First name
Matteo
Last name
Biancospino
Year of Study
Research Center
Thesis Title
Functional and structural characterization of myosin VI isoforms
Thesis Abstract
Myosin
VI
is
unique
among
the
many
members
of
the
myosin
superfamily.
The
peculiarities
of
this
motor
protein
reside
on
its
ability
to
travel
along
actin
microfilaments
towards
their
pointed-‐
end,
as
well
as
its
capacity
to
act
either
as
anchor
or
as
processive
motor.
As
a
consequence,
myosin
VI
has
been
implicated
in
clathrin-‐mediated
endocytosis,
vesicle
trafficking,
autophagy,
cell
migration
and
tumorigenesis.
The
current
understanding
of
the
myosin
VI
protein
does
not
explain
how
it
carries
out
these
diverse
processes,
as
functional
mechanistic
studies
are
lacking.
Alternative
splicing
in
the
tail
region
generates
myosin
VI
molecules
with
different
features
(myosin
VIshort
and
myosin
VIlong)
but
little
is
known
about
the
impact
of
the
variable
region
on
physiological
and
pathological
functions
of
myosin
VI.
In
this
study,
we
have
analysed
the
myosin
VI
isoforms
from
a
molecular
and
a
functional
perspective.
Using
quantitative
mass
spectrometry
approach
and
modern
NMR
we
identified
and
structurally
characterized
clathrin
light
chain
as
novel
and
isoform-‐specific
interactor.
Within
the
novel
clathrin-‐binding
domain
that
is
unique
to
myosin
VIlong,
an
isoform-‐specific
regulatory
helix,
named
α2-‐linker,
defines
a
specific
myosin
VIlong
conformation.
Its
presence
or
absence
determines
the
target
selectivity
of
myosin
VIshort
and
myosin
VIlong
isoforms,
acting
like
a
molecular
switch
that
regulates
their
functional
involvement
in
migratory
or
endocytic
pathways,
respectively.
The
adaptor-‐binding
RRL
motif
is
embedded
in
the
clathrin-‐binding
domain
and
is
masked
in
the
structural
configuration
adopted
by
the
myosin
VIlong.
Consequently,
the
previously
identified
RRL
interactors
show
selective
binding
to
myosin
VIshort.
Thus,
we
can
provide
for
the
first
time
a
mechanistic
explanation
of
why
the
various
isoforms
show
different
localization
and
function
(i.e.
myosin
VIlong
selectively
involved
in
clathrin-‐mediated
endocytosis).
We
also
found
that
alternative
myosin
VI
splicing
is
aberrantly
regulated
in
ovarian
cancers,
where
exon
skipping
dictates
myosin
VIshort-‐only
expression.
Importantly,
cancer
cell
lines
selectively
expressing
the
myosin
VIshort
isoform
exhibit
severe
migration
defects
when
myosin
VI
is
knocked
down.
VI
is
unique
among
the
many
members
of
the
myosin
superfamily.
The
peculiarities
of
this
motor
protein
reside
on
its
ability
to
travel
along
actin
microfilaments
towards
their
pointed-‐
end,
as
well
as
its
capacity
to
act
either
as
anchor
or
as
processive
motor.
As
a
consequence,
myosin
VI
has
been
implicated
in
clathrin-‐mediated
endocytosis,
vesicle
trafficking,
autophagy,
cell
migration
and
tumorigenesis.
The
current
understanding
of
the
myosin
VI
protein
does
not
explain
how
it
carries
out
these
diverse
processes,
as
functional
mechanistic
studies
are
lacking.
Alternative
splicing
in
the
tail
region
generates
myosin
VI
molecules
with
different
features
(myosin
VIshort
and
myosin
VIlong)
but
little
is
known
about
the
impact
of
the
variable
region
on
physiological
and
pathological
functions
of
myosin
VI.
In
this
study,
we
have
analysed
the
myosin
VI
isoforms
from
a
molecular
and
a
functional
perspective.
Using
quantitative
mass
spectrometry
approach
and
modern
NMR
we
identified
and
structurally
characterized
clathrin
light
chain
as
novel
and
isoform-‐specific
interactor.
Within
the
novel
clathrin-‐binding
domain
that
is
unique
to
myosin
VIlong,
an
isoform-‐specific
regulatory
helix,
named
α2-‐linker,
defines
a
specific
myosin
VIlong
conformation.
Its
presence
or
absence
determines
the
target
selectivity
of
myosin
VIshort
and
myosin
VIlong
isoforms,
acting
like
a
molecular
switch
that
regulates
their
functional
involvement
in
migratory
or
endocytic
pathways,
respectively.
The
adaptor-‐binding
RRL
motif
is
embedded
in
the
clathrin-‐binding
domain
and
is
masked
in
the
structural
configuration
adopted
by
the
myosin
VIlong.
Consequently,
the
previously
identified
RRL
interactors
show
selective
binding
to
myosin
VIshort.
Thus,
we
can
provide
for
the
first
time
a
mechanistic
explanation
of
why
the
various
isoforms
show
different
localization
and
function
(i.e.
myosin
VIlong
selectively
involved
in
clathrin-‐mediated
endocytosis).
We
also
found
that
alternative
myosin
VI
splicing
is
aberrantly
regulated
in
ovarian
cancers,
where
exon
skipping
dictates
myosin
VIshort-‐only
expression.
Importantly,
cancer
cell
lines
selectively
expressing
the
myosin
VIshort
isoform
exhibit
severe
migration
defects
when
myosin
VI
is
knocked
down.
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