And that thanks to Jennifer and
louder cannot be for inviting me so

I am a professor of physics I know
surprising because I look like I'm twelve

but I'm not well five promise and so

because I'm a professor of physics I mean
I do a lot of teaching and I was and so

you know this is these are the things that
you do and I really enjoy teaching and

so of course I'm going to start off my
lecture with a quiz Are you ready OK so

you've got one two three four movies
here and what we're going to do is

don't worry about this is just an opinion
poll to start off with this quiz so

we want you to do is you're going to vote
on whether you think these are these

movies or from life living life cells or
if you think there's something that

we recreate in the laboratory OK So
first we're going to do this movie here so

raise your hand if you
think it's a live cell.

OK if you but I RAISE YOUR HAND IF
YOU THINK IT'S recreated thing in

the laboratory OK that was about fifty
fifty it was about this one in the middle

this is actually the same thing red green
and red green overlay if you're color

blind don't look here look here all right
so raising a visa from a live cell.

OK Raise your hand if you think it's
only recreate in the laboratory for

a more of you thought it was recreated
again what do these two together

to raise your hand if you
think these are live cells.

OK most of you raise your hand if you
think it's something we recreate in

the laboratory OK if you
feel that if you cheat.

If you've been to my if you've been to my
Web site this quiz list on my web site and

so you could have cheated by already
taking it I said you one of the answers

OK here they go all right so

actually most of you got fifty percent of
you were right on this one it was alive so

there's something about this one it looks
fake I think because it everybody always

thinks it's reconstituted and
it is but this one is the one.

Always full of everyone or a mostly
everybody and these two systems have been

reconstituted in the microscope
from small component parts and

they do very much look like cells and I'm
going to do another set of questions for

you so why do these look like life cells.

But now.

It looks it looks a guy has
some kind of disorder but

it's a completely disorder.

When he actually has order a I mean it
has structure some kind of structure.

Plus I you know here's the thing let
me ask you a question What's this.

What is this a nose OK you have a nose and
you have a nose and

human nose most people have a nose not
everybody but most people OK So and

when I look if I were to take all of
our noses and average them together

they would make an average nose and
you would know it was a nose right but

if I would take that the noise right is
the standard deviation of those noses

there'd be a very large standard
deviation gets to be both in those and

some people take notice that's what we
expect from biology we expect to have

an average shape plus a lot of noise
on top right and that's what I

think you're noticing here that's what we
expect from things that are living right

there's another thing about it most people
say maybe one of the students would one of

my just look alive.

It moves them well that's not cool.

Are you five is what I was about
a fabulous in the front two rows right.

All right so
you know it's a dynamic rate so

we also expect that living things
are dynamic that they move right and

actually it's one of the first things
you do when you come across a dead body

where you do they have that dead body
thing or somewhere in Georgia to learn

about how a corpse is decay I think
it's somewhere here right it's hearsay.

Where I don't know where it is but

the first thing you would do if you came
across it I would like lift up the hand

and I'd make sure it doesn't
move autonomy Asli right and

that's why zombies are creepy because
they're dead and they move so

actually I call the zombie systems
right because they actually move but

they're not alive I mean
they're not living cells but

they're something that we recreated.

Yes right you have the walking dead here
that's very very exciting All right so

so OK so this is a big open questions that
I'm not going to claim to solve our answer

at all so let me something look alive
now this is something we might be able

to quantify this and that something maybe
a physicist might be able to answer

this Miss Middleton is probably not
something maybe well maybe physics

could that contribute to answering it so
well make something actually be alive so

what's interesting looking alive and
actually being a life right and

so you know there's probably some There's
probably some philosophy philosophers need

to way remember we are all of science
started as philosophy right we're trying

to explain the world so I think it's OK
to have these big open questions here but

you know so I think maybe in twenty years
if we've made a dent on any of these

things it would be nice perhaps.

Let me put it a little bit of a plug for
bio physics I don't really have to in this

group because there's many
biophysicist here but

I think that reconstitution of functional
biological systems in whatever way you

want to reconstitute is a very important
grand challenge of biological physics so

in my case it's these things in the test
too right but in the case of somebody else

here could be making robots that do things
the way that animals move right and so but

we have to if in order to understand
things we build things right this

is all fine and quote right and I didn't
quote him I just say he has a quote I

don't know what it is but it's about
how are you in order to understand

something you must build it and so this
this is actually the way Fineman proposed

to study biology he wanted to move on to
biology towards the end of his career and

you propose that we study it from the
bottom up that we recreate these things

and that we will truly understand them and
in order.

Really after you've recreated that it's
not quite enough to under to recreate and

we also have to measure them right and
so quantitative biology or

bio physics it might in my mind go hand in
hand right we we want to measure things

and so for instance here you have this X.
on we can recreate something similar to it

in vitro with say you know so there's
cargo transport up and down this X.

on and we can recreate that by putting
these kind of the cargo transport motors

on these beaches and walking them
around and see these things but

what's actually more interesting than
that is if we track our is a function of

time we might be able to start something
about half as a function of time and

this is what we really want to
do as physicists write in so

everything in physics started by particle
tracking OK So planets particles and

proteins right all of them started
with particle tracking and then from

there we were backing out forces ready and
that's what we want to do and all

the REAL LIFE IS biology so one biology
is really hard because it's young OK so

I know we're not used to think about
biology as being a young field

because we've been naming things for
a long time but that's not really biology

right it's not understanding mechanism but
like true molecular and

biochemistry these kinds of things this is
a very young field microtubules thing I'm

going to tell you about there are women
who graduated from my alma mater

with biology degrees in the fifty's
who did not know what this protein was

this protein didn't exist to them and they
did an entire degree in biology right so

this is this is very this is it's
a very young field and we and

I should also add here animals
cells right all of these things and

I just wanted to have in the literature
and so I put proteins there but

obviously with everything in order to
learn the rules behind it the first step

is to track it right OK so what we're
interested in my lab is interest so

organization so
I'm motivated by pictures like this so

here's a plant cell a neuron so we added
cells like you might find in your guts and

then this is a cell a cell culture
dish is going through my ptosis and

what's being highlighted in ever.

A single one of these movie everything
one of these images are these long

filamentous things and these long
filaments of things are microtubules and

what I find amusing is that this
sequence the genes that are coding for

these proteins are the same they're
like almost the same between plants and

you and your guts and whatever
sheep yeast there are they're very

very similar in fact I can take used to be
alive and do all my experiments we used to

have only minor biochemical perturbations
right the majority of what of way used

to really works is the same as the
majority of the human two and works and

so what I'm amazed at is how this
same protein can exist in all these

different cells and it's a very vital for
almost every cell in your body and

yet it looks completely different in
a nerve cell than it does and I got so

right it's it's organized in a completely
different way how does it do that

there is no micromanager in there
right telling it where to go

right is also forgiveness ation right and
so this is a place in physics that we're

not very good at right so we're not very
good at understanding how you inject

energy into systems Anderson and how they
or things can organize themselves and

so that's what that's what I am I want to
learn that the biology and we can learn

some physics from that right because we
can learn from the non equilibria active

matter type of physics from us so
who knows about microtubules already but

not that many are right so
we'll go through it a little bit slowly so

this is a microchip a real
cartoon it's not a real one

they're not this big and they're.

Circles so I am so it's mostly so but

it's a it's a schematic of what a model
not the kind of model that we write down

in physics but but
more like a model ship All right so so

it is also a cartoon so those are my she
was twenty five millimeters in outer

diameter seventeen animators
an inner diameter it is a tube and

it's made out of these two real and
proteins that bind head to head the.

An Alpha and a beta they come in
a dime are always a dime are right so

you never find them just Alpha or
just beta always alpha beta together after

the beta bind to each other head to tail
head to tail head to tail head to tail and

they form kind of one of the substructures
here's the thing we call a proto film and

the reason why we call this the proto
foam it obviously is it's a clear lattice

spacing that we can identify but
in addition we can classify the different

types of materials by the number of
filaments they have inside of your cells

you have mostly thirteen proto filament
microtubules you see this part of this

dimer is eight nanometers high is about
four nanometers around and they bind and

these these diaries always bind head to
tail you can never flip a dime around and

stick it in it always binds in the same
orientation within the part of Solomon

because these proto filaments then
are bind together in the same way than

one end always is kept with an alpha the
other end is always kept with the beta and

we give these different and
different names this is the plus and

with the beta and the minus and
with the elf are you paying attention

because there's going to be a quiz later
OK so you have to pay attention OK So so

the plus and so this person minus
It has nothing to do with charge OK

it has to do with the fact that the plus
and actually adds new sub units faster so

the way you make these guys
is you nucleate something and

if you're looking for a really outstanding
problem in bio physics how microtubules

nucleate is completely unknown OK So
there you go I just gave you the next

Nobel Prize seriously we don't understand
how much your two rows are nucleated but

so once you have a little bitty
microtubule then you can add individual

dimmers on to the end they add faster on
the plus and on the minus and and the hair

structure has this overall polarity and
again I don't want to get into semantics

about this is not charge I should say
that my travels are charged these

Kobach see terminal tails which actually
come into play tomorrow tomorrow so

talk are highly negatively charged and

every single one of the monomers Alpha and
Beta has one of those and so

they they are actually a really
important part of the structure.

But they're an intrinsically
disordered part of this protein yes.

Could I I wasn't going to point that
out but you're absolutely right

there's a defect right here it's called
the seam and that's because the when all

these got guys to come together
there's thirteen proto filaments and

when you wrap thirteen performance around
with this particular kind of Cairo ality

then you have to reconnect with a seam
where instead of a bit attaching a bit

a sideways or an alpha touching an affair
you get an alpha touching a beta and

it is not known exactly what the seam
is doing nor if it's a weak point

mechanically in the microtubule but it is
thought that perhaps it could be weak.

Good i All right so in our lab over
the last eight years we've been studying

a variety of aspects of microtubules So we
have some work on my kid to go mechanics

to Mars talk is about these
microtubule severing enzymes and

we're also going to touch on my good she
will dynamics in that talk and then but

today I'm going to tell you about self
organize ation of microtubules into

the power of molecular Motors we will flip
side of that those sorts of experiments

where we actually look at organizations
that are already made and

then try to understand how motors
navigate across complex networks

I'm not talking to you about that but
I did just submit a proposal to see bits

of any of you review that
think it's not in the stock.

So I'm So we're very again you know
influenced by movies like this so

this is a movie of a cell dividing this
is it was taken by the IT WAS WORTH

who's actually my cross
the street neighbor at U.

Mass to pick up a few of those are going
through my toes this is a G.F.P.

labels to realign cell line and you can
see it's going through like these various

stages which everybody gives names to so
there's this interface is very and

in this case at the very before my toes
says than the microtubules all arranged

in an astral array where there minus ends
are gathered next to the nucleus and

their persons extend out throughout
the whole maker the whole cell and

then they completely rearrange and
form this my.

Todd expendable and that this
arrangement is the kind of the topic

of many people who study my ptosis they're
very interested in how this forms and

then you as after you've created
the perfect Mike are my top expendable

the chromosomes are lined in the middle
you separate into tonight new cells and

what you're left with is a microtubule
bundle that's between the two

daughter cells and then you kind
of restart the whole process and

there's obviously other side of skeletal
filaments and here are motor proteins and

things like this allowing this to happen
but this is what the process looks

like from the micro to go point of view
and when I look at something like this

I think about phases that these could
be different phases in this case

it's all energetically driven so instead
of phases I think of study States but

it makes me think of something like this
right and so on if you had this thermal

phase diagram for let's say water you
could go some liquid gas or liquid gas and

you know once a day you could go through
this cycle just like i said i once

a day goes through its cycle but
instead of again this is a phase diagram

where you have pressure and
temperature on the axes but

instead what we want to know about is a
state diagram and instead of pressure and

temperature instead of temperature we have
activity right or A.T.P.'s activity or

enzyme activity of many many proteins we
don't even know how many necessarily and

we have number of objects and so you
can think of each of these as analogous

number of objects a similar pressure
activity is similar to temperature and so

the question is that and

this is obviously fake don't this is not
real data right I just drew these lines in

here with the idea that you know could we
dance around something like this in order

to go between these different phases is
this is this some way we could understand

the way that cells pattern
themselves based on physics and

chemistry physical science understanding.

So a lot of people have been studying
this these studies States and

what they do is they start with like
a bag of goo of all the things and

then they go turned on and
then they watch.

It and it evolves into some state and
that's cool but

actually cells don't do
that very often right so

only when cells are like egg cells to they
start is a big bag of goo that then turns

into something actually most of the time
so a star in one of these states rate and

then they they go from
one state to another so

although it's important to understand
these states from the homogeneous state

right it's actually also important to
understand how these states can flip from

one to another this is a huge huge open
question I'm not going to answer OK I'm

going to keep telling you about really
cool problems that I can't answer yet and

the hope that some are you will
be very excited about it and

want to join my team and
want to work on these things with me but

I really would love us to
get into a steady state and

then mechanically or
chemically trigger into other states and

what I really what I think is
that you know the importance of

this steady state is not to have you know
the state itself it's actually to position

yourself to transition because when
you have not equilibrium phenomena

right you're injecting energy when you
have a phase transition a state transition

the energy injection could take you
crazy in a crazy place the initial

conditions are very important and so
I think actually the initial conditions of

those city states why there's so
many checkpoints on the my top expendible

it's because if you don't have
the perfect my toy expander

you're not going to transition
into the next state properly and

you're not going to divide the cell right
so these initial states are important but

the transitions are really important in
the states are just setting you up to

transition properly and

the transitions are important because
this is where force is being produced

it's where the actual process is occurring
and perhaps even the essential step for

life certainly if you sat in my talks
panels really pretty to look at but

if you sat in my top expend all day
long you're not dividing yourself so

it's actually the transition that's
important All right so we need in order

to do this well we're going to start
off with just first the structured

organized which is microtubules and

then we're going to use we need some kind
of energy and action so the system so

we're going to use are these
again I'm just showing you a.

Two of these motor proteins the motor
protein that we're going to look at is can

use in one motors and they use A.T.P.
as an energy source I'm going

to give you like two seconds I can use
a motor proteins OK so they're made out of

a dime of heavy chains I'm showing you
a blue and pink here just so that you can

distinguish them but they're actually
the same protein they bind together so

there's this big Robbie Parker is about
foreign enemy Toure's and size and

it's where the enzyme is it's where A.T.P.
binds It's where binds to microtubule and

large scale conformational changes
occur upon binding of A.T.P.

or hydrolysis that make it change shape so
that it actually takes steps OK And

then it's it's a dime of these
two heavy chains and it locks and

they hand over here
manner like this ready.

OK so right here you guys ready to be kind
Hughson because it's about time you got

falsely so now you're to wake up so
put your arms in the air

OK now it will make your hands in the fist
because these are the motor proteins

now cross at the wrists and they're trying
to swing on monkey bars OK so if you swing

a monkey wrench this is how the rock so
I used to do a funny walk I'll do it for

you now where I try to walk with my ankle
side together OK it is really hard and

sometimes I fall down so it's
embarrassing so I stopped doing that and

I started just making you guys do
this all right so it only takes to

these little tiny steps because it
actually goes on a lot of spacing OK let's

remember what was the other spacing
of the microtubule remember.

Close double that.

Eight Yeah the monomer was four there
you go all right so goes forward so

it takes even a meter steps uses one
A.T.P. first step and it's a process of

meaning it can take multiple steps in
a row where of for about one micron

on average is an exponential decay because
at any step there's a constant probability

it'll fall off and so you know one might
go for three steps and one might go for

ten thousand steps and then there's
some the average is one micron and

they can go top speeds of about one micron
per second so this is an impressive

machine OK but it's only it's only
fair only in a meter is it sticky.

Reader steps in it can go one micron a
second that's that's really fast many body

lengths per second you can't go
many body lengths per second.

Right this is faster than you with
you re skilled you all right so

I'm going to do another quiz
Are you ready you OK so

this made a protein and
it walks towards the plus sense OK so

now I have this is when is your automatic
She was right it was for us and

I'm going to flip the whole system around
we're going to put the motors on a glass

surface and it's going to it's going to
move and I get to know what direction will

move the microtubule I want
us to vote with your hands.

OK so it looks like most of you got it and

some of you are are are abstaining from
voting and many of the people in the back

have abstained from voting that's fine
OK so it's when I get to the process and

these motors of the bottom rocks where is
the person which moves the material so

minus I am for those of my friends who
are in high energy this is relativity it's

just it is Galileo and relativity is the
classical right is not it's not it's not

it's not general relativity so special OK
so what is meant to be because you can

either be in the my God She rose reference
frame and see the motor proteins walk or

you can be in the motors reference frame
and see them are going to really go and

in fact you can do both of these
sides of experiments right so

here in the Maker she was reference
frame and you see the motors walking and

here we have is a set I have
of a gliding of of sorry it's

a ban on of motor proteins and the
individual microtubules are now on top and

they're moving around and
I don't know I was supposed to hand out my

thing earlier OK so
I headed out now because this

is another time tis you're going to fall
asleep so these are my city bills so

if you want to see what my good she rose
looks like they look like Twizzlers.

Fruit Well take a second and

inspect the mice are still there past
some around I guess individually

wrapped land because apparently I
don't care about the environment.

I was worried that you guys were
very upset because your hand.

Well each other's can there
should be more of that.

Crane has some back.

In them for him some tax money.

Because have enough.

That's around.

OK.

So.

So if.

You've got to bite the ends
off to see that it's hollow

if you buy see ends off
it is actually had.

Well.

It doesn't have enough credit film it so

that actually it's very difficult to
find a perfect microtubule Candy.

We have a mutant show this was
made at a high salt lake hair

new hair doesn't have hair.

Right and that way when I bring them
together they're appalled as I always say.

If you look at these guys they are left
handed he was to use like Mike or

two girls I saw Sam the other
day that were mutants and

they were right handed I didn't I didn't
get those if you buy of the end they do

have a hole in the middle and they're fun
to eat tomorrow I'm going to talk about

severing enzymes I show you how severing
enzymes work like this more of an.

All right.

Now is so
these guys are you there right in there or

you can have them by around where we
just did was kind of similar to this

right I just passed out a bunch of
microtubules also this is similar to so

how many of you like music your music
I mean if you go to concerts still

grownups OK And how many of you have
been to a concert with a crowd surfer

where biophysical Society meeting
actually if you go to that

come to my residence Society meeting go to
the dance there's always a crowd so for

the end of the DIDN'T night after
everybody has had several drinks.

So that is it so that's basically what
these guys are doing right there's motor

proteins all down right if I had
you guys put your hands up again

to be the most proteins and
then I just like jumped on you and

then you guys can push me around that's
precisely what's going on here and

so we can visualize these things
actually with we can come across but

they're actually space time pots just like
high energy so you draw a line along your

path and then we just put images
of this path side by side and

it creates our kind of graph where space
time plot now anything that's moving

with a slope has a velocity anything
that's vertical does not and

this I this is like terrible Right like
when you want time here in like space here

but this is how Image day makes a so
this is so I was sure like this but

I always also come and
this is not great but

anything here that's vertical is not
moving anything that has a trajectory is

moving it's a little bit more difficult to
do one extra step to actually figure out

what the velocity of the microtubules are
the first thing we do is make a standard

deviation map so for for an entire
movie we collapse the movie and and

report the standard deviation of the city
Vientiane is low then it's dark so

you can see in the background
there's only noise fluctuations so

it's dark and if the C.B.
isn't as high but only.

If so also if Sorry if there's a high
signal but it's always constant

you can see here see it's dark here
because this is a piece on the background

that just stays there and so here it just
turns black and so the standard deviation

is also low there but anywhere where you
had dark and then light or light and

then dark the seriousness high and so
now you have a trail and so now all this

little snake trails that you're seeing
are where microtubules when we can draw

a line along one of those trails move
it up to our Mike or to our movie and

now take make our camera graph of
the movie and you can see the velocity.

Over time right so in fact you could even
see velocity changes or loss of mass or

anything like that over time.

All right so what we are interested
in is how can we have a very

small group of reconstituted things and

how to start to make things that look
like cells right and so we're going to

we're going to like I said the micro tools
are the things we're going to organize and

then we're going to we're going to add
these motors and they're going to to

move in and give us our motion in order
to cause organization and so on the first

thing we're going to do is just change
the density of the microtubules and

we'll keep the motors the same and
to see what happens OK so so

our first question is can just crowding
alone stare kender and sorry biting into

each other like a at a very high density
like a liquid crystal perhaps calls

organization right and so we have
a very high density of microtubules and

we're labeling only one to three percent
of them and so in these images here what

you're seeing is actually only one in
a hundred most microtubules labeled so

there's a hundred times as many
microtubules down and so of course this

is what you have seen
assistive this work was all

done by Lynn Lu when she was an undergrad
in my lab she had a paper come out and

she is now a grad student at Yale in
the biophysics program there I am going to

play the movie and I want you guys to
look at them and tell me what you see and

what's interesting this is what we do in
my lab a lot I go do that thing and then

the soon cuz I go what's interesting OK so
now you're in the lab what's interesting.

Circles Yeah.

Yes you see collisions and use some
circles there's a lot of crossing that

doesn't result in anything right but
then there are these kind of circles that

come about so this is what Lynn also
noticed and she noticed it was it was

not just obvious from these craft movies
but was actually obvious from the whole

movie if you had ten times as many of
these loops as you do normally you

you sometimes get loops just in
a regular lighting I say but but not so.

Many of them and so she started
she tried to characterize them.

And so what we are seeing is no long range
order only the short range bumping and

this was this was just her being because
when people do the same thing with

acting in my ascent they see like these
beautiful long range order like always

flock eating it works perfectly with all
the theory from John toner and I didn't

see any of that and like all great I say I
am like our system doesn't do that right

if you want to talk to me more about why
I think this doesn't and that does and

other things that I think we could do
to make it do that I can do that but

it's kind of long and drawn out so
I'm happy to chat more about how I think

motors are actually the the actual
biology of the motors are affecting this.

So I added this actually for

Harold because we talked earlier
today about bending and so

these microtubules have a persistent
length and a half a half a millimeter and

yet the rate loop radius that we're
measuring is one to two microns.

OK so if we use this bending energy
equation as the contour length

you know anywhere from less than five to
all the way up to twenty five you see

the majority of them actually have this
same radius of curvature no matter what

their length is and these radio courage
are actually which mean one in ten which

is what I'm showing you here but actually
mostly a one so if we use one on one

micron we actually see the and the bending
energy is is twenty five hundred K.T.

that's really that's a lot of
energy right and so you know so

what's keeping these it while it's being
buffered around by the other buffeted

around by the other microtubules but then
also you know there's motors on here and

these motors each motor can make five
people Newton's of force taking eight

nanometers steps so they can they make a
lot of force and there's many many motors

right there the whole surface is coded
in these motors and so the motors

we estimated based on the surface
density based on how many we add that.

They're actually contributing
to about half of this energy and

the other half is actually coming from
bumping into your neighbors very and so so

I know this is actually published in
this paper and it was it was awesome for

an undergrad but it did not it did not
make any large scale pattern formation

like I want it there are so we decided
that we need to add something else we

need to drive condensation by
adding in cross linkers OK so

the cross the thing we're going to do is
we're going to add these cross-link has to

make microtubules like each other they
don't like each other right they just they

just cross over each other they don't care
so if we can drive condensation maybe and

make some see each other and interact with
each other more that maybe we'll have

to get more patterns
Yeah that's what we want.

Our site yes I'm going to go through them
OK so the cross-link areas that we're

going to add is map sixty five which is
a plant will also see a protein which is

sixty five killed omes it's a really
exciting name All right unfortunately

to add some excitement to it they're
also called a swan if you're looking and

fission yeast over there P.R.C. one if
you're looking in a million cells so

although this name is very
boring they have three names so

that makes a lot more exciting and
also confusing but

if you look at this protein is found where
microtubules antiparallel overlap so

there is an anti parallel overlapping
cross-linking protein so what I mean by

that is so if microtubules where they have
a memory of a percentage of Meissen So

if your fingers or
the person's Why do those doing activity

then I refer you to the president
this is hair and that's antiparallel.

OK so this one is antiparallel so
it wants to cross them so that they go

like this OK and you can see that in the
cells I want to point out that it's used

this protein is used in a million stars
for my ptosis to set up the my top expend

all of this overlapping inner polar ray as
well the end in sight OK reason to have

this kind of a string you bundle that's
between the two new daughter cells and.

So all those states earlier right those
are two of the states that I pointed out.

So first thing we did was assume if it
was diagram OK so no motors I mean for

that someone were to know in
motors OK this is just thermal and

this was work done by a Muslim or to
Kumar does her last name look familiar to

anybody Yeah that's a move to his
daughter and so she was in our lab and

she did this all by herself because she's
moved his daughter there I was like just

makes the stuff together and
she came back with a phase diagram and

I was like Wow OK So and it was
repeated by many people in the lab so

you know if you can should worry about
a high school student did all this work.

Everybody actually repeated it so

what she found were there were three
phases there was this kind of gas phase

where they don't interact there was
a small bundle phase these small

bundles didn't interact with each other
they kind of just had a finite size and

then there was a could bundle phase
where the bundles of these bundles

would buy into each other there was enough
microtubules in there was a cross-link or

that everything would get kind of snarled
up together in the literature about this

protein only this had never been published
before no one had ever seen anything

like this because they were all biologists
they were like let's be physiological So

what happens when you're
not physiologically.

Right is it OK So but interestingly So
it was so then to my undergrads Amanda and

Josh just wanted to see what would happen
to a blighting as a sort of guide you

might get chills so the measure of
the not here and then they added in then

the interacting cross-linking protein and
then they measured the V. After OK and

then they just wanted to compare those
velocities How did they how do they look

so they plotted the relative velocity
as a function of cross-link or and

they found this really cool thing so when
you have this phase you have high velocity

and when you have this phase you slow down
and when you have this phase you saw.

Right and
it perfectly followed this phase diagram.

I'm not showing you the rest because they
were all pink which means there was no

velocity and if it matched at all of
the microtubule densities that we tried it

also matched at different velocities
initial Rossi's So if we had less A.T.P.

and had slower interactions we still
saw this if we had more E.T.P.

and had faster interactions
we still solve this.

But I thought that was really cool
All right so Josh was not happy

though because he could not see what
was going on that was it getting slower

right is it just that it's like acting
like stuck to the glass or is it actually

doing something like its own function and
so we did as we had a green for

us in protein version of map sixty five
this is a movie that I'm going to play and

the kind of graphs are going to pop up
over here as it plays OK but what you're

going to look at is there's a microchip
all here with Arrowhead microtubule here

and these are gliding at each other like
this OK and what you're going to I want

you to look at the measured She rose and
the green channel and again if you're if

you're a green color blind look up
here OK All right so here goes.

All right so
your my right again what do you notice.

They get stuck they slow down so

here you see the velocity is
high the last of these low.

What I'll see you notice.

But not the green channel what's
happening in the green channel.

When now.

It's binding it's actually binding I
mean you can I guess you can think of

a specific biting as opposed to
nonspecific aggregation I guess or

Gratian for using that as a negative term
and so my protein they stop working.

But yeah so it's binding So look at the
map sixty five first of all look at it on

the part of my teeth when they're not
overlapping it barely binds at all it's

kind of a crappy binder OK but as soon as
they get within range of each other to

overlap this thing by Is it biased
co-operatively really really fast.

That's right that's very and so Josh did
a really nice job of quantifying all of

this in this paper that came out two years
ago and what's really cool so this slower

velocity is actually due to the protein
interacting with tween two microtubules

right and so that the vases I was showing
you were an average velocity right so

obviously we can have sparks so even the
same will go faster and slower but overall

the average goes down and of course if you
look later times you're going to get more

and more of them that are stuck to each
other OK now I know this was beautiful and

there's even more data that I'm
not even showing you here but

I said to Joshua like josh All that's
really great but none of the six like

cells I want to see some pattern
formation let's do something and so

we thought how can we do this and so we
decided let's take the things we'd already

bundled the thermal bundles an atom to
a gliding asset so all we're doing in this

case is everything's the same as these
assays that we just showed you except for

We've added the crossing our first OK so

it's just the order of that we've added
things into the into the microscope and

when we do that we get these things that
you've already told me look like cells so

to.

So you know you go to a go that doesn't
really look like a cell too late

you already said so.

All right so so this to me looks like
different parts of the my top expand and

I'm going to show you here there's
actually three microtubule bundles of

people identify in the spindle
there's the the astral already is at

the end of the poles that helps to keep
the spindle in the middle of the cell

there's the Connecticut fibers that helps
to align the chromosomes in the middle of

the spindle and then ultimately to
hold on to them as they fall apart and

pulls the two chromosomes
apart from each other and

then finally there is this
overlapping Interpol ray and

that overlapping Interpol Ray is actually
what this looks like to me and it's

actually where map sixty five is found
in heal itself so that's pretty cool.

And.

There he also found structures or exist
in the same chamber this one looks like

the end states of my my toe says when
you're going through cider can you says

You basically have two daughters cells
right which look like these photos and

then there's a stroppy
bundle in the middle and

that's where you see again we have
sixty five buying and He La cells so

I was really excited about that and
then we saw things like this.

And this is that these are only three
things that we saw we saw him all over

the chamber and some nice products so

that we took the pictures in the green
channel to buy you didn't see anything

different as Looks like this so
what does this look like to you.

It looks like a gel for Jell-O.
or cilia that's ago I've had the Allen and

body else and I want to hear something
crazy and you neuronal head that So

to me I agree I think that's what I see
but what does anybody have anything crazy

here doesn't matter OK I have
I right when I want to talk

as if I have my dark matter friends I want
to write a paper a review article called

intrinsically disordered proteins the Dark
matter of biology because we can't see

them because they're not structured it's
perfect OK anyways i'm Yeah so so so

one time an undergrad said it looks like
sperm which is kind of like the sewing or

flagella but unfortunately the sperm
is moving in the wrong direction so

I told him that if this was
a sperm he has troubles and

used to go to see that one thing.

But I bet I could actually about
the neurons I think it looks like very

early time points of neurons in fact
here's a movie that I just found on

the Internet from this paper and these
are green fluorescent neurons so they're

making green source and proteins are the
whole neuron very early time points and

they're actually crawling along
you know these things in red are.

They well I guess this is in a brain
this is in the brain first of all

as I help you know these red things are.

The blood vessels so
these neurons are burned in the middle.

Your brain and they actually migrate out
to the edges and so this whole process

is happening here when you're when you're
a baby when you're in the world OK so

you've got these cells in here they get
born all in the middle part and then they

migrate out to the edges and that gives
your brain extra mass at the edge and

that's what causes these
kind of lesions and

these credit lesions are what
makes you smart and.

So actually my star have as
many incredible Asians for

instance as humans do but I should also
point out that there is no mutation so

if you have listened subtly
it's a it's a birth defect

where you have a smooth brain that's
what Listen listen smoothed so fully and

subtly as brain and what you see in these
patients and this is a child because

people don't live very long with us and
they have severe mental defects and but

when you see look at all this mass here
right those cells did not migrate out to

the edge here on the cortex right and
when I put normal brain I put it in quotes

because it's actually my brain that was
the reason for the pressure of my brain

in an M.R.I. when I was in grad school so
it's I don't you know who's to say right

OK They said it was normal they said it
was in our I don't know but you can see so

this was brain fever type where you have
all these extra Saarland here right and

so what's clear is that now we have a
hypothesis that biologist contests and so

Peter boss is this really
cool neurobiologists and

he's actually already shown that my target
proteins have a role in early stages and

rural development and so I called
them up and I sent in my paper and

I was I do think mob sixty five or P.R.C.
one could have an effect and

he was like come down we have to talk
about this yes absolutely this is a this

is a thing I think those things are like
great I knew you were a person.

He thinks outside the box.

That is a good question I think that
it's occurring over your development

of the brain but I think for an individual
one to do it it's on the order if it's

like a child's brain it's on the order
of days to get to get through and.

What's interesting too is actually
this process is dining driven so

the protein I studied as a grad student so
what's happening is the front of the axon

is moving forward but the back has
the nucleus at the back of the cell.

And it has to pull it up behind it and in
order to do that it actually docks dining

onto the onto the nucleus and actually
Toni's them up the axon to actually pull

the back up with dining while the front is
forward actually actually moves the hole

so it's a it's a it's so they're dying
mutations that cause listen softly and

proteins are associated with dining
that causes me Taishan as well.

So.

That's the thought.

Yeah.

Yeah but but yes exactly so

it's like a it's like a it buckles
It's like a sin film buckling and so

I think you know these concepts right from
soft matter these other concepts from self

matter I think have especially
in development have a ladder of.

Have a lot of consequences for

organ development in particular
brain development since you and

these are actually you know the shapes
of these Crennel lesions are something

that you can theoretically predict
from certain buckling patterns.

All right so we did this and I showed you
this really really cool movies but I only

did it at one concentration and just like
a one off experiment because I frankly

I just wanted to like I'm just mark my
territory a little bit on this problem so

I threw this in at the end of that paper
and so that we can be like we are working

on this OK so far but we've been and
we've been working on it since then so so

the next thing to do is actually do a nice
like systematic study where we change

the microtubule concentration away to have
an animation would say and we also want to

change the cost think or concentration
into it systematically and understand.

What happens and when do you get these
patterns versus other patterns or

activities OK so.

We have to we have to turn
it up to eleven right so

you have to be a to have a separate knob
but we don't want volume one in volume

two we actually want cross-link
a density and filament a C.

So who's old enough to actually know
what this is a reference to Oregon.

I also want to point out I picture
this front car of Georgia I

just want to let you know I
was in honor of you all OK.

And I also want to point out that this is
our world premiere of this so I haven't

shown it anywhere else and so if it's
totally crappy please just let me know and

tell me what to change because we haven't
written the sub or anything it's brand new

so what we're doing is we're
going to change the cross-link or

density we're going to keep the filament
density at the intermediate level or

level pretty much a low level and this
work was done by talky stand hope she was

an undergrad in my lab who stayed on for a
fifth year master and is probably going to

stand to be my lab manager and and so
what you see a little cross-link or

density is this what's happening here
what do you notice when you see it.

It is just dissolving the motors
just pull the whole thing apart.

Yes So it starts out as well John it's
really good to use the word OK So

basically you have motors on the bottom
and then we put it in the bundle.

So this is whatever these these
big craggy bundles right.

With cost linger well one without so
busy or it's just nothing we

actually did it with Peg to make it
kind of want to be cohesive and it just

just gets torn apart right so using
depletion to make a bundle at zero OK So

when you and then this thing falls right
over time and then it gets pulled apart OK

it's good to know I will add that slug had
a slight head and I didn't put it in but

if we increase the Chrysler density was
to get the same thing even at really like

we're starting to get now intermediate
here and then at some level you get this.

And I made this twice as fast so these are
these are actually really sped up movies

they're they take about an hour each and
the reason why I sped up this one even

more is because I want you know Lucy the
loop to see what's happening on the loop.

Pop you see it that was the restart OK so

that what does that mean if when it when
it restarts it gets bigger because I mean

it was during during the movie it was
contracting Yeah so we can so and

higher crossing against the we start to
get contraction it doesn't look great so

for any of you who've ever seen active
minus in contraction this looks like

poop compared to that I mean this is
nothing almost nothing but it is contracts

right and then if we have if we turn up to
eleven but this is actually a movie and

that's hard sell as a nothing's happening.

It's not actually it's just I
can see it in focus here but

it doesn't look it's a little
the resolution isn't that great but

also it is really not changing over
time it's a frozen state there's so

much cost linger that the motors
can't overpower and this makes

sense with Joshua's work remember because
a very high cost linker everything from so

that makes sense OK So
this is now being tracked

by I have a new post doc he's
a soft matter person from our side.

Not.

Control these labs and
so what he's done and

he's been a real blessing because he
had already studied filaments but

he was studying coffee servers right when
he was well because he was saying granular

matter and they were large things and
now he's studying filaments but

they're actually microtubules But what
he's really great at is data analysis so

we had this this data sitting in hand for
a long time in and really didn't have

the expertise to handle it or the time and
so so he came in and he has not

been tracking the boundary has actually
an automated algorithm these are not his

boundaries I drew them in for your eyeball
OK but he actually has real boundaries and

so again we're talking about tracking
are is a function of T. Where are is this.

Boundary and then we can also
quantify the center of mass mew and

now we can calculate the standard
deviation as a function of time so

it's a standard deviations basically
if this is the center of mass it's

the standard deviation of this object so
that the distance to this.

OK And then we can take that data
normalize it by its initial with and

then plot it over time these error bars
are not error bars are there to give you

an idea of what the width of this
to sure looks like and so you can

see it's a fairly wide distribution
some some move faster than others but

what I really really like there is that
it's clear the trends are very clear

right coming out because there's enough
data over time that you're actually able

to pull out a real trend and
what you see is that little cross-link or

density right you spread over
time that's what you just saw

if you go to an intermediate crossing
you're density in the green then you

actually contract slightly and then if
you're a very high concentration density

then you are frozen you're
in a frozen state and so.

The expression is linear in
time why what did you expect.

Because you thought it was diffusion But
this is motor driven

right motor driven exactly excellent
That's what I thought too at first and

I was totally wrong and he would he
said this and I was like PA isn't it but

I didn't understand it and he was like
because it's all motor driven and

this is the velocity of the motors and
I was like.

Yes you're awesome good job
you guys think all right so

we can now take the speed which the speed
has little error bars on it their little

bodies are there because obviously
the slope is the same I'm sorry and

now we can see in extenso apart
a contract top are in a frozen part and

we can do this for a variety of different
experiments and we're actually still

working filling out this part in this part
but we cannot have a phase diagram and

you see these very sharp transitions
between extends our contract.

Well in frozen parts of the phase
diagram we're working with the fear is

to actually model this and he was able
to actually see extend sorrow and

contract on the part that's probably not

that surprising because these models
have been used before to see to see this

what I think is going to happen is
that he's not going to be able to fit.

With what we have here for instance at
a very high cross-link I think you have to

go all the way to infinity right in your
cranial cross-linking in order to get

frozen state and because we have
a cooperative system it's non-linear and

obviously biology is often non-linear
right because it works to be cooperative

on purpose it's tuned and so I think
actually these transitions are going to be

much much sharper than what the model is
going to say but we do have more to do and

particularly in these areas we want to
fill in some more data and Cerro of that

brings us to the end Sara I'm just in
summary I think in visual reconstitution

can be very informative and we can build
structures from minimal components and

I think most importantly the take home is
that non equilibrium behavior of the city

state and the transitions to the in
from those two states are important and

biology is using it and we need to
understand it in a systematic way and

before you go I just want
to say thanks to you for

listening and thanks to the people
in my lab they do the literal

and figurative heavy
lifting of the lab and

thanks to all of our funding agencies
here and I'm happy to take any questions.

Thank.

You.

So we're currently working on
the encapsulation because I think so

are the systems a surgery and everything
in IT systems and it's clear that you.

Like condemn states that
are smaller than you know.

The system.

Valjean calculates itself for a reason so
they can control its chemistry and

if we want to get we want to be able
to make transitions we have to be

able to control the chemistry locally.

And so we're working on putting these
systems into motion droplets and

we're also working on then couple in those
emotion droplets with ion channels so

we can for Turbo and then have
the chemicals go in and out on the

other.

It is it is it using
the contractility I mean I think it.

So I actually so.

This is actually the first example of
contract Filippi seen in microtubule

systems in Acton systems it happens all
the time because you can buckle the acting

so it's not as stiff so part of reason why
I showed you that part with the stiffness

of front was to show you really how stiff
microtubules are they really do are able

to push out against compressive loads and
that's what they're designed to do so

we were surprised I was actually surprised
we saw a contract to Lety at all

there's one other system that people
are starting to see contractility also in

microtubules and that's where you take a
Zen oppose extract you add Taxol to it so

that you make a ton more microtubules
than you normally do and

then when that kind of activates where the
A.T.P. dining motor proteins come in and

they actually contract the whole like a
like a big just like an active gel system

and then they end up making these
weird like like pineapples Ray And so

again that's also not physiological
because you don't make that many

microtubules normally but it does
show you that you actually do if you

increase the amount of polymer
you can get a contract all state

which is not normal and even in even
in the literature probably local

contractionary even forming
the my top expendable

needs local contraction of some sort in
the sense that you glide microtubule.

Relative to each other and

you can get this just through depletion
interactions if you have to make it two

goals of the same length they'll come
together by depletion interactions and

then the depletion reaction is you
know the the energy is minimized if

there are overlapping You
know one hundred percent and

so if you put him apart like this they'll
actually pull back together like this and

so armored Odjick is doing a lot of
stuff looking at that stuff right now.

Because the Army has people actually
came to our lab and got some of that

like they made sixty five in our labs
they wanted to test that same thing but

with cross-linking proteins in the

middle.

That's a good question
we actually measured it

there is a slight depression of the run
length when you have the cross-link or

on a single mike or
two Bill and you add Neeson.

And you look at the can use and
run lengths but

it's not it's not huge I thought
it would be bigger I you know so

because especially because these cross
linkers they kind of like forming a ray

by people seeing that they
kind of form an array and

so I would have thought that it would
have been more but it's not super huge so

we have a paper that came out inside
a skeleton last year where we looked at.

Chinese and
when moving on bundles of microtubules and

of course we always compare it
to the non bundled state so

all that data is published in that
paper came out and last fall.