Rescue of infectious particles from preassembled alphavirus nucleocapsid cores.

Alphaviruses are small, spherical, enveloped, positive-sense, single-stranded, RNA viruses responsible for considerable human and animal disease. Using microinjection of preassembled cores as a tool, a system has been established to study the assembly and budding process of Sindbis virus, the type member of the alphaviruses. We demonstrate the release of infectious virus-like particles from cells expressing Sindbis virus envelope glycoproteins following microinjection of Sindbis virus nucleocapsids purified from the cytoplasm of infected cells. Furthermore, it is shown that nucleocapsids assembled in vitro mimic those isolated in the cytoplasm of infected cells with respect to their ability to be incorporated into enveloped virions following microinjection. This system allows for the study of the alphavirus budding process independent of an authentic infection and provides a platform to study viral and host requirements for budding.

Different lineage contexts direct common pro-neural factors to specify distinct retinal cell subtypes.

How astounding neuronal diversity arises from variable cell lineages in vertebrates remains mostly elusive. By in vivo lineage tracing of ∼1,000 single zebrafish retinal progenitors, we identified a repertoire of subtype-specific stereotyped neurogenic lineages. Remarkably, within these stereotyped lineages, GABAergic amacrine cells were born with photoreceptor cells, whereas glycinergic amacrine cells were born with OFF bipolar cells. More interestingly, post-mitotic differentiation blockage of GABAergic and glycinergic amacrine cells resulted in their respecification into photoreceptor and bipolar cells, respectively, suggesting lineage constraint in cell subtype specification. Using single-cell RNA-seq and ATAC-seq analyses, we further identified lineage-specific progenitors, each defined by specific transcription factors that exhibited characteristic chromatin accessibility dynamics. Finally, single pro-neural factors could specify different neuron types/subtypes in a lineage-dependent manner. Our findings reveal the importance of lineage context in defining neuronal subtypes and provide a demonstration of in vivo lineage-dependent induction of unique retinal neuron subtypes for treatment purposes.

Topography and nanomechanics of live neuronal growth cones analyzed by atomic force microscopy.

Neuronal growth cones are motile structures located at the end of axons that translate extracellular guidance information into directional movements. Despite the important role of growth cones in neuronal development and regeneration, relatively little is known about the topography and mechanical properties of distinct subcellular growth cone regions under live conditions. In this study, we used the AFM to study the P domain, T zone, and C domain of live Aplysia growth cones. The average height of these regions was calculated from contact mode AFM images to be 183 +/- 33, 690 +/- 274, and 1322 +/- 164 nm, respectively. These findings are consistent with data derived from dynamic mode images of live and contact mode images of fixed growth cones. Nano-indentation measurements indicate that the elastic moduli of the C domain and T zone ruffling region ranged between 3-7 and 7-23 kPa, respectively. The range of the measured elastic modulus of the P domain was 10-40 kPa. High resolution images of the P domain suggest its relatively high elastic modulus results from a dense meshwork of actin filaments in lamellipodia and from actin bundles in the filopodia. The increased mechanical stiffness of the P and T domains is likely important to support and transduce tension that develops during growth cone steering.

Cortactin colocalizes with filopodial actin and accumulates at IgCAM adhesion sites in Aplysia growth cones.

Both IgCAMs and the actin cytoskeleton play critical roles in neuronal growth cone motility and guidance. However, it is unclear how IgCAM receptors transduce signals from the plasma membrane to induce actin remodeling. Previous studies have shown that local clustering and immobilization of apCAM, the Aplysia homolog of NCAM, induces Src kinase activity and F-actin polymerization in the peripheral domain of cultured Aplysia bag cell growth cones. Therefore, we wanted to test whether the Src kinase substrate and actin regulator cortactin could be a molecular link between Src activity and actin assembly during apCAM-mediated growth cone guidance. Here, we cloned Aplysia cortactin and showed that it is abundant in the nervous system. Immunostaining of growth cones revealed a strong colocalization of cortactin with F-actin in filopodial bundles and at the leading edge of lamellipodia. Perturbation of the cytoskeleton indicated that cortactin distribution largely depends on actin filaments. Furthermore, active Src colocalized with cortactin in regions of actin assembly, including leading edge and filopodia tips. Finally, we observed that cortactin, like F-actin, localizes to apCAM adhesion sites mediating growth cone guidance. Altogether, these data suggest that cortactin is a mediator of IgCAM-triggered actin assembly involved in growth cone motility and guidance.

A low-cost microwell device for high-resolution imaging of neurite outgrowth in 3D.

OBJECTIVE: Current neuronal cell culture is mostly performed on two-dimensional (2D) surfaces, which lack many of the important features of the native environment of neurons, including topographical cues, deformable extracellular matrix, and spatial isotropy or anisotropy in three dimensions. Although three-dimensional (3D) cell culture systems provide a more physiologically relevant environment than 2D systems, their popularity is greatly hampered by the lack of easy-to-make-and-use devices. We aim to develop a widely applicable 3D culture procedure to facilitate the transition of neuronal cultures from 2D to 3D. APPROACH: We made a simple microwell device for 3D neuronal cell culture that is inexpensive, easy to assemble, and fully compatible with commonly used imaging techniques, including super-resolution microscopy. MAIN RESULTS: We developed a novel gel mixture to support 3D neurite regeneration of Aplysia bag cell neurons, a system that has been extensively used for quantitative analysis of growth cone dynamics in 2D. We found that the morphology and growth pattern of bag cell growth cones in 3D culture closely resemble the ones of growth cones observed in vivo. We demonstrated the capability of our device for high-resolution imaging of cytoskeletal and signaling proteins as well as organelles. SIGNIFICANCE: Neuronal cell culture has been a valuable tool for neuroscientists to study the behavior of neurons in a controlled environment. Compared to 2D, neurons cultured in 3D retain the majority of their native characteristics, while offering higher accessibility, control, and repeatability. We expect that our microwell device will facilitate a wider adoption of 3D neuronal cultures to study the mechanisms of neurite regeneration.

Src and cortactin promote lamellipodia protrusion and filopodia formation and stability in growth cones.

Src tyrosine kinases have been implicated in axonal growth and guidance; however, the underlying cellular mechanisms are not well understood. Specifically, it is unclear which aspects of actin organization and dynamics are regulated by Src in neuronal growth cones. Here, we investigated the function of Src2 and one of its substrates, cortactin, in lamellipodia and filopodia of Aplysia growth cones. We found that up-regulation of Src2 activation state or cortactin increased lamellipodial length, protrusion time, and actin network density, whereas down-regulation had opposite effects. Furthermore, Src2 or cortactin up-regulation increased filopodial density, length, and protrusion time, whereas down-regulation promoted lateral movements of filopodia. Fluorescent speckle microscopy revealed that rates of actin assembly and retrograde flow were not affected in either case. In summary, our results support a model in which Src and cortactin regulate growth cone motility by increasing actin network density and protrusion persistence of lamellipodia by controlling the state of actin-driven protrusion versus retraction. In addition, both proteins promote the formation and stability of actin bundles in filopodia.

Differential requirement of F-actin and microtubule cytoskeleton in cue-induced local protein synthesis in axonal growth cones.

BACKGROUND: Local protein synthesis (LPS) via receptor-mediated signaling plays a role in the directional responses of axons to extrinsic cues. An intact cytoskeleton is critical to enact these responses, but it is not known whether the two major cytoskeletal elements, F-actin and microtubules, have any roles in regulating axonal protein synthesis. RESULTS: Here, we show that pharmacological disruption of either microtubules or actin filaments in growth cones blocks netrin-1-induced de novo synthesis of proteins, as measured by metabolic incorporation of labeled amino acids, implicating both elements in axonal synthesis. However, comparative analysis of the activated translation initiation regulator, eIF4E-BP1, revealed a striking difference in the point of action of the two elements: actin disruption completely inhibited netrin-1-induced eIF4E-BP1 phosphorylation while microtubule disruption had no effect. An intact F-actin, but not microtubule, cytoskeleton was also required for netrin-1-induced activation of the PI3K/Akt/mTOR pathway, upstream of translation initiation. Downstream of translation initiation, microtubules were required for netrin-1-induced activation of eukaryotic elongation factor 2 kinase (eEF2K) and eEF2. CONCLUSIONS: Taken together, our results show that while actin and microtubules are both crucial for cue-induced axonal protein synthesis, they serve distinct roles with F-actin being required for the initiation of translation and microtubules acting later at the elongation step.

Coupling of NF-protocadherin signaling to axon guidance by cue-induced translation.

Cell adhesion molecules and diffusible cues both regulate axon pathfinding, yet how these two modes of signaling interact is poorly understood. The homophilic cell adhesion molecule NF-protocadherin (NFPC) is expressed in the mid-dorsal optic tract neuroepithelium and in the axons of developing retinal ganglion cells (RGC) in Xenopus laevis. Here we report that targeted disruption of NFPC function in RGC axons or the optic tract neuroepithelium results in unexpectedly localized pathfinding defects at the caudal turn in the mid-optic tract. Semaphorin 3A (Sema3A), which lies adjacent to this turn, stimulates rapid, protein synthesis-dependent increases in growth cone NFPC and its cofactor, TAF1, in vitro. In vivo, growth cones exhibit marked increases in NFPC translation reporter activity in this mid-optic tract region that are attenuated by blocking neuropilin-1 function. Our results suggest that translation-linked coupling between regionally localized diffusible cues and cell adhesion can help axons navigate discrete segments of the pathway.

Neuronal cell cultures from aplysia for high-resolution imaging of growth cones.

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones.

Quantitative analysis of microtubule dynamics during adhesion-mediated growth cone guidance.

During adhesion-mediated neuronal growth cone guidance microtubules undergo major rearrangements. However, it is unknown whether microtubules extend to adhesion sites because of changes in plus-end polymerization and/or translocation dynamics, because of changes in actin-microtubule interactions, or because they follow the reorganization of the actin cytoskeleton. Here, we used fluorescent speckle microscopy to directly quantify microtubule and actin dynamics in Aplysia growth cones as they turn towards beads coated with the cell adhesion molecule apCAM. During the initial phase of adhesion formation, dynamic microtubules in the peripheral domain preferentially explore apCAM-beads prior to changes in growth cone morphology and retrograde actin flow. Interestingly, these early microtubules have unchanged polymerization rates but spend less time in retrograde translocation due to uncoupling from actin flow. Furthermore, microtubules exploring the adhesion site spend less time in depolymerization. During the later phase of traction force generation, the central domain advances and more microtubules in the peripheral domain extend because of attenuation of actin flow and clearance of F-actin structures. Microtubules in the transition zone and central domain, however, translocate towards the adhesion site in concert with actin arcs and bundles, respectively. We conclude that adhesion molecules guide neuronal growth cones and underlying microtubule rearrangements largely by differentially regulating microtubule-actin coupling and actin movements according to growth cone region and not by controlling plus-end polymerization rates.

High-resolution analysis of neuronal growth cone morphology by comparative atomic force and optical microscopy.

Neuronal growth cones are motile sensory structures at the tip of axons, transducing guidance information into directional movements towards target cells. The morphology and dynamics of neuronal growth cones have been well characterized with optical techniques; however, very little quantitative information is available on the three-dimensional structure and mechanical properties of distinct subregions. In the present study, we imaged the large Aplysia growth cones after chemical fixation with the atomic force microscope (AFM) and directly compared our data with images acquired by light microscopy methods. Constant force imaging in contact mode in combination with force-distant measurements revealed an average height of 200 nm for the peripheral (P) domain, 800 nm for the transition (T) zone, and 1200 nm for the central (C) domain, respectively. The AFM images show that the filopodial F-actin bundles are stiffer than surrounding F-actin networks. Enlarged filopodia tips are 60 nm higher than the corresponding shafts. Measurements of the mechanical properties of the specific growth cone regions with the AFM revealed that the T zone is stiffer than the P and the C domain. Direct comparison of AFM and optical data acquired by differential interference contrast and fluorescence microscopy revealed a good correlation between these imaging methods. However, the AFM provides height and volume information at higher resolution than fluorescence methods frequently used to estimate the volume of cellular compartments. These findings suggest that AFM measurements on live growth cones will provide a quantitative understanding of how proteins can move between different growth cone regions.