5 research outputs found
<i>In Vivo</i> Fast Nonlinear Microscopy Reveals Impairment of Fast Axonal Transport Induced by Molecular Motor Imbalances in the Brain of Zebrafish Larvae
Cargo transport by molecular motors along microtubules
is essential
for the function of eukaryotic cells, in particular neurons in which
axonal transport defects constitute the early pathological features
of neurodegenerative diseases. Mainly studied in motor and sensory
neurons, axonal transport is still difficult to characterize in neurons
of the brain in absence of appropriate in vivo tools.
Here, we measured fast axonal transport by tracing the second harmonic
generation (SHG) signal of potassium titanyl phosphate (KTP) nanocrystals
(nanoKTP) endocytosed by brain neurons of zebrafish (Zf) larvae. Thanks
to the optical translucency of Zf larvae and to the perfect photostability
of nanoKTP SHG, we achieved a high scanning speed of 20 frames (of
≈90 μm × 60 μm size) per second in Zf brain.
We focused our study on endolysosomal vesicle transport in axons of
known polarization, separately analyzing kinesin and dynein motor-driven
displacements. To validate our assay, we used either loss-of-function
mutations of dynein or kinesin 1 or the dynein inhibitor dynapyrazole
and quantified several transport parameters. We successfully demonstrated
that dynapyrazole reduces the nanoKTP mobile fraction and retrograde
run length consistently, while the retrograde run length increased
in kinesin 1 mutants. Taking advantage of nanoKTP SHG directional
emission, we also quantified fluctuations of vesicle orientation.
Thus, by combining endocytosis of nanocrystals having a nonlinear
response, fast two-photon microscopy, and high-throughput analysis,
we are able to finely monitor fast axonal transport in vivo in the brain of a vertebrate and reveal subtle axonal transport
alterations. The high spatiotemporal resolution achieved in our model
may be relevant to precisely investigate axonal transport impairment
associated with disease models
<i>In Vivo</i> Fast Nonlinear Microscopy Reveals Impairment of Fast Axonal Transport Induced by Molecular Motor Imbalances in the Brain of Zebrafish Larvae
Cargo transport by molecular motors along microtubules
is essential
for the function of eukaryotic cells, in particular neurons in which
axonal transport defects constitute the early pathological features
of neurodegenerative diseases. Mainly studied in motor and sensory
neurons, axonal transport is still difficult to characterize in neurons
of the brain in absence of appropriate in vivo tools.
Here, we measured fast axonal transport by tracing the second harmonic
generation (SHG) signal of potassium titanyl phosphate (KTP) nanocrystals
(nanoKTP) endocytosed by brain neurons of zebrafish (Zf) larvae. Thanks
to the optical translucency of Zf larvae and to the perfect photostability
of nanoKTP SHG, we achieved a high scanning speed of 20 frames (of
≈90 μm × 60 μm size) per second in Zf brain.
We focused our study on endolysosomal vesicle transport in axons of
known polarization, separately analyzing kinesin and dynein motor-driven
displacements. To validate our assay, we used either loss-of-function
mutations of dynein or kinesin 1 or the dynein inhibitor dynapyrazole
and quantified several transport parameters. We successfully demonstrated
that dynapyrazole reduces the nanoKTP mobile fraction and retrograde
run length consistently, while the retrograde run length increased
in kinesin 1 mutants. Taking advantage of nanoKTP SHG directional
emission, we also quantified fluctuations of vesicle orientation.
Thus, by combining endocytosis of nanocrystals having a nonlinear
response, fast two-photon microscopy, and high-throughput analysis,
we are able to finely monitor fast axonal transport in vivo in the brain of a vertebrate and reveal subtle axonal transport
alterations. The high spatiotemporal resolution achieved in our model
may be relevant to precisely investigate axonal transport impairment
associated with disease models
<i>In Vivo</i> Fast Nonlinear Microscopy Reveals Impairment of Fast Axonal Transport Induced by Molecular Motor Imbalances in the Brain of Zebrafish Larvae
Cargo transport by molecular motors along microtubules
is essential
for the function of eukaryotic cells, in particular neurons in which
axonal transport defects constitute the early pathological features
of neurodegenerative diseases. Mainly studied in motor and sensory
neurons, axonal transport is still difficult to characterize in neurons
of the brain in absence of appropriate in vivo tools.
Here, we measured fast axonal transport by tracing the second harmonic
generation (SHG) signal of potassium titanyl phosphate (KTP) nanocrystals
(nanoKTP) endocytosed by brain neurons of zebrafish (Zf) larvae. Thanks
to the optical translucency of Zf larvae and to the perfect photostability
of nanoKTP SHG, we achieved a high scanning speed of 20 frames (of
≈90 μm × 60 μm size) per second in Zf brain.
We focused our study on endolysosomal vesicle transport in axons of
known polarization, separately analyzing kinesin and dynein motor-driven
displacements. To validate our assay, we used either loss-of-function
mutations of dynein or kinesin 1 or the dynein inhibitor dynapyrazole
and quantified several transport parameters. We successfully demonstrated
that dynapyrazole reduces the nanoKTP mobile fraction and retrograde
run length consistently, while the retrograde run length increased
in kinesin 1 mutants. Taking advantage of nanoKTP SHG directional
emission, we also quantified fluctuations of vesicle orientation.
Thus, by combining endocytosis of nanocrystals having a nonlinear
response, fast two-photon microscopy, and high-throughput analysis,
we are able to finely monitor fast axonal transport in vivo in the brain of a vertebrate and reveal subtle axonal transport
alterations. The high spatiotemporal resolution achieved in our model
may be relevant to precisely investigate axonal transport impairment
associated with disease models
<i>In Vivo</i> Fast Nonlinear Microscopy Reveals Impairment of Fast Axonal Transport Induced by Molecular Motor Imbalances in the Brain of Zebrafish Larvae
Cargo transport by molecular motors along microtubules
is essential
for the function of eukaryotic cells, in particular neurons in which
axonal transport defects constitute the early pathological features
of neurodegenerative diseases. Mainly studied in motor and sensory
neurons, axonal transport is still difficult to characterize in neurons
of the brain in absence of appropriate in vivo tools.
Here, we measured fast axonal transport by tracing the second harmonic
generation (SHG) signal of potassium titanyl phosphate (KTP) nanocrystals
(nanoKTP) endocytosed by brain neurons of zebrafish (Zf) larvae. Thanks
to the optical translucency of Zf larvae and to the perfect photostability
of nanoKTP SHG, we achieved a high scanning speed of 20 frames (of
≈90 μm × 60 μm size) per second in Zf brain.
We focused our study on endolysosomal vesicle transport in axons of
known polarization, separately analyzing kinesin and dynein motor-driven
displacements. To validate our assay, we used either loss-of-function
mutations of dynein or kinesin 1 or the dynein inhibitor dynapyrazole
and quantified several transport parameters. We successfully demonstrated
that dynapyrazole reduces the nanoKTP mobile fraction and retrograde
run length consistently, while the retrograde run length increased
in kinesin 1 mutants. Taking advantage of nanoKTP SHG directional
emission, we also quantified fluctuations of vesicle orientation.
Thus, by combining endocytosis of nanocrystals having a nonlinear
response, fast two-photon microscopy, and high-throughput analysis,
we are able to finely monitor fast axonal transport in vivo in the brain of a vertebrate and reveal subtle axonal transport
alterations. The high spatiotemporal resolution achieved in our model
may be relevant to precisely investigate axonal transport impairment
associated with disease models
In-vivo fast non-linear microscopy reveals impairment of fast axonal transport induced by molecular motor imbalances in the brain of zebrafish larvae
Abstract Cargo transport by molecular motors along microtubules is essential for the function of eucaryotic cells, in particular neurons in which axonal transport defects constitute the early pathological features of neurodegenerative diseases. Mainly studied in motor and sensory neurons, axonal transport is still difficult to characterize in neurons of the brain in absence of appropriate in vivo tools. Here, we measured fast axonal transport by tracing the second harmonic generation (SHG) signal of potassium titanyl phosphate (KTP) nanocrystals endocytosed by brain neurons of zebrafish (Zf) larvae. Thanks to the optical translucency of Zf larvae and of the perfect photostability of nanoKTP SHG, we achieved a high scanning speed of 20 frames (of ≈ 90 μ m×60 μ m size) per second in Zf brain. We focused our study on endolysosomal vesicle transport in axons of known polarization, separately analyzing kinesin and dynein motor-driven displacements. To validate our assay, we used either loss-of-function mutations of dynein or kinesin 1 or the dynein inhibitor dynapyrazole, and quantified several transport parameters. We successfully demonstrated that dynapyrazole reduces nanoKTP mobile fraction and retrograde run length consistently, while the retrograde run length increased in kinesin 1 mutants. Taking advantage of nanoKTP SHG directional emission, we also quantified fluctuations of vesicle orientation. Thus, by combining endocytosis of nanocrystals having non-linear response, fast two-photon microscopy, and high-throughput analysis, we are able to finely monitor fast axonal transport in vivo in the brain of a vertebrate, and reveal subtle axonal transport alterations. The high spatiotemporal resolution achieved in our model may be relevant to precisely investigate axonal transport impairment associated to disease models