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