Research Article
Single-particle fluorescence tracking combined with TrackMate assay revealshighly heterogeneous and discontinuouslysosomal transport in freely orientated axons
Yongyang Liu1,2,#, Yaxin Lu1,2,#, Zhiyong Tang1,2, Yuheng Cao2,3, Dehua Huang1, Feng Wu1, Yejun Zhang1, Chunyan Li1,2, Guangcun Chen1,2,*, Qiangbin Wang1,2,3,4
1School of Nano-Tech and Nano-Bionics, University of Science and Technology of China, Hefei 230026, China
2CAS Key Laboratory of Nano-Bio Interface, Suzhou Key Laboratory of Functional Molecular Imaging Technology, Division of Nanobiomedicine and i -Lab, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123, China
3School of Physical Science and Technology, ShanghaiTech University, Shanghai 201210, China
4College of Materials Sciences and Opto-Electronic Technology, University of Chinese Academy of Sciences, Beijing 100049, China
#These authors contributed equally to this work.
Correspondence: Prof. Guangcun Chen, Division of Nanobiomedicine and i -Lab, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123, China.
E-mail: gcchen2011@sinano.ac.cn
Keywords: fluorescence imaging, axonal transport, lysosomal transport, TrackMate, single-particle tracking
Abstract
Axonal transport plays a significant role in the establishment of neuronal polarity, axon growth, and synapse formation during neuronal development. The axon of a naturally growing neuron is a highly complex and multifurcated structure with a large number of bends and branches. Nowadays, the study of dynamic axonal transport in morphologically complex neurons is greatly limited by the technological barrier. Here, a sparse gene transfection strategy was developed to locate fluorescent mCherry in the lysosome of primary neurons, thus enabling us to track the lysosome-based axonal transport with a single-particle resolution. Thereby, several axonal transport models were observed, including forward or backward transport model, stop-and-go model, repeated back-and-forth transport model, and cross-branch transport model. Then, the accurate single-particle velocity quantification by TrackMate revealed a highly heterogeneous and discontinuous transportation process of lysosome-based axonal transport in freely orientated axons. And, multiple physical factors, such as the axonal structure and the size of particles, were disclosed to affect the velocity of particle transporting in freely orientated axons. The combined single-particle fluorescence tracking and TrackMate assay can be served as a facile tool for evaluating axonal transport in neuronal development and axonal transport-related diseases.
1. Introduction
Axonal transport serves as a cytoplasm trafficker during neuronal development, playing a significant role in the establishment of neuronal polarity, axon growth and stability, and synapse formation.[1] The cytoplasm trafficker represents cargoes varying from every type of membranous organelle and vesicles such as lysosome, mitochondria, endosome, to non-membranous cargoes like cytosolic protein complexes, mRNAs, cytoskeletal polymers, and ribosomes.[2-4]Lysosomes have long been viewed as housekeeping organelles responsible for endocytic and autophagic component degradation, thus maintaining cellular homeostasis in the neuron. The latest research indicates that a small rat sarcoma virus-relatedĀ GTPase (Rab2) mediates dense core vesicles biogenesis and endosome-lysosome fusion,[5]via Rab2, activated by the upstream regulator endosomal membrane protein, directly or indirectly assisting lysosomal motility factor Arl8,[6]activated by the upstream regulator BORC,[7] to recruit kinesin motor proteins.[8]Moreover, not like any other organelle transport, lysosomes are found in all neuronal domains, such as the soma, dendrites, and axon. How lysosomes are transported in these domains, however, has not been fully understood.
For a long time, the study of axonal transport-associated dynamic process in multifurcated neuron has greatly limited by the technological barrier.[9] Generally, to simplify the axonal transport model, neurons were cultured in a type of microchannel, which was pre-processed into a linear groove with a nanometer width to guide the axon to elongate straight along the groove.[10] Though it is convenient to observe a relatively stable transport along the linear axon, the related conclusion is not enough to be applied to multifurcated complex axons. If this kind of freely orientated multifurcated neuron is cultured on a coverslip pre-treated with cell adhesion molecules, then no space limitation will exist while growth cones move ahead,[11] which provides a naturally morphogenetic neuron model for studying the mechanism of complex axonal transport. Due to the highly complex structure of freely orientated axons with a large number of bends and branches, evaluating the axonal transport principle concerning cargoes passing by the complex axon structure and axon branch point is still challenging.[12,13] Previously, the researcher mainly gathers information via imaging those linear axons and extract data by the means of analyzing fluorescence intensity[14] or kymograph tracking,[15,16] which is not enough to reveal quantitative motions of axonal transport from the point of anfractuous tracking and instantaneous velocity analysis. Thus, optimized imaging as well as data analysis techniques are still needed to understand axonal transport in freely orientated axons.
In this study, a sparse gene transfection strategy was developed to express and locate fluorescent mCherry in the lysosome of primary neurons. In this way, a single neuron with a mature axon structure can be visualized in the neural network culture derived from the primary neurons. Moreover, the mCherry protein-loaded lysosome can offer a good signal-to-noise ratio for tracking the lysosome-based axonal transport with a single-particle resolution, thus offering a possibility for imaging axonal transport in freely orientated axons. Then, a concise and accurate quantitative algorithm of single-particle velocity, TrackMate, was applied to evaluate the axon transport modes and their reasonable biological interpretations. The combined single-particle tracking and accurate velocity assay revealed a highly heterogeneous and discontinuous transportation process in freely orientated axons. Furthermore, it was also disclosed that multiple physical factors affected the velocity of particle transporting, including the axonal structure and particle size. We believe that combining the advanced single-particle tracking strategy with a flexible and accurate particle velocity analysis will promote a comprehensive understanding of the freely orientated axonal transport mechanism and its biological functions during neuronal development.
2. Material and Methods