Bidirectional actin transport is influenced by microtubule and actin stability.

Local and long-distance transport of cytoskeletal proteins is vital to neuronal maintenance and growth. Though recent progress has provided insight into the movement of microtubules and neurofilaments, mechanisms underlying the movement of actin remain elusive, in large part due to rapid transitions between its filament states and its diverse cellular localization and function. In this work, we integrated live imaging of rat sensory neurons, image processing, multiple regression analysis, and mathematical modeling to perform the first quantitative, high-resolution investigation of GFP-actin identity and movement in individual axons. Our data revealed that filamentous actin densities arise along the length of the axon and move short but significant distances bidirectionally, with a net anterograde bias. We directly tested the role of actin and microtubules in this movement. We also confirmed a role for actin densities in extension of axonal filopodia, and demonstrated intermittent correlation of actin and mitochondrial movement. Our results support a novel mechanism underlying slow component axonal transport, in which the stability of both microtubule and actin cytoskeletal components influence the mobility of filamentous actin.

A comparative quantitative assessment of axonal and dendritic mRNA transport in maturing hippocampal neurons.

Translation of mRNA in axons and dendrites enables a rapid supply of proteins to specific sites of localization within the neuron. Distinct mRNA-containing cargoes, including granules and mitochondrial mRNA, are transported within neuronal projections. The distributions of these cargoes appear to change during neuronal development, but details on the dynamics of mRNA transport during these transitions remain to be elucidated. For this study, we have developed imaging and image processing methods to quantify several transport parameters that can define the dynamics of RNA transport and localization. Using these methods, we characterized the transport of mitochondrial and non-mitochondrial mRNA in differentiated axons and dendrites of cultured hippocampal neurons varying in developmental maturity. Our results suggest differences in the transport profiles of mitochondrial and non-mitochondrial mRNA, and differences in transport parameters at different time points, and between axons and dendrites. Furthermore, within the non-mitochondrial mRNA pool, we observed two distinct populations that differed in their fluorescence intensity and velocity. The net axonal velocity of the brighter pool was highest at day 7 (0.002±0.001 µm/s, mean ± SEM), raising the possibility of a presynaptic requirement for mRNA during early stages of synapse formation. In contrast, the net dendritic velocity of the brighter pool increased steadily as neurons matured, with a significant difference between day 12 (0.0013±0.0006 µm/s ) and day 4 (-0.003±0.001 µm/s) suggesting a postsynaptic role for mRNAs in more mature neurons. The dim population showed similar trends, though velocities were two orders of magnitude higher than of the bright particles. This study provides a baseline for further studies on mRNA transport, and has important implications for the regulation of neuronal plasticity during neuronal development and in response to neuronal injury.

A novel algorithm to generate kymographs from dynamic axons for the quantitative analysis of axonal transport.

The biological and clinical relevance of axonal transport has driven the development of a variety of new approaches to its study, including the generation of fluorescence or brightfield movies of moving cargoes within axons. Kymograph analysis is a simple and effective tool used to analyze axonal transport in neurons. Typically, kymographs are built by having a user trace the path of the axon in one frame of a time-lapse movie and extracting intensity profiles from subsequent frames along that path. This method cannot accommodate movies in which translation of the axon, or changes in axonal orientation or geometry, occur. Both are frequently observed in long-term movies of neurons, both in vitro and in vivo. To solve this problem and automate the creation of kymographs from these movies, we developed a two step algorithm. The first step implemented a simple image registration algorithm that aligned axons based on identification of a reference point on the axon in each image. The second step used a Hough transformation (HT) to automatically detect the axonal contour in each frame. Intensity profiles along this contour were then used to construct a kymograph. This algorithm was able to build an accurate kymograph of mitochondrial and actin transport in dynamic cultured sensory neurons, which were not amenable to previously used analytical methods. Although developed as a tool for analyzing transport, this algorithm is easily modified to analyze movies for the directionality and speed of axonal outgrowth, another metric of interest to neuroscientists.

Porous EH and EH-PEG scaffolds as gene delivery vehicles to skeletal muscle.

PURPOSE: Synthetic biomaterials are widely used in an attempt to control the cellular behavior of regenerative tissues. This can be done by altering the chemical and physical properties of the polymeric scaffold to guide tissue repair. This paper addresses the use of a polymeric scaffold (EH network) made from the cyclic acetal monomer, 5-ethyl-5-(hydroxymethyl)-ß,ß-dimethyl-1,3-dioxane-2-ethanol diacrylate (EHD), as a release device for a therapeutic plasmid encoding for an insulin-like growth factor-1 green fluorescent protein fusion protein (IGF-1 GFP). METHODS: Scaffolds were designed to have different porous architectures, and the impact of these architectures on plasmid release was determined. We hypothesized that IGF-1 could be delivered more effectively using a porous scaffold to allow for the release of IGF-1. RESULTS: We showed that by altering the number of pores exposed to the surface of the network, faster plasmid loading and release were achieved. In addition, the IGF-1 GFP plasmids were found to be effective in producing IGF-1 and GFP within human skeletal muscle myoblast cell cultures. CONCLUSIONS: This work aims to show the utility of EH biomaterials for plasmid delivery for potentially localized skeletal muscle regeneration.

Cytoskeletal dynamics in response to tensile loading of mammalian axons.

In response to an applied tensile load, axons of cultured neurons exhibit a number of morphological responses. We designed and implemented a cell stretching device to study the cellular mechanisms governing these responses. Rat sensory neurons were seeded onto a flexible silicone substrate and imaged during substrate stretch. The positions of stationary mitochondria, docked to the axonal cytoskeleton, were determined before and after 10% stretch, and used to calculate the resulting "instantaneous" strain in regions of the axon. There was dramatic heterogeneity in strain along the length of the stretched axons, particularly in regions shorter than 20 µm. The substrate was then held at 10% strain and the axons imaged for 20 min during "relaxation." Both strain magnitude and variability were larger at small lengths in stretched axons during the initial phase of relaxation, but after 14 min, decreased to levels smaller than those seen in unstretched axons. Mitochondrial pairs in stretched axons showed uncoordinated movement with each other at all lengths, suggesting that cytoskeletal cohesion is reduced after stretch. Collectively, these data present the axonal cytoskeleton as a dynamic structure, which responds to stretch rapidly and locally. Globally, the axon behaves as a viscoelastic continuum. Below a characteristic length, though, it appears to behave as a series of independent linked elements, each with unique mechanical properties which suggests a length scale within which cytoskeletal structural elements may be altered to modulate the biomechanical response of the axon. Finally, testable hypotheses of strain accomodation in the axon are suggested.