In the conventional fluorescence images ( Fig.
To identify axons and dendrites, we immunolabeled MAP2, a microtubule associate protein enriched in dendrites, or NrCAM, a cell adhesion molecule found in the initial segments of axons ( 15), using a dye of a different color ( Fig. To image actin in neurons, we fixed cultured rat hippocampal neurons at various days in vitro (DIV), and labeled actin filaments with phalloidin conjugated to a photoswitchable dye, Alexa Fluor 647 ( Fig. In this work, we studied the three-dimensional (3D) ultrastructural organization of actin and spectrin in neurons using a super-resolution fluorescence imaging method, stochastic optical reconstruction microscopy (STORM) ( 23– 27). In particular, super-resolution studies of neurons have provided valuable structural and dynamic information of actin in dendritic spines ( 19– 22). Recent advances in super-resolution fluorescence microscopy ( 17, 18) allow resolutions down to ~10 nm to be achieved with molecular specificity, providing a promising solution to the above challenges. However, the ultrastructural organization of spectrin in non-erythrocyte cells is largely unknown due to similar challenges in imaging. An erythrocyte-like, polygonal lattice structure has been observed for spectrin in the drosophila neuromuscular junction ( 16), and models similar to the erythrocyte cytoskeleton have also been proposed for other systems ( 10). They play important roles, ranging from the regulation of the heartbeat, to the stabilization of axons, the formation of axon initial segments and nodes of Ranvier, and the stabilization of synapses in neurons ( 9, 10, 15). Spectrin analogues have been found in many other animal cells ( 9, 10), including neurons ( 13, 14). Hence, resolving the organization of actin in axons and dendrites is challenging, requiring imaging tools with both high spatial resolution and molecular specificity.Ī prototypical actin-spectrin-based cytoskeleton structure is found in red blood cells (erythrocytes) ( 9, 10), where actin, spectrin and associated proteins form a two-dimensional (2D) polygonal network (mostly comprised of hexagons and pentagons) underneath the erythrocyte membrane ( 11, 12). Though neurites often have sub-micrometer diameters, they contain a high density of different types of cytoskeletal filaments, such as microtubules and neurofilaments ( 6– 8). Electron microscopy has provided detailed actin ultrastructure in growth cones and dendritic spines ( 5, 6), where actin is the dominant cytoskeletal protein, but little is known about the organization of actin in the axonal and dendritic shafts ( 4). Despite its importance, our understanding of actin structures in neurons remains incomplete.
In neurons, actin is essential for the establishment of neuronal polarity, the transport of cargos, the growth of neurites, and the stabilization of synaptic structures ( 2– 4). Actin plays critical roles in shaping and maintaining cell morphology, as well as in supporting cellular various functions, including cell motility, cell division, and intracellular transport ( 1).
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