Abstract
Visualizing
the
subcellular
localization
of
presynaptic
proteins
with
fluorescent
is
a
powerful
tool
to
dissect
genetic
and
molecular
mechanisms
underlying
synapse
formation
patterning
in
live
animals.
Here,
we
utilize
split
green
red
visualize
endogenously
expressed
at
single
neuron
resolution
Caenorhabditis
elegans.
By
using
CRISPR/Cas9
genome
editing,
generated
collection
C.
elegans
strains
which
(RAB-3/Rab3,
SNG-1/Synaptogyrin,
CLA-1/Piccolo,
SYD-2/Liprin-α,
UNC-10/RIM,
RIMB-1/RIM-BP,
ELKS-1/ELKS)
are
tagged
tandem
repeats
GFP11
and/or
wrmScarlet11.
We
show
that
expression
GFP1-10
wrmScarlet1-10
under
neuron-specific
promoters
can
robustly
label
different
types.
believe
combination
our
knock-in
plasmids
versatile
modular
system
useful
for
neuroscientists
examine
endogenous
any
type
Autophagy,
Год журнала:
2023,
Номер
19(10), С. 2807 - 2808
Опубликована: Июнь 30, 2023
In
neuronal
synapses,
autophagosome
biogenesis
is
coupled
with
the
activity-dependent
synaptic
vesicle
cycle
via
ATG-9.
How
vesicles
containing
ATG-9
are
sorted
at
presynapse
unknown.
We
performed
forward
genetic
screens
single
synapses
of
C.
elegans
neurons
for
mutants
that
disrupt
presynaptic
localization,
and
identified
long
isoform
active
zone
protein
CLA-1
(Clarinet;
L).
find
disrupting
L
results
in
abnormal
accumulation
ATG-9-containing
enriched
clathrin.
The
adaptor
complexes
proteins
periactive
genetically
interact
sorting.
Moreover,
phenotype
cla-1(L)
was
not
observed
integral
proteins,
suggesting
distinct
mechanisms
regulate
sorting
vesicles.
Our
findings
reveal
novel
roles
macroautophagy/autophagy.
bioRxiv (Cold Spring Harbor Laboratory),
Год журнала:
2024,
Номер
unknown
Опубликована: Апрель 23, 2024
Abstract
To
enable
transmission
of
information
in
the
brain,
synaptic
vesicles
fuse
to
presynaptic
membranes,
liberating
their
content
and
exposing
transiently
a
myriad
vesicular
transmembrane
proteins.
However,
versatile
methods
for
quantifying
translocation
endogenous
proteins
during
neuronal
activity
remain
unavailable,
as
fast
dynamics
vesicle
cycling
difficult
specific
isolation
trafficking
such
transient
surface
exposure.
Here
we
developed
novel
approach
using
cleft
proximity
labeling
capture
quantify
activity-driven
at
synapse.
We
show
that
accelerating
biotinylation
times
match
exocytosis
allows
capturing
exposed
neural
activity,
enabling
first
time
study
nearly
every
protein.
As
proof-of-concept,
further
applied
this
technology
obtain
direct
evidence
non-canonical
proteins,
ATG9A
NPTX1,
which
had
been
proposed
traffic
but
proof
not
yet
shown.
The
technological
advancement
presented
here
will
facilitate
future
studies
dissecting
molecular
identity
exocytosed
synapse
helping
define
machinery
sustains
neurotransmission
mammalian
brain.
Significance
statement
Synaptic
is
critical
neurons
communicate
sustain
brain
function.
Pascual-Caro
de
Juan-Sanz
develop
pioneering
method
any
Coordinating
clefts
just
few
seconds,
authors
visualize
This
work
provides
framework
uncover
complex
choreography
events
occurring
within
firing
synapses,
deeper
control
communication
circuit
physiology
bioRxiv (Cold Spring Harbor Laboratory),
Год журнала:
2024,
Номер
unknown
Опубликована: Июль 30, 2024
Abstract
Visualizing
the
subcellular
localization
of
presynaptic
proteins
with
fluorescent
is
a
powerful
tool
to
dissect
genetic
and
molecular
mechanisms
underlying
synapse
formation
patterning
in
live
animals.
Here,
we
utilize
split
green
red
visualize
endogenously
expressed
at
single
neuron
resolution
Caenorhabditis
elegans.
By
using
CRISPR/Cas9
genome
editing,
generated
collection
C.
elegans
strains
which
(RAB-3/Rab3,
CLA-1/Piccolo,
SYD-2/Liprin-α,
UNC-10/RIM
ELKS-1/ELKS)
are
tagged
tandem
repeats
GFP
11
and/or
wrmScarlet
.
We
show
that
expression
1-10
under
neuron-specific
promoters
can
robustly
label
different
types.
believe
combinations
knock-in
plasmids
versatile
modular
system
examine
endogenous
any
type.
PLoS Biology,
Год журнала:
2024,
Номер
22(10), С. e3002860 - e3002860
Опубликована: Окт. 28, 2024
To
enable
transmission
of
information
in
the
brain,
synaptic
vesicles
fuse
to
presynaptic
membranes,
liberating
their
content
and
exposing
transiently
a
myriad
vesicular
transmembrane
proteins.
However,
versatile
methods
for
quantifying
translocation
endogenous
proteins
during
neuronal
activity
remain
unavailable,
as
fast
dynamics
vesicle
cycling
difficult
specific
isolation
trafficking
such
transient
surface
exposure.
Here,
we
developed
novel
approach
using
cleft
proximity
labeling
capture
quantify
activity-driven
at
synapse.
We
show
that
accelerating
biotinylation
times
match
exocytosis
allows
capturing
exposed
neural
activity,
enabling
first
time
study
nearly
every
protein.
As
proof-of-concept,
further
applied
this
technology
obtain
direct
evidence
noncanonical
proteins,
ATG9A
NPTX1,
which
had
been
proposed
traffic
but
proof
not
yet
shown.
The
technological
advancement
presented
here
will
facilitate
future
studies
dissecting
molecular
identity
exocytosed
synapse
helping
define
machinery
sustains
neurotransmission
mammalian
brain.
Abstract
Visualizing
the
subcellular
localization
of
presynaptic
proteins
with
fluorescent
is
a
powerful
tool
to
dissect
genetic
and
molecular
mechanisms
underlying
synapse
formation
patterning
in
live
animals.
Here,
we
utilize
split
green
red
visualize
endogenously
expressed
at
single
neuron
resolution
Caenorhabditis
elegans.
By
using
CRISPR/Cas9
genome
editing,
generated
collection
C.
elegans
strains
which
(RAB-3/Rab3,
SNG-1/Synaptogyrin,
CLA-1/Piccolo,
SYD-2/Liprin-α,
UNC-10/RIM,
RIMB-1/RIM-BP,
ELKS-1/ELKS)
are
tagged
tandem
repeats
GFP11
and/or
wrmScarlet11.
We
show
that
expression
GFP1-10
wrmScarlet1-10
under
neuron-specific
promoters
can
robustly
label
different
types.
believe
combination
our
knock-in
plasmids
versatile
modular
system
useful
for
neuroscientists
examine
endogenous
any
type