SUMMARY Translation is under tight spatial and temporal controls to ensure protein production in the right time and place in cells. Methods that allow real-time, high-resolution visualization of translation in live cells are essential for understanding the spatiotemporal dynamics of translation regulation. Based on multivalent fluorescence amplification of the nascent polypeptide signal, we develop a method to image translation on individual mRNA molecules in real time in live cells, allowing direct visualization of translation events at the translation sites. Using this approach, we monitor transient changes of translation dynamics in responses to environmental stresses, capture distinct mobilities of individual polysomes in different subcellular compartments, and detect 3’UTR-dependent local translation and active transport of polysomes in dendrites of primary neurons.

Actin, spectrin, and associated molecules form a membraneassociated periodic skeleton (MPS) in neurons. In the MPS, short actin filaments, capped by actin-capping proteins, form ring-like structures that wrap around the circumference of neurites, and these rings are periodically spaced along the neurite by spectrin tetramers, forming a quasi-1D lattice structure. This 1D MPS structure was initially observed in axons and exists extensively in axons, spanning nearly the entire axonal shaft of mature neurons. Such 1D MPS was also observed in dendrites, but the extent to which it exists and how it develops in dendrites remain unclear. It is also unclear whether other structural forms of the membrane skeleton are present in neurons. Here, we investigated the spatial organizations of spectrin, actin, and adducin, an actin-capping protein, in the dendrites and soma of cultured hippocampal neurons at different developmental stages, and compared results with those obtained in axons, using superresolution imaging. We observed that the 1D MPS exists in a substantial fraction of dendritic regions in relatively mature neurons, but this structure develops slower and forms with a lower propensity in dendrites than in axons. In addition, we observed that spectrin, actin, and adducin also form a 2D polygonal lattice structure, resembling the expanded erythrocyte membrane skeleton structure, in the somatodendritic compartment. This 2D lattice structure also develops substantially more slowly in the soma and dendrites than the development of the 1D MPS in axons. These results suggest membrane skeleton structures are differentially regulated across different subcompartments of neurons.actin | spectrin | adducin | super-resolution imaging | STORM I t was recently discovered that actin, spectrin, and associated molecules form a membrane-associated periodic skeleton (MPS) structure in neurons (1). As revealed by superresolution stochastic optical reconstruction microscopy (STORM) (1), this structure contains molecules homologous to those present in the erythrocyte membrane skeleton, including spectrin (2-4), actin, and actincapping proteins such as adducin (5, 6), but adopts a structural organization that is distinct from the polygonal lattice structure of the erythrocyte membrane skeleton (7,8). In this structure, short actin filaments capped by adducin are organized into repetitive, ring-like structures that wrap around the circumference of the axon underneath the axonal membrane, and adjacent actin rings are connected by spectrin tetramers, forming a long-range quasi-1D periodic structure with a periodicity of ∼190 nm (1). This periodic structure is formed extensively throughout the axonal shaft, including the axon initial segment and the unmyelinated distal axons, as well as the nodes of Ranvier and internodal segments in myelinated axons (1, 9-14), but appears to be perturbed at synaptic sites (13,15,16). It is a highly prevalent structure observed in many different types of neurons, including both excitatory and inhibitory neuro...

Actin, spectrin, and associated molecules form a periodic, submembrane cytoskeleton in the axons of neurons. For a better understanding of this membrane-associated periodic skeleton (MPS), it is important to address how prevalent this structure is in different neuronal types, different subcellular compartments, and across different animal species. Here, we investigated the organization of spectrin in a variety of neuronal-and glial-cell types. We observed the presence of MPS in all of the tested neuronal types cultured from mouse central and peripheral nervous systems, including excitatory and inhibitory neurons from several brain regions, as well as sensory and motor neurons. Quantitative analyses show that MPS is preferentially formed in axons in all neuronal types tested here: Spectrin shows a long-range, periodic distribution throughout all axons but appears periodic only in a small fraction of dendrites, typically in the form of isolated patches in subregions of these dendrites. As in dendrites, we also observed patches of periodic spectrin structures in a small fraction of glial-cell processes in four types of glial cells cultured from rodent tissues. Interestingly, despite its strong presence in the axonal shaft, MPS is disrupted in most presynaptic boutons but is present in an appreciable fraction of dendritic spine necks, including some projecting from dendrites where such a periodic structure is not observed in the shaft. Finally, we found that spectrin is capable of adopting a similar periodic organization in neurons of a variety of animal species, including Caenorhabditis elegans, Drosophila, Gallus gallus, Mus musculus, and Homo sapiens.ctin is critically involved in the regulation of neuronal polarization, differentiation, and growth of neuronal processes, cargo trafficking, and plasticity of synapses (1-3). Spectrin is an actin-binding protein that is important for the development and stabilization of axons and maintenance of neuronal polarization (4-6). In Caenorhabditis elegans, spectrin is important for the stability and integrity of axons under mechanical stress (4, 6) and for mechanosensation (6), and spectrin depletion results in axon breakage during animal locomotion (4). In Drosophila, spectrin has been shown to be involved in axonal path finding (7) and stabilization of presynaptic terminals (8). In mice, spectrin null mutations are embryonically lethal, and neurons with spectrin knockdown display defects in axonal initial segment assembly (5, 9, 10).Actin and spectrin form a 2D polygonal lattice structure underneath the membrane of erythrocytes (11). Recently, a novel form of actin-spectrin-based submembrane skeleton structure was discovered in neuronal axons (12) using superresolution STORM imaging (13,14). This membrane-associated periodic skeleton (MPS) has been observed in both fixed and live cultured neurons (12, 15, 16) and in brain tissue sections (12). In this structure, short actin filaments are organized into repetitive, ring-like structures that wrap around the circumference o...