along the axon is determined by the temporal pattern of the ON and OFF of the delivery. To examine the course of localization, I performed time-lapse recordings of growing neurons that express Robo3-EGFP fusion proteins. When axons just started to extend, Robo3-EGFP was detected only in cell bodies. At around 8 hrs from the onset of axonal elongation, strong GFP signals appeared at the distal segment of the axons. Localization of Robo3-EGFP at the distal axon segment occurred after axonal elongation but not together with axonal elongation. In addition, when the fluorescent signal of Robo3-EGFP in the distal axon segment was eliminated by intense 488 nm laser illumination, Robo3-EGFP signal reappeared to the distal axon segment without further axonal elongation (13% recovery in 40 minutes). Thus, it is likely that the localization of molecules and axonal elongation are uncoupled processes.<br />　Next, I examined how the localization of transmembrane molecules such as Robo receptors in the axonal membrane is maintained. One possibility is that molecules are immobilized along the axon by being anchored to the membrane cytoskeleton and therefore the localization patterns can be retained. To test this possibility, I assessed the dynamics of molecules that are localized to the distal segment of the axon using a fluorescence recovery after photobleaching (FRAP) method. In FRAP experiments, a discrete region of the cell is photobleached, and then the recovery of fluorescence over time is measured, which gives information about the mobility of molecules. When FRAP analysis of Robo3-EGFP was performed in the distal axon segment, fluorescent signals recovered to the photobleached area in an exponential manner, suggesting that Robo3-EGFP is diffusible along the axon. Thus, anchoring model seems to be insufficient to explain the persisted localization of Robo3 in the axon.<br />　Given that Robo3-EGFP is mobile in the axonal membrane, how is Robo3-EGFP kept from uniformly distributing along the axon? One possibility is that there exists a diffusion barrier between the distal and proximal segments of the axon, and that the barrier prevents Robo3-EGFP from entering into the proximal segment. I reasoned that if there exists a diffusion barrier at the boundary between the distal and proximal segments (Robo3-EGFP localization defines the distal segment), then the mobility of Robo-EGFP across the boundary could also be restricted by the diffusion barrier. In this case, FRAP experiments should result in that fluorescent signals recovering more slowly from the side close to the boundary compared to the side away from the boundary. Conversely, if there is no diffusion barrier at the boundary, fluorescent signals should recover equally from both ends of the photobleached area. When the proximal part of the axon from the boundary was photobleached, less Robo-EGFP signal recovered from the side close to the boundary compared to the side close to the cell body. Thus, it is likely that the mobility of molecules across the boundary of segments is restricted by a diffusion barrier mechanism.<br />　Finally, I tested whether this diffusion barrier acts specifically to Robo family of receptors or acts generally to transmembrane proteins. The mobility of CD8-GFP, a transmembrane molecule that has no similarity to Robo receptors, was tested by FRAP experiments. The mobility of CD8-GFP across the boundary was also restricted. This result suggests that the boundary between the proximal and distal compartments acts as a general diffusion barrier to transmembrane proteins.<br />　Using a primary cell culture system of Drosophila, here I have demonstrated that neurons have an intrinsic property to create the sub-axonal localization of Robo2 and Robo3. FRAP experiments revealed that the mobility of transmembrane proteins was restricted at the boundary between the segments. I propose that neurons possess an intrinsic property to compartmentalize their axons by a diffusion barrier mechanism, and that the compartment boundary regulates the localization of Robo2 and Robo3. The result that Robo was uniformly distributed along the axons in cultured neurons suggests that neurons might use extrinsic factor(s) to generate the localization of Robo. It would be also interesting to examine whether the localization of Robo2 and Robo3 could be further adjusted by extrinsic factors. Robo receptors would provide insights into how intrinsic properties of neurons and extrinsic factors participate in regulating the distribution of guidance receptors.","subitem_description_type":"Other"}]},"item_1_description_7":{"attribute_name":"学位記番号","attribute_value_mlt":[{"subitem_description":"総研大甲第867号","subitem_description_type":"Other"}]},"item_1_select_14":{"attribute_name":"所蔵","attribute_value_mlt":[{"subitem_select_item":"有"}]},"item_1_select_8":{"attribute_name":"研究科","attribute_value_mlt":[{"subitem_select_item":"生命科学研究科"}]},"item_1_select_9":{"attribute_name":"専攻","attribute_value_mlt":[{"subitem_select_item":"18 遺伝学専攻"}]},"item_1_text_10":{"attribute_name":"学位授与年度","attribute_value_mlt":[{"subitem_text_value":"2004"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"KATSUKI, Takeo","creatorNameLang":"en"}],"nameIdentifiers":[{}]}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2016-02-17"}],"displaytype":"simple","filename":"甲867_要旨.pdf","filesize":[{"value":"365.5 kB"}],"format":"application/pdf","licensetype":"license_11","mimetype":"application/pdf","url":{"label":"要旨・審査要旨","url":"https://ir.soken.ac.jp/record/1009/files/甲867_要旨.pdf"},"version_id":"b8d7e195-da6c-4b3a-b6d5-69664d52269c"}]},"item_language":{"attribute_name":"言語","attribute_value_mlt":[{"subitem_language":"eng"}]},"item_resource_type":{"attribute_name":"資源タイプ","attribute_value_mlt":[{"resourcetype":"thesis","resourceuri":"http://purl.org/coar/resource_type/c_46ec"}]},"item_title":"Intrinsic sub-axonal patterning in Drosophila Neurons: Compartment boundaries in axons regulate the localization of Robo receptors","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"Intrinsic sub-axonal patterning in Drosophila Neurons: Compartment boundaries in axons regulate the localization of Robo receptors"},{"subitem_title":"Intrinsic sub-axonal patterning in Drosophila Neurons: Compartment boundaries in axons regulate the localization of Robo receptors","subitem_title_language":"en"}]},"item_type_id":"1","owner":"1","path":["20"],"pubdate":{"attribute_name":"公開日","attribute_value":"2010-02-22"},"publish_date":"2010-02-22","publish_status":"0","recid":"1009","relation_version_is_last":true,"title":["Intrinsic sub-axonal patterning in Drosophila Neurons: Compartment boundaries in axons regulate the localization of Robo receptors"],"weko_creator_id":"1","weko_shared_id":-1},"updated":"2023-06-20T16:09:15.597243+00:00"}