Journal of Cell Science,
Год журнала:
2020,
Номер
133(21)
Опубликована: Ноя. 1, 2020
ABSTRACT
One
of
the
challenges
in
modern
fluorescence
microscopy
is
to
reconcile
conventional
utilization
microscopes
as
exploratory
instruments
with
their
emerging
and
rapidly
expanding
role
a
quantitative
tools.
The
contribution
observational
biology
will
remain
enormous
owing
improvements
acquisition
speed,
imaging
depth,
resolution
biocompatibility
instruments.
However,
use
facilitate
measurements
necessary
challenge
hypotheses
relatively
recent
concept,
made
possible
by
advanced
optics,
functional
probes
increasing
computational
power.
We
argue
here
that
fully
leverage
evolving
application
hypothesis-driven
biology,
we
not
only
need
ensure
images
are
acquired
quantitatively
but
must
also
re-evaluate
how
microscopy-based
experiments
designed.
In
this
Opinion,
present
reverse
logic
guides
design
experiments.
This
unique
approach
starts
from
identifying
results
would
inform
hypothesis
map
process
backward
microscope
selection.
ensures
aspects
testing
central
focus
entire
experimental
design.
Nature Reviews Methods Primers,
Год журнала:
2021,
Номер
1(1)
Опубликована: Июнь 3, 2021
Single-molecule
localization
microscopy
(SMLM)
describes
a
family
of
powerful
imaging
techniques
that
dramatically
improve
spatial
resolution
over
standard,
diffraction-limited
and
can
image
biological
structures
at
the
molecular
scale.
In
SMLM,
individual
fluorescent
molecules
are
computationally
localized
from
sequences
localizations
used
to
generate
super-resolution
or
time
course
images,
define
trajectories.
this
Primer,
we
introduce
basic
principles
SMLM
before
describing
main
experimental
considerations
when
performing
including
labelling,
sample
preparation,
hardware
requirements
acquisition
in
fixed
live
cells.
We
then
explain
how
low-resolution
processed
reconstruct
images
and/or
extract
quantitative
information,
highlight
selection
discoveries
enabled
by
closely
related
methods.
discuss
some
limitations
potential
artefacts
as
well
ways
alleviate
them.
Finally,
present
an
outlook
on
advanced
promising
new
developments
fast-evolving
field
SMLM.
hope
Primer
will
be
useful
reference
for
both
newcomers
practitioners
Chemical Reviews,
Год журнала:
2018,
Номер
118(21), С. 10659 - 10709
Опубликована: Июль 9, 2018
Sensory
photoreceptors
underpin
light-dependent
adaptations
of
organismal
physiology,
development,
and
behavior
in
nature.
Adapted
for
optogenetics,
sensory
become
genetically
encoded
actuators
reporters
to
enable
the
noninvasive,
spatiotemporally
accurate
reversible
control
by
light
cellular
processes.
Rooted
a
mechanistic
understanding
natural
photoreceptors,
artificial
with
customized
light-gated
function
have
been
engineered
that
greatly
expand
scope
optogenetics
beyond
original
application
light-controlled
ion
flow.
As
we
survey
presently,
UV/blue-light-sensitive
particularly
allowed
transcend
its
initial
neuroscience
applications
unlocking
numerous
additional
processes
parameters
optogenetic
intervention,
including
gene
expression,
DNA
recombination,
subcellular
localization,
cytoskeleton
dynamics,
intracellular
protein
stability,
signal
transduction
cascades,
apoptosis,
enzyme
activity.
The
engineering
novel
benefits
from
powerful
reusable
design
strategies,
most
importantly
association
(un)folding
reactions.
Additionally,
modified
versions
these
same
serve
as
fluorescent
proteins
generators
singlet
oxygen,
thereby
further
enriching
toolkit.
available
upcoming
detailed
quantitative
interrogation
networks
increasingly
more
precise
illuminating
manners.
Proceedings of the National Academy of Sciences,
Год журнала:
2018,
Номер
115(5)
Опубликована: Янв. 16, 2018
Significance
Bardet–Biedl
syndrome
(BBS)
is
a
rare
disease
caused
by
dysfunctional
cilia.
In
bbs
mutants,
the
composition
of
ciliary
membrane
altered
due
to
defects
in
BBSome,
conserved
complex
BBS
proteins.
To
determine
molecular
function
we
used
single
particle
vivo
imaging.
Transport
protein
phospholipase
D
(PLD)
BBSome-dependent,
and
PLD
comigrates
with
BBSomes
on
intraflagellar
transport
(IFT)
trains.
accumulates
inside
cilia
after
removal
its
export
sequence
(CES)
or
absence
BBSomes.
conclusion,
BBSome
participates
directly
serving
as
an
adapter
allowing
proteins
that
alone
are
unable
bind
IFT
be
exported
from
Analytical Sciences,
Год журнала:
2018,
Номер
35(1), С. 5 - 27
Опубликована: Окт. 11, 2018
Chemical
modification
of
proteins
is
important
for
creating
a
myriad
engineered
and
elucidating
the
function
dynamics
in
live
cells.
A
wide
variety
chemical
protein
methods
have
been
developed
can
be
categorized
into
three
classes:
(i)
using
reactivity
naturally
occurring
amino
acids;
(ii)
by
bioorthogonal
reactions
unnatural
acids,
most
which
site-
selectively
incorporated
proteins-of-interest
genetic
codon
expansion
techniques;
(iii)
recognition
driven
modification,
only
approach
that
allows
endogenous
without
any
manipulation
even
under
heavily
crowded
multi-molecular
conditions,
as
cells
organisms.
All
these
approaches
merits
limitations.
In
this
review,
we
summarize
discuss
their
characteristics
with
respect
to
specificity,
reaction
rate
versatility.
Biotechnology for Biofuels,
Год журнала:
2021,
Номер
14(1)
Опубликована: Янв. 25, 2021
Abstract
Non-model
microorganisms
often
possess
complex
phenotypes
that
could
be
important
for
the
future
of
biofuel
and
chemical
production.
They
have
received
significant
interest
last
several
years,
but
advancement
is
still
slow
due
to
lack
a
robust
genetic
toolbox
in
most
organisms.
Typically,
“domestication”
new
non-model
microorganism
has
been
done
on
an
ad
hoc
basis,
historically,
it
can
take
years
develop
transformation
basic
tools.
Here,
we
review
barriers
solutions
rapid
development
tools
hosts,
with
major
focus
Restriction-Modification
systems,
which
are
well-known
barrier
efficient
transformation.
We
further
explore
approaches
used
gene
deletion,
DNA
insertion,
heterologous
expression.
Finally,
more
advanced
high-throughput
now
being
developed
diverse
microbes,
paving
way
multiplexed
genome
engineering
biotechnology.
Chemical Reviews,
Год журнала:
2021,
Номер
121(19), С. 11971 - 12015
Опубликована: Март 22, 2021
Fluorescence
imaging
techniques
play
a
pivotal
role
in
our
understanding
of
the
nervous
system.
The
emergence
various
super-resolution
microscopy
methods
and
specialized
fluorescent
probes
enables
direct
insight
into
neuronal
structure
protein
arrangements
cellular
subcompartments
with
so
far
unmatched
resolution.
Super-resolving
visualization
neurons
unveil
novel
cytoskeletal
composition,
distribution,
motility,
signaling
membrane
proteins,
subsynaptic
function,
neuron-glia
interaction.
Well-defined
molecular
targets
autoimmune
neurodegenerative
disease
models
provide
excellent
starting
points
for
in-depth
investigation
pathophysiology
using
innovative
methodology.
Application
human
brain
samples
testing
clinical
biomarkers
is
still
its
infancy
but
opens
new
opportunities
translational
research
neurology
neuroscience.
In
this
review,
we
describe
how
super-resolving
has
improved
function
dysfunction
last
two
decades.
Journal of the American Chemical Society,
Год журнала:
2021,
Номер
143(48), С. 20095 - 20108
Опубликована: Ноя. 24, 2021
Chemical
modifications
of
native
proteins
can
affect
their
stability,
activity,
interactions,
localization,
and
more.
However,
there
are
few
nongenetic
methods
for
the
installation
chemical
at
a
specific
protein
site
in
cells.
Here
we
report
covalent
ligand
directed
release
(CoLDR)
site-specific
labeling
strategy,
which
enables
variety
functional
tags
on
target
while
releasing
directing
ligand.
Using
this
approach,
were
able
to
label
various
such
as
BTK,
K-RasG12C,
SARS-CoV-2
PLpro
with
different
tags.
For
BTK
have
shown
selective
cells
both
alkyne
fluorophores
Protein
by
traditional
affinity
often
inhibits
activity
since
permanently
occupies
binding
pocket.
We
that
using
CoLDR
chemistry,
modification
these
probes
preserves
its
activity.
demonstrated
several
applications
approach
including
determining
half-life
environment
minimal
perturbation,
well
quantification
degradation
noncovalent
proteolysis
targeting
chimera
(PROTAC)
in-gel
fluorescence.
an
environment-sensitive
"turn-on"
fluorescent
probe,
monitor
active
BTK.
Finally,
efficient
CoLDR-based
PROTACs
(DC50
<
100
nM),
installed
CRBN
binder
onto
This
joins
very
available
strategies
maintain
thus
makes
important
addition
toolbox
biology.
