Abstract
Transport of two types of organelles in Drosophila neurons
in culture and in vivo
Velocity (µm/s)
0.5
0.4
0.3
40
30
20
10
0
0 sec
Histogram of velocity of Syt-GFP in culture
1 3 5 7 9 1 3 5 7 9 1 3
1 3 5 7 9 1 3 5 7 9 1 3
0. 0. 0. 0. 0. 1. 1. 1. 1. 1. 2. 2. 0. 0. 0. 0. 0. 1. 1. 1. 1. 1. 2. 2.
Velocity (µm/s)
0.0
0.5
1.0
1.5
2.0
***
0.0
0.2
0.4
0.6
0.8
1.0
Syt-GFP anterograde
Syt-GFP retrograde
120 sec
Transport of synaptic vesicles in culture
***
N=591
2
2
1
N=184
50
40
30
20
10
0
0 sec
120 sec
1
2
Histogram of velocity of Mito-GFP in culture
0.1 0.3 0.5 0.7 0.9 1.1 1.3 1.5 1.7 1.9 2.1
Velocity (µm/s)
0.1 0.3 0.5 0.7 0.9 1.1 1.3 1.5 1.7 1.9 2.1
0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
Mito-GFP anterograde
Mito-GFP retrograde
Transport of mitochondria in culture
N=560
1
N=190
0.4
0.3
0.2
0.1
0.0
0.6
0.4
0.2
0.0
N=18
50
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
Velocity (µm/s)
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
Histogram of velocity of Syt-GFP in vivo
40
30
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10
0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1
Velocity (µm/s)
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1
Histogram of velocity of Mito-GFP in vivo
50
40
30
20
10
0
1.5
1.0
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0.0
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1.0
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0 sec
0 sec
1.0
0.8
0.6
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1.0
180 sec
Transport of mitochondria in vivo
N=125
90 sec
Mito-GFP anterograde
Mito-GFP retrograde
Syt-GFP anterograde
Syt-GFP retrograde
Transport of synaptic vesicles in vivo
N=71
N=145
Based on our quantification data, we found that:
(1) in culture synaptic vesicles enter all processes, suggesting that some factor(s)
for axonal/dendritic differentiation are lacking in vitro;
(2) both organelle types move bidirectionally, in neurites both in culture and
in vivo, suggesting that they have both plus-end and minus-end motors attached
at the same time;
(3) in culture the retrograde velocities of synaptic vesicles are higher than in vivo,
suggesting that the in culture some regulation factor of synaptic vesicle retrograde
transport is missing;
(4) surprisingly in vivo more synaptic vesicles move retrogradely than
anterogradely.
Conclusions:
Velocity (µm/s)
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0.1
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0.1
0.0
Acknowledgements:
We thank Timo Zimmermann and Raul Gomez Riera for help with CRG
microscopes and Zimin Foundation for support of of SMTB 2016 in general and
the Drosophila lab in particular
Velocity (µm/s)
Olga Khasina*, Ksenia Meteleva*, Ekaterina Podruzhko*, Sasha Rogachevskaya*, Anna Rozina*, Mila Zudina, Wen Lu, Vladimir Gelfand
Moving
vesicles
Rainbow
Velocity (µm/s)
School of Molecular and Theoretical Biology, Barcelona; Northwestern University, Chicago
* Equal contribution
+
Neurons are the core components of animal nervous system. They are the most
polarized cells with long processes (axons and dendrites) in the animal bodies.
Transport of specific cargoes into the longest process, the axon, is highly
regulated. Misregulation of axonal cargo trafficking is linked to multiple
neurodegenerative diseases. Microtubules serve as the tracks for cargo transport
and microtubule motor proteins are ATPases that carry various cargoes along
microtubule tracks in highly polarized neurons. In this laboratory, we employed
genetic tools to apply fluorescent markers to two types of neuronal cargoes,
axonal-specific synaptic vesicles and general cargo, mitochondria, and study
their transport in cultured neurons and motor neurons in live Drosophila larvae.
We used fluorescent total internal reflection (TIRF) microscopy and confocal
scanning microscopy to examine transport in the axons, and
quantified the velocities and directions of transport both in cultures and in vivo.
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TIRF microscopy
Stationary
vesicles
Temporal-color code
Distance
Projection
White dot
Fractions
Fractions
Top 10%Velocity (µm/s)
Top 10% Velocity (µm/s)
Top 10%Velocity (µm/s)
Top 10% Velocity (µm/s)
Percentage %
Percentage %
Percentage %
Percentage %
Fractions
Fractions
Methods
Culture of 3rd instar larval brain neurons
Imaging of 3rd instar larval motor neurons
Time