Маркировка F-актин Колючая Заканчивается родамин-актина в проницаемыми нейронов конусы роста
Summary
Метод для визуализации и количественной оценки F-актин колючей заканчивается в нейронных конусы роста описывается. После культивирования нейронов на стекле покровные, клетки с проницаемыми сапонина содержащего решение. Затем, короткий инкубационный с буфера, содержащего сапонин родамин-актина включает в себя люминесцентные актина на свободные актина колючей концах.
Abstract
The motile tips of growing axons are called growth cones. Growth cones lead navigating axons through developing tissues by interacting with locally expressed molecular guidance cues that bind growth cone receptors and regulate the dynamics and organization of the growth cone cytoskeleton3-6. The main target of these navigational signals is the actin filament meshwork that fills the growth cone periphery and that drives growth cone motility through continual actin polymerization and dynamic remodeling7. Positive or attractive guidance cues induce growth cone turning by stimulating actin filament (F-actin) polymerization in the region of the growth cone periphery that is nearer the source of the attractant cue. This actin polymerization drives local growth cone protrusion, adhesion of the leading margin and axonal elongation toward the attractant.
Actin filament polymerization depends on the availability of sufficient actin monomer and on polymerization nuclei or actin filament barbed ends for the addition of monomer. Actin monomer is abundantly available in chick retinal and dorsal root ganglion (DRG) growth cones. Consequently, polymerization increases rapidly when free F-actin barbed ends become available for monomer addition. This occurs in chick DRG and retinal growth cones via the local activation of the F-actin severing protein actin depolymerizing factor (ADF/cofilin) in the growth cone region closer to an attractant8-10. This heightened ADF/cofilin activity severs actin filaments to create new F-actin barbed ends for polymerization. The following method demonstrates this mechanism. Total content of F-actin is visualized by staining with fluorescent phalloidin. F-actin barbed ends are visualized by the incorporation of rhodamine-actin within growth cones that are permeabilized with the procedure described in the following, which is adapted from previous studies of other motile cells11, 12. When rhodamine-actin is added at a concentration above the critical concentration for actin monomer addition to barbed ends, rhodamine-actin assembles onto free barbed ends. If the attractive cue is presented in a gradient, such as being released from a micropipette positioned to one side of a growth cone, the incorporation of rhodamine-actin onto F-actin barbed ends will be greater in the growth cone side toward the micropipette10.
Growth cones are small and delicate cell structures. The procedures of permeabilization, rhodamine-actin incorporation, fixation and fluorescence visualization are all carefully done and can be conducted on the stage of an inverted microscope. These methods can be applied to studying local actin polymerization in migrating neurons, other primary tissue cells or cell lines.
Protocol
Discussion
Методы, представленные здесь, позволяют временным и пространственным разрешением клеточных компонентов, которые участвуют в динамической реконструкции актина цитоскелета на переднем крае миграции роста конусов. Действие аттрактанта молекул, как и NGF или netrin, быстро стимулировать по?…
Declarações
The authors have nothing to disclose.
Acknowledgements
Авторы выражают благодарность д-р Джеймс Bamburg и члены его лаборатории для сотрудничества в этих исследованиях. Эта работа финансировалась за счет грантов NIH HD19950, EY07133 и за счет субсидий из Миннесоты Медицинский фонд.
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| 18×18 mm coverslip | Gold Seal | 3305 | ||
| 35mm Petri dishes | Falcon | 351008 | ||
| Aquarium cement | DAP 100% silicone aquarium sealant | Any hardware store | ||
| Poly-D-lysine (mw > 300,000) | Sigma | P1024 | ||
| Natural mouse laminin | Invitrogen | 23017-015 | ||
| L1 CAM | R & D systems | 777-NC | ||
| Alexa-fluor 350 phalloidin | Invitrogen (Molecular Probes) | A22281 | ||
| Rhodamine non-muscle actin | Cytoskeleton, Inc. | APHR-A | ||
| F12 Culture medium | Invitrogen (Gibco) | 21700-075 | ||
| B27 | Invitrogen (Gibco) | 17504-044 | ||
| Slowfade | Invitrogen | 536937 |
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