hello I'm Ron Vale and today I'd like to
tell you about experiments that led to
the discovery of a new type of
microtubule motor protein called kinesin
and in this talk I'd like to highlight
this experiment shown in this video
where kinesin motors are attached onto a
glass coverslip and are transporting
these microtubules across the glass and
I'll explain to you why this experiment
came as a complete and utter surprise to
us and also how this experiment changed
the course of the research project but
also changed the course of my life and
career as well but before I go into
these experiments I just like to share
some general thoughts with you about
scientific discovery we often teach that
the way scientific discoveries are made
is through a process called the
scientific method and you can see many
different versions of them here but they
mostly reduce to a similar theme a first
coming up with a question then
developing a hypothesis and then doing
experiments to test that hypothesis
analyzing the data and then coming to a
conclusion now many of the scientific
projects that I've been involved with
haven't followed such a linear path and
been so predictable oftentimes you get
started with an idea but then the
experiments take you in all kinds of
different directions and there are
various twists and turns that happen in
a project and then maybe if you're very
lucky you come across in one of your
experiments some really unexpected
result that tells you something very
important and very new about biological
systems and I'd like to illustrate that
with the story that I'm going to tell
you and this story I think is very much
like a detective who done it's story
where we started a following in fact the
most obvious suspect but only found out
later that that suspect was not correct
and we were on the wrong track so then
we had to go back and find new clues
develop new pieces of evidence put
things together in a new way that got us
back on the right track and allowed us
to come up with the correct answer and
indeed many scientific projects are
really like detective stories so today
I'd like to share with you my detective
story that started off when I was an MD
PhD student in the lab of Eric shooter
and I was interested in this problem of
how material is transported inside of
nerve cells in particular I was studying
this one hormone called nerve growth
factor which interacts with receptors at
the nerve terminal and then some message
must get transmitted transmitted back
through this axon back to the cell body
but this axon in fact can be
fantastically long even in a mature
motor neuron in your body the distal tip
of that nerve terminal may be at the
tips of your toe that axon from that one
nerve cell travels all the way up your
leg and then the spinal then the cell
body would be in your spinal cord so to
transport material over this long
distance there had to be some machinery
that was moving material inside the axon
but in 1983 very little was known about
what that machinery was but an
opportunity to begin to understand this
problem came about actually one floor
below me at Stanford in the laboratory
of Jim's Budaj where a very exciting
experiment was going on between Jim and
a sabbatical professor Mike sheets and
what Jim and Mike were trying to do was
to develop in vitro assay to study
muscle contraction and what they did in
their experiment was first of all to
purify muscle myosin and then add that
purify muscle myosin onto plastic beads
and then they added these myosin coated
beads onto actin cables which were
derived from a plant cell might Ella and
you can see these
beads actually clumps of beads being
transported along these actin cables and
from watching these movies that I saw
one floor below me downstairs it was
very tempting to think that maybe a
similar process was going on the axon
perhaps myosin was coating the surface
of membrane organelles and it was
transporting these membrane organelles
along actin filaments inside the axon so
Mike and I wanted to test this idea with
a very simple experiment which was to
inject these myosin coated beads inside
of an axon and see if they would move
see if they would be transported along
actin filaments so to do this we had to
find a good experimental organism and we
chose the granddaddy of all axons which
is the giant axon from the squid which
has an axon about 50 times bigger in
diameter
dennah mammalian axon so the next step
was to get some squid so I called the
Hopkins marine station in Monterey and
asked them if they would be able to
provide some squid for these experiments
and they should sure we'd be very happy
to do that we'll give you a call when we
make a catch of squid well weeks went by
in fact months went by and we never got
a phone call from Hopkins marine station
so eventually I called them in June and
I said well you know where's our squid
and they said well you know we've been
trying to catch them but it's been a
very unusual year we we've been trying
to fish for them simply haven't been
able to catch any and indeed that was an
unusual year as shown in this graph of
the amount of squid that was caught in
California over various years and that
year was 1983 where there were there was
a precipitous decline in the amount of
squid that were caught and we now know
why 1983 there was an unusual
meteorological phenomenon which is
called El Nino that warmed the coast of
California and when the water warm the
squid just disappeared so this seemed
like incredibly bad luck
however this
turned into in fact good luck in the end
as he'll describe to you and in fact is
it's funny to think but perhaps Oh a lot
of the luck that I've had in my
scientific career to this El Nino
phenomenon because what El Nino did was
to force us to change our plans and move
and do the experimental work at this
beautiful location which is the Marine
Biological Laboratory at Woods Hole now
not only was there a lot of squid at the
MBL in Massachusetts but there was also
incredibly exciting research going on at
the time indeed an entire new revolution
in microscopy that was developed
independently by Robert Allen and shiney
in a way and what these scientists did
was instead of looking through the
microscope with with your eyes they
attached a video camera and also used
computers to enhance the contrast of
these images and saw details inside of
cells that were never seen before by
microscopy and Alan Brady and lassic
used this kind of microscopy technique
to investigate axonal transport in the
squid and they made these amazing movies
such as shown here where you can see now
all of these fine details of small
organelles being transported up and down
in this axon so the for the first time
axonal transport could be directly
visualized so this was all very exciting
but again we had some bad news right
when we arrived at Woods Hole and I
opened up this issue of nature and found
out to my great surprise that someone
else did the experiment that we wanted
to do they took foreign particles were
to in fact eat small plastic beads and
micro injected them into an axon it
found that they were rapidly transported
inside the axon except it wasn't exactly
the experiment that we wanted to do
because these were not myosin coated
beads these were inert beads that had
nothing on them and in fact this was a
mystery and the investigators themselves
explain it they thought well maybe there
was a flow of fluid going on inside the
axon and these bees got caught up in
this fluid flow or perhaps myosin got
picked up by these beads or maybe they
were just piggybacking on membrane
organelles so they didn't have a good
idea of what why this happened undaunted
we went ahead and then did our
experiment with myosin coated beads so
we took our myosin coated beads added it
to the squid acts of plasm and the
moment of truth came and we saw nothing
the these myosin coated beads did not
move at all ah so that was disappointing
we then still had these control beads
that didn't have any myosin on them and
tried them and surprisingly these beads
moved so we got this same strange result
that these other investigators did which
we couldn't explain either but what we
did know was that our original idea that
of these myosin coated beads finding and
moving along acting cables in the squid
axon was somehow incorrect so we had to
go back to the drawing board and develop
a new plan of Investigation and Mike and
I did that by joining up with Tom Reese
and Bruce schnap Tom had a laboratory at
Woods Hole and both Tom and Bruce were
exceptional electron microscopy and
Bruce also developed a video microscope
in Tom's lab and the four of us worked
as a team to do all the experiments that
I'm going to describe to you next so
first of all to probe the system deeper
we took the the inside of the squid axon
out the axe' plasm kind of broke it
apart and then found out in this
dissociated AXA plasm transport still
continue but now you could see that
there were individual tracks that were
supporting this axonal transport however
with the lower resolution of this video
microscope we didn't know what these
tracks were so we had to go to the
electron microscope to get that detail
and Tom and Bruce did this brilliant
experiment where
they first looked at a field of view
under the video microscope and then
found the same field of view under
electron microscope to find out what
these filaments were and indeed when
they went to the electron microscope
they found organelles on single
filaments that had the diameter and the
features of microtubules so the main
tracks were axonal transport the long
distance tracks were not actin they were
microtubules but the question then was
well the the motor could be myosin which
only worked on actin so some other motor
had to be at play here and we originally
had some hypothesis what that motor
would be but those hypotheses also
turned out to be wrong so we had to take
an unbiased approach to understand the
motor and the machinery that was driving
this transport so one way to take this
approach was to reconstitute the system
essentially take it apart put it back
together again and find out what was the
essential machinery and perhaps the
motor so we did this by introducing
tracks which were here made from
purified tubulin we then also took the
cargo which were the membrane organelles
purified from the squid giant axon and
added ATP as an energy source and we
figured that the motors were going to be
bound to these membrane organelles and
when they had ATP they would move along
these microtubules but again
disappointingly the experiment was
negative we didn't see any movement well
then we rationalized okay maybe
something is missing here something else
is needed so we added to this mix a
soluble protein fraction can handing
hundreds of different kinds of proteins
and to our joy this worked we now saw
lots of membranes moving along these
microtubule tracks and this happened
late at night but before going home we
decided to do what was kind of a boring
control experiment just to make sure
that the things that we saw that we're
moving were not particles coming from
the soluble protein fraction but in fact
we're indeed the organelles we mixed
everything together but now without the
organelle so we just added the
microtubules
ATP and the soluble protein fraction
expecting nothing to happen at all well
what happened blew our mind what we saw
under the microscope was that these
microtubules were attaching themselves
onto the glass coverslip and they were
crawling across the surface of the glass
like little worms and this was as I said
completely unexpected we had no
anticipation that this was going to
happen at all but then in thinking about
this in more detail what we thought was
happening here was that in the soluble
protein fraction there were motor
proteins that were just floating around
in the cytosol and they attached onto
the glass surface and they were grabbing
hold of these microtubules and then
transporting these microtubules across
the glass surface so suddenly this old
experiment that we did with these
control beads made sense
maybe these control beads were picking
up this soluble motor protein and then
moving along these microtubule tracks so
we now tested this again using our
soluble protein fraction and indeed
these beads picked up a motor from the
soluble protein mix and we're moving
beautifully along these microtubules so
why was this a surprise well the reason
is that most people in the field
including us thought that the motor for
this transport would be permanently
bound either to the membrane organelles
which were the cargo or onto the
filamentous track like the microtubule
but no one expected there to be a lot of
soluble motor floating around in the
cytoplasm but the fact that there was a
lot of soluble motor gave us an
opportunity to think about now purifying
the motor protein from this complex
mixture of proteins the problem was this
experiment happened at the very end of
August and I was supposed to go back to
medical school in two weeks and start my
clerkships and it would probably be
another three years until I was going to
get back to research again so I called
up Stanford and I said a very exciting
experiment I needed to keep working on
it can't I postpone my medical school
clerkships and they said yes so I
that fall and winter I spent trying to
purify this motor protein now Woods Hole
is a very exciting place lots of people
there in the summer and particularly in
the 80s there was really very few people
at Woods Hole in the winter so I kind of
had this interesting monastic
environment of just really trying to
focus on this problem of purifying the
motor and of course it was really one of
the most rewarding and exciting times in
my life so the experiment was really to
use this motility assay in combination
with protein purification techniques to
identify which protein of these hundreds
of proteins that were in this soluble
fraction was the motor and so you could
use this motility assay for this purpose
so here are different fractions coming
off of a protein column and here each of
these fractions now was assayed for
motor activity you can see this peak of
microtubule motor activity here
corresponding to this polypeptide which
had never been described before and
since it was a new type of protein a new
type of motor we needed a name and a lot
of people ask me where'd the name
kinesin come from well I could tell you
that it came from a phone call with my
mother and indeed my mother from the
greek root kenny came up with the name
kinesin since 1985 it's also been
amazing to see how the whole feel of
kinesin has developed in 1985 we knew
almost nothing about how this motor
moved objects along microtubules but
since that time single molecule acids
were developed it was possible to get
x-rays and e/m structures of kinesin all
kinds of protein engineering experiments
you know these things done in our lab
but many laboratories around the world
and now we actually have a pretty good
idea of how this motor is actually
moving along a microtubule track the
same thing happened in biology at the
beginning 1985 we at one kinesin motor
but since then many kinesin motors have
been discovered it's now known to be a
large super family they're known to be
involved
many different roles in cell biology and
several neuro degenerative nerve
degenerative diseases have been linked
now to mutations in kinesin motors as
well so it's been a remarkable adventure
a remarkable journey this detective
story that I told you about had moments
of frustration when we couldn't get
squid we had times when we had results
we simply couldn't explain but there
were also these fantastic Eureka moments
that you really will remember for the
rest of your life and also an
opportunity to really dig into a problem
and emerge with a satisfying mechanism
now what about my career in medicine
that I never did go back to medical
school I never did complete my
clerkships and that was all all for the
best I've enjoyed being a scientific
detective and I have enjoyed all the
problems I've worked on and I hope that
you will enjoy your scientific detective
work as well thank you
you