GTP-tubulin
is
preferentially
incorporated
at
growing
microtubule
ends,
but
the
biochemical
mechanism
by
which
bound
nucleotide
regulates
strength
of
tubulin:tubulin
interactions
debated.
The
'self-acting'
(cis)
model
posits
that
(GTP
or
GDP)
to
a
particular
tubulin
dictates
how
strongly
interacts,
whereas
'interface-acting'
(trans)
interface
two
dimers
determinant.
We
identified
testable
difference
between
these
mechanisms
using
mixed
simulations
elongation:
with
self-acting
nucleotide,
plus-
and
minus-end
growth
rates
decreased
in
same
proportion
amount
GDP-tubulin,
interface-acting
plus-end
disproportionately.
then
experimentally
measured
elongation
nucleotides
observed
disproportionate
effect
GDP-tubulin
on
rates.
Simulations
were
consistent
binding
'poisoning'
plus-ends
not
minus-ends.
Quantitative
agreement
experiments
required
exchange
terminal
subunits
mitigate
poisoning
there.
Our
results
indicate
interfacial
determines
interaction
strength,
thereby
settling
longstanding
debate
over
state
dynamics.
Cell Cycle,
Год журнала:
2025,
Номер
unknown, С. 1 - 20
Опубликована: Апрель 22, 2025
Microtubules
are
polymers
of
α/β
tubulin
dimers
that
build
the
mitotic
spindle,
which
segregates
duplicated
chromosomes
during
cell
division.
Microtubule
function
is
governed
by
dynamic
instability,
whereby
cycles
growth
and
shrinkage
contribute
to
forces
necessary
for
chromosome
movement.
Regulation
microtubule
velocity
requires
cycle-dependent
changes
in
expression,
localization
activity
microtubule-associated
proteins
(MAPs)
as
well
post-translational
modifications
modulate
dynamics.
It
has
become
clear
optimal
velocities
required
proper
segregation
ploidy
maintenance.
Suboptimal
rates
can
result
from
altered
MAPs
could
lead
aneuploidy,
possibly
disrupting
establishment
bundles
at
kinetochores
altering
mechanical
sister
chromatid
segregation.
Future
work
using
high-resolution,
low-phototoxicity
microscopy
novel
fluorescent
markers
will
be
invaluable
obtaining
deeper
mechanistic
insights
into
how
processes
The Journal of Cell Biology,
Год журнала:
2023,
Номер
222(7)
Опубликована: Май 15, 2023
Cytoplasmic
linker-associated
proteins
(CLASPs)
regulate
microtubules
in
fundamental
cellular
processes.
CLASPs
stabilize
dynamic
by
suppressing
microtubule
catastrophe
and
promoting
rescue,
the
switch-like
transitions
between
growth
shrinkage.
How
specifically
modulate
is
not
understood.
Here,
we
investigate
effects
of
on
pre-catastrophe
intermediate
state
dynamics,
employing
distinct
substrates
to
mimic
state.
Surprisingly,
find
that
CLASP1
promotes
depolymerization
stabilized
presence
GTP,
but
absence
nucleotide.
This
activity
also
observed
for
CLASP2
family
members
a
minimal
TOG2-domain
construct.
Conversely,
stabilizes
unstable
upon
tubulin
dilution
GTP.
Strikingly,
our
results
reveal
drives
with
vastly
different
inherent
stabilities
into
same
slowly
depolymerizing
nucleotide-dependent
manner.
We
interpret
this
as
Therefore,
conclude
suppress
stabilizing
bioRxiv (Cold Spring Harbor Laboratory),
Год журнала:
2024,
Номер
unknown
Опубликована: Май 2, 2024
Summary
Microtubule
cytoskeletons
play
pivotal
roles
in
various
cellular
processes,
including
cell
division
and
locomotion,
by
dynamically
changing
their
length
distribution
cells
through
tubulin
polymerization/depolymerization.
Recent
structural
studies
have
revealed
the
polymorphic
lattice
structure
of
microtubules
closely
correlate
with
microtubule
dynamics,
but
were
limited
to
averaged
structures.
To
reveal
transient
localized
structures,
such
as
GTP-cap,
we
developed
several
non-averaging
methods
for
cryogenic
electron
tomography
precisely
measure
longitudinal
spacing
helical
twisting
individual
lattices
at
subunit
level.
Our
analysis
that
polymerizing
depolymerizing
ends
share
a
similar
regards
spacing.
The
most
distinctive
property
specific
plus
end
was
left-handed
inter-dimer
interface,
suggesting
might
accelerate
fast
polymerization
ends.
uncovered
heterogeneity
native
will
be
indispensable
study
dynamics
under
physiological
contexts
or
during
events.
bioRxiv (Cold Spring Harbor Laboratory),
Год журнала:
2022,
Номер
unknown
Опубликована: Дек. 4, 2022
ABSTRACT
CLASPs
regulate
microtubules
in
many
fundamental
cellular
processes.
stabilize
dynamic
by
suppressing
catastrophe
and
promoting
rescue,
the
switch-like
transitions
between
microtubule
growth
shrinkage.
However,
molecular
mechanisms
underlying
CLASP’s
activity
are
not
understood.
Here,
we
investigate
effects
of
on
distinct
substrates
absence
tubulin
to
gain
insight
into
how
dynamics.
Surprisingly,
find
that
human
CLASP1
depolymerizes
stable
presence
GTP,
but
nucleotide.
Conversely,
stabilizes
upon
dilution
GTP.
Our
results
demonstrate
drives
with
different
inherent
stabilities
same
slowly-depolymerizing
state
a
nucleotide-dependent
manner.
We
interpret
this
as
pre-catastrophe
intermediate
Thus,
conclude
shrinkage
suppress
promote
rescue.
GTP-tubulin
is
preferentially
incorporated
at
growing
microtubule
ends,
but
the
biochemical
mechanism
by
which
bound
nucleotide
regulates
strength
of
tubulin:tubulin
interactions
debated.
The
'self-acting'
(cis)
model
posits
that
(GTP
or
GDP)
to
a
particular
tubulin
dictates
how
strongly
interacts,
whereas
'interface-acting'
(trans)
interface
two
dimers
determinant.
We
identified
testable
difference
between
these
mechanisms
using
mixed
simulations
elongation:
with
self-acting
nucleotide,
plus-
and
minus-end
growth
rates
decreased
in
same
proportion
amount
GDP-tubulin,
interface-acting
plus-end
disproportionately.
then
experimentally
measured
elongation
nucleotides
observed
disproportionate
effect
GDP-tubulin
on
rates.
Simulations
were
consistent
binding
'poisoning'
plus-ends
not
minus-ends.
Quantitative
agreement
experiments
required
exchange
terminal
subunits
mitigate
poisoning
there.
Our
results
indicate
interfacial
determines
interaction
strength,
thereby
settling
longstanding
debate
over
state
dynamics.
GTP-tubulin
is
preferentially
incorporated
at
growing
microtubule
ends,
but
the
biochemical
mechanism
by
which
bound
nucleotide
regulates
strength
of
tubulin:tubulin
interactions
debated.
The
‘self-acting’
(cis)
model
posits
that
(GTP
or
GDP)
to
a
particular
tubulin
dictates
how
strongly
interacts,
whereas
‘interface-acting’
(trans)
interface
two
dimers
determinant.
We
identified
testable
difference
between
these
mechanisms
using
mixed
simulations
elongation:
with
self-acting
nucleotide,
plus-
and
minus-end
growth
rates
decreased
in
same
proportion
amount
GDP-tubulin,
interface-acting
plus-end
disproportionately.
then
experimentally
measured
elongation
nucleotides
observed
disproportionate
effect
GDP-tubulin
on
rates.
Simulations
were
consistent
binding
‘poisoning’
plus-ends
not
minus-ends.
Quantitative
agreement
experiments
required
exchange
terminal
subunits
mitigate
poisoning
there.
Our
results
indicate
interfacial
determines
interaction
strength,
thereby
settling
longstanding
debate
over
state
dynamics.
bioRxiv (Cold Spring Harbor Laboratory),
Год журнала:
2024,
Номер
unknown
Опубликована: Июнь 26, 2024
ABSTRACT
The
microtubule
cytoskeleton
is
a
dynamic
network
essential
for
many
cellular
processes,
influenced
by
physicochemical
factor
such
as
temperature,
pH,
dimer
concentration
and
ionic
environment.
In
this
study,
we
used
in
vitro
reconstitution
assays
to
examine
the
effects
of
four
monovalent
ions
(Na
+
,
K
Cl
-
Ac
)
on
dynamics,
uncovering
distinct
each
ion.
Na
was
found
increase
dynamicity
raising
catastrophe
frequency,
polymerization
depolymerization
speeds,
ultimately
reducing
lifetime
80
%.
Conversely,
boosts
nucleation
stabilizes
microtubules
increasing
rescue
frequency
preventing
breakages,
resulting
longer
with
extended
lifetimes.
appeared
potentiate
while
had
minimal
impact
parameters.
These
findings
demonstrate
that
have
opposing
destabilizing
stabilizing
structure.
This
mainly
through
modulation
tubulin-tubulin
interactions
rather
than
affecting
hydrolysis
rate.
conclusion,
ion
identity
plays
crucial
role
modulating
dynamics.
Understanding
environment
microtubule-related
research,
it
significantly
influences
behavior,
stability,
other
proteins.
SIGNIFICANCE
STATEMENT
vital
processes
concentration,
how
these
factors
regulate
elucidating
dynamics
stability.
Our
reveal
increases
%,
enhances
microtubules.
potentiates
has
impact.
highlight
crucially
modulates
influencing
stability
interactions.
