My name is Andrew Murray.
I am a faculty member
at the Department of Molecular and Cellular Biology
and the Center for Systems Biology
at Harvard University,
and my plan today is to give you
a talk about sex,
not in its full gory, X-rated version,
but the sort of sex that a model organism,
the budding yeast Saccharomyces cerevisiae
This talk is a general introduction -
it's going to last about half an hour.
They'll be a longer talk
which gives more detail
and talks about how scientists run through
and discriminate between different possible explanations
of how something works by doing experiments.
So, to get started, there's a famous saying,
"A loaf of bread, a jug of wind, and thou",
but in fact everyone except thou
is provided by our friend the budding yeast.
So, here is a pitcher of beer.
Here are two bottles of wine.
If you look carefully, this says,
"Andrew Murray Vineyards".
That's not me.
That's just someone who happens to have the same name
and work on the same organism,
but in a slightly more productive way.
And here is the loaf of bread.
And here is the guy who produces it.
So, these are budding yeast cells
-- they're about 5 thousandths of a millimeter,
that's 5 microns in diameter --
and as you can see from this guy,
right down here,
they divide by budding.
So, there is mom and here is...
right there is her little daughter.
And so those are the guys we're going to talk about,
and you might want to ask,
why work on something crazy like this
when we can study sex in humans in bedrooms
and stuff like that?
And the answer is very simple:
people are complicated,
and yeast are simple.
And I'm just going to go through
peeps versus yeasties...
so, these are the Latin names.
We're Homo sapiens,
the actors of today's drama
are called Saccharomyces cerevisiae,
and here are some differences.
It takes about 20 years to double
a human being.
It take 90 minutes -- WAY shorter --
to double a yeast cell.
We are made up of about 50 trillion cells.
So, you started out with
the fusion of your mother's egg and your dad's sperm,
and then there have been many, many, many
billions and billions of cell divisions to make you.
A yeast cell is a single organism.
So, each time the cell divides,
that's two new organisms.
So, everything is simpler and faster.
We have about 6 billion bases of DNA
in each one of our cells.
If you're a yeast cell
it's only 12 [million],
so it's about 500-fold less DNA.
It's about 5- or 6-fold fewer genes.
But here's the big deal.
There's only so much experimentation
you can do on humans.
We're not allowed to do forced matings on anyone.
We're not allowed to do genetic manipulation.
In yeast, the bottom line is,
That means genetic manipulation,
it means forced mating,
and it means a version of Survivor
that you will never see on FOX,
where almost everyone dies
and only the fittest survive.
So, we can actually look at evolution
in the laboratory
in this organism.
And last but not least,
everyone has sex, including yeast.
And so what we're interested in doing
is understanding how cells find each other
to mate in this much simpler system.
So, to talk you through that,
I have to start out with the very basics
of the cell division cycle,
the process by which you turn one cell into two.
So, here we go.
This is the diagram of the simplest possible way
of imagining the cell.
So, the circle here is the outline of the cell.
This line is a chromosome
and that little black dot in the middle
is a specialized part of the chromosome
that's going to attach to the machinery
that will ultimately separate
two sister chromosomes from each other.
So, the first thing you have to do
is you have to replicate your DNA,
so now there are two chromosomes, here,
where there used to be one.
Then you need some sort of machinery
to separate the chromosomes from each other.
So, we're going to generate two genetically identical daughter cells.
And last but not least,
the cell is going to have to divide.
So, here we are, back at the beginning of the cell cycle,
but now instead of one cell we have one... two.
And one of the things that you'll notice is that
during this whole process,
and this is true of almost every cell division,
but not quite all of them,
these cells are growing and getting bigger,
so that the daughters that get produced
are the same size that their moms were
when they were born.
So, now what we're going to do...
that's the simplest possible cartoon,
so now we're going to do 90 minutes
in the life of our friend, the budding yeast.
So, we're just going to walk our way round
a single cell cycle.
So, here's a yeast cell,
down on the bottom.
It's an ovoid cell.
It has a nucleus, which is highlighted here.
The cell is going to grow,
and it's going to ask a bunch of questions at this point.
So, these questions include:
am I big enough to be allowed
to keep going?
Have I got food?
And in particular, the thing that we'll talk a little bit about today,
are there any partners?
Can I smell anyone of the opposite sex around?
And the reason that this happens only once in this cycle
is when two cells fuse with each other,
you want them both to have the same number of chromosomes.
So, if one of the two cells
had replicated its chromosomes
and the other hadn't,
you'd have some weird mixed-up cell,
which would be unable to decide which chromosomes
should get replicated and which not.
So, in budding yeast, mating occurs
at one point in the cell cycle.
This period, as the cells are born and growing,
but before they get up here,
where they switch from growing uniformly
-- just blowing up like a beach ball --
to growing nonuniformly,
because here's the little bud pooching out.
And before this happens,
this part down here,
is the only time when you can mate.
Okay? We get bigger,
we set up the machinery to segregate
but before we segregate them we get to ask another question,
are my chromosomes all lined up properly?
Because if they're not,
I'm not going to make genetically identical daughters.
If they are,
we can break one nucleus into two,
the cell divides,
and we're all the way around again.
So, the next thing we want to talk about...
so that was the cell division cycle of one cell.
One yeast makes two yeasts.
Now we want to go through the whole life cycle.
If it this was people,
this would be like taking your family tree
and starting with mom and pop,
going to you,
meeting someone cute,
and so on.
So, here are...
so, there are two mating types,
the equivalent of our two sexes,
male and female,
but in yeast they're just called a and α (alpha),
and this is a haploid cell,
meaning it has 16 chromosomes,
one of each sort,
and it's going to bud,
get bigger, and divide,
just like I showed you on the last slide.
And here is an α haploid.
All it differs in is
a single small piece of DNA
on chromosome 3
-- so, there's no X and Y chromosome --
and these α cells make a perfume
and the a cells have a receptor
that detects that pheromone.
The a cell make their pheromone or perfume,
which is called a-factor,
and the α cells, here,
detect that a-factor and respond to it.
And when they respond,
the cells don't bud,
but they make these little protuberances.
They grow in a non-uniform way,
so this is a different sort of non-uniform growth
and these little things are called shmoos,
and I'll tell you where that name comes from in a second.
They eventually... their tips touch, they fuse...
this is the first diploid cell,
so now we have 32 chromosomes
-- 16 from mom-ish yeast,
and 16 from pop-ish yeast --
and these cells can once again divide.
They can go around this cell division cycle
and they can keep doing that again and again and again,
unless you starve them,
in which case they go through these specialized cell divisions
that are called meiosis.
They make four spores,
two a's and two α's,
and if you feed them again,
these spores will germinate
and we get right back to the beginning again.
So, that's the whole life cycle
and it's this part here, this business of mating,
that we're interested in.
And so what I'm going to do now is to
show you a night at the singles bar,
and what we're going to see...
this is a movie and there are two cells.
Here's an a cell
and here's an α cell.
You're going to watch them grow
towards each other,
meet in the middle, here.
They're going to fuse
and they're going to produce their first daughter,
right over here.
So, here we go.
You watch them grow and grow and grow
and they're going to touch... boom...
and here comes daughter, right there.
And I'll play that for you one more time.
Here we go.
You want to watch this guy grow and grow,
and we're going to see fusion, right there,
and there comes the daughter.
That whole process there,
which you saw,
takes about an hour and a half in real life,
so it's been sped up, like, a lot.
So, what I want to do now for you
is define different sorts of ways that cells move
when they feel chemical gradients.
So, the first, which you might be more familiar with,
is something called chemotaxis.
That means that the whole cell moves,
just the way that I'm walking towards the screen,
towards some gradient,
and I can give you some examples of that.
So, one of them is a bacterium
swimming up or down a gradient of attractants.
If it smells sugar, for example,
it can swim up a gradient
of increasing sugar concentration.
If they get inside your body,
they can swim in various directions.
We don't like bacteria inside our bodies,
and so there are specialized cells called neutrophils,
and what they do
-- they're some of the white blood cells in our body --
is they literally hone in on the vapor trail
of waste products that the bacteria leave,
which are different from the sorts of waste products
that our bodies make,
and they literally zoom around
and catch the bacteria.
And last but not least,
boy moths can smell,
from astonishingly long distances,
on the order of a mile,
the pheromones, the perfumes,
that are emitted by female moths,
and they can fly up the gradients
of those concentrations
to mate with each other.
So that's distinct from what scientists
and in chemotropism
it's cells that can't move.
So, a yeast cell doesn't have legs,
it doesn't have a propeller,
it can't crawl...
the cells just sit there,
but what they can do is they can do non-uniform growth,
so they're growing predominantly
in the direction of some chemical gradient,
and some examples of that...
as you were developing
inside your mother
and as you grew up, even,
neurons that were born in your body,
they had to send out processes
to find things like muscles
to go from your spinal cord to your brain,
and when they do that they also
follow gradients of chemicals,
but they're growing, not moving.
In plant sex,
you have a plant flower
and an insect lands
and it deposits pollen on it.
which is like a sperm in some sense,
but it doesn't move.
It sends out a tube and that tube has to grow and grow and grow,
and find the plant equivalent of the egg,
technically called an ovule,
and that's how you get fertilization in plants.
And we're going to talk about,
and I just showed you in the movie,
where a and α cells grow towards each other,
their tips touch,
and they fuse.
So, that reminds me to saw something important,
which is that just is an example
of what biologists call symmetry breaking,
a state where cells were growing uniformly,
like blowing up a beach ball
-- it's the same shape, it's just getting bigger and bigger --
and that's what happens
just after a baby yeast cell has been born
and it's growing bigger and bigger.
Eventually, when it gets big enough,
it's allowed to do non-uniform growth,
which means that on this point right on the tip of this cell here,
a little part of the cell will pooch out
because the wall that surrounds the cell
has gotten softer,
and this is how you make the bud,
and the bud grows bigger, like this.
And during mating,
what happens is you have a similar sort of non-uniform growth,
but now instead of a bud,
the cell grows out this tip
and the tip gets bigger
as it grows towards its prospective partner,
and the reason they're called shmoos
is there's a cartoon strip called L'il Abner,
and there were creatures in this strip
which were called shmoos.
They look a little bit... a little bit...
like a mating yeast cell,
they had certain interesting properties:
"According to the shmoo legend, the lovable create laid eggs,
and died of sheer ecstasy
when looked at with hunger."
And so, one of the things that I want to talk about,
and in the talk that goes into more details
about how testing ideas works,
I can come back to is...
which direction cells pick to shmoo in.
So, this is a diagram,
it's a cartoon.
Here's a gradient of pheromone,
low at the top, high at the bottom.
Here are five cells.
When they make shmoos,
they will make them incredibly reliably
in the direction of higher pheromone concentrations.
So, this is a classical example of chemotropism -
you're growing down a concentration gradient
of something you see as an attractant.
But here are five cells,
and instead of being put in a gradient of pheromone,
the pheromone concentration is the same
But even those cells
will eventually break symmetry
and they'll make shmoos.
They'll make them in all sorts of random directions,
and this is, like I said,
an example of something called symmetry breaking,
which happens very often in biology.
It's not particularly well understood,
and it's one of the things that we're interested in in this studying.
to help put the framework for things,
I want to point out the differences
between chemotaxis and mating.
So, if you are a bacteria
swimming up a concentration gradient
or you're one of your white blood cells
chasing that bacteria
and trying to move faster,
things happen fast --
cells move several times the lengths of their own bodies
within a second --
whereas mating is slow.
That movie I showed you
took place over two hours,
so it's sort of slow, stately courtship,
a bit like courtly love in the 13th century.
Chemotaxis is a long-range process.
If you are a male moth
and there's a female moth
half a mile away,
you have to find that concentration gradient
and you have to keep flying up it
for a long, long time,
whereas mating is a profoundly local process,
a little bit like villages
in the 15th and 16th, 17th, 18th centuries.
Unless two yeast cells
are less than one cell diameter apart,
they don't even know each other exist.
The pheromone concentration is too low.
And the last thing that's different
is when cells mate with each other
they have to pick a target.
If you imagine yourself,
or someone you know,
in a bar sitting on a bar stool
flanked by two very attractive people,
whatever sex you prefer,
and you're drinking,
at the end of the evening the goal is
to turn towards one of them or the other
and not to just topple, drunken,
off the bar stool exactly halfway in between them.
In the end, you have to pick a target
and there are lots and lots of examples in biology,
especially in what's called developmental biology
-- the question of how you build organisms --
that involve this sort of picking a target,
and we see this as a simple example
of a general class of problems.
So, now I have to tell you how we study
how cells polarize,
so we're going to start right over here.
This is a yeast cell that hasn't yet budded,
and we've colored some things.
This red thing is actually
telling the cell where it should bud.
It hasn't budded yet,
but we can tell from this red color
that it is going to bud there,
and that's because we've marked something called
with a fluorescent protein,
so we can see that fluorescent protein
underneath the microscope.
Behind it, in the green color, here,
there's a ring of stuff laid down,
which helps to strengthen the cell wall
except where the bud is going to pooch out.
The proteins that make that up are called septins,
so we can label them in a different color.
And last but not least,
these straight lines that you see going back here
are actin cables,
so they're made up...
they're polymers of a small protein called actin,
and they are the cell's railroad tracks.
So, stuff gets transported by motors,
which are seriously analogous to locomotives,
and it will come down this railroad track, here,
be deposited here,
and this is the new material
that's used to grow cell membrane and cell walls.
And last but not least,
and not labeled here,
the blue thing is the nucleus of the cell,
where the DNA is.
And so we can watch how these markers behave,
in this case,
as we go around the circle this way,
as the cell buds.
So, as I told you,
that accumulation of red, the polarisome,
tells you where the cell is going to bud,
and later on,
when the cell is going to divide,
right down here,
those same polarity proteins,
and especially the septin ring,
is required in order for the cell to divide successfully.
Those same proteins get recycled, if you like,
when the cells are treated with pheromone.
So, this is an example of an example of a cell
that's been treated with α-factor,
so you're fooling an a cell into believing
that an α cell is somewhere nearby,
and here's the tip of the shmoo,
which is going to grow up, like this.
The polarisome is right there.
There's a distribution of septins behind it,
which is required to keep that cell wall
strong behind the growing tip,
so that the cell just doesn't blow up like a beach ball,
and the actin cables stream back
from the tip of the cell,
because this is all the stuff
that's going to make new cell.
It's going to go up these filaments
and get deposited here,
so that the cell can grow.
So, now what I'm going to do,
and this is probably going to be
the hardest part to follow
and I will try and go slowly,
is give you a molecular view
of how all this signaling machinery works.
You can still get a lot out of this talk
if you don't follow all these little details.
So, here we go.
The most important thing to say is
this is the outside of the cell,
this is the inside of the cell, here,
and right here,
this railroad track, this grey line
with the thicker grey lines on top,
is the plasma membrane.
This is what separates the inside from the outside.
Here comes α-factor,
this little black horseshoe.
It's going to bind to this protein
that snakes back and forth through the membrane,
the α-factor receptor,
and that protein, in turn,
is going to talk to this protein here.
This is what's called a G protein,
and it's called a G
because it stands for guanine nucleotide binding,
and what you can see...
where it says GDP,
is this molecule has a molecule of
guanine nucleotide diphosphate bound to it.
So, what's going to happen...
the pheromone binds to the receptor.
The receptor changes its shape.
Its change in shape
causes it to interact physically
with this G protein,
and it makes the G protein
let go of its GDP,
so the GDP comes off,
GTP, guanine nucleotide triphosphate,
and that causes a change in the structure
of one of the three subunits,
the one called α subunit.
You can think of it as the surface getting bumpy.
The β and 𝛾 subunits float off,
and they're going to be the business end
of how this cell communicates
from the outside to the inside
that it's seen the mating factor.
And the way they do that is
they recruit scaffold proteins,
so this light blue color
is a scaffold protein that's called Far1.
Far stands for pheromone arrest,
so this was found by geneticists.
Cdc42 stands for cell division cycle,
so this was found by people
working on how the cell cycle works.
This protein stimulates, indirectly,
the formation of those actin filaments
that are going to drag everything the cell needs
to the surface.
It does something else -
it activates a class of molecules called protein kinases.
This one is called sterile 20.
Again, geneticists call these sterile
because mutations in these genes
keep cells from mating.
This is a class of protein called a protein kinase.
It chemically modifies
and affects the activity of other proteins.
So, sterile 20
modifies sterile 11,
which in turn modifies sterile 7,
which in turn modifies this guy,
and this all ultimately leads to
gene transcription and it's responsible
for arresting the cell cycle
so that the two cells that mate with each other
are in the same part of the cell cycle.
So, there's still some more stuff to go.
So, one of the things that's important
is for the cell to polarize in one direction,
to break symmetry,
something has to differentiate some parts of the cell surface
from other parts of the cell surface,
and to do that successfully
the rich have to get richer
and the poor have to get poorer
-- this is a little bit like certain governmental philosophies
in our country --
and these green arrows indicate
what biologists would call a positive feedback loop.
So, if we follow the arrows around,
Cdc42 stimulates actin polymerization.
Everything that's associated with the membrane,
the pheromone receptor itself,
has to be delivered to the surface of the cell
along these filaments,
so the more filaments you have
the more of this molecule you deliver.
The more of this molecule you have,
the more G protein you activate,
the more Cdc42 gets turned on,
the more actin you make,
the more pheromone receptor you get...
and you keep going around this circle,
as things rev up basically,
and so it can cause
little random fluctuations to get amplified
into big differences
between different parts of the cell.
So, that's how you turn things on.
You also need, at the end of the day,
to turn things off.
So, to give you an analogy,
your visual system adapts
to the ambient light intensity,
so if you walk into a dark room,
you get more sensitive to dim light.
If you walk out of a movie theatre
in the middle of the afternoon into the
blinding California sunlight,
your visual system...
everything temporarily looks blindingly bright,
and you adapt.
If you didn't adapt and recover,
you would still be looking at the blinding white light
of the delivery room
that a doctor put you in.
So you need ways to turn things off,
so one way to turn things off
is this protein, which is called Sst2,
the Sst stands for super sensitive to pheromone,
because these mutations don't recover well
from seeing pheromone,
and what this does is
it hydrolyzes, or helps to hydrolyze,
this GTP back to GDP,
the bumps go away on this α subunit,
it binds back to the β and 𝛾 subunits,
the scaffolds fall off,
and signaling stops.
You also need a way,
if your courtship partner has disappeared,
or gets too far away,
to turn things off,
and in particular you need to deal
with the fact that there's still this α-factor
on the outside of the cell.
So, if you're an a cell,
you make this molecule, here,
and Bar1 is drawn as a little pacman here,
and what this is...
this is what's called a protease,
a protein that cuts other proteins,
and it literally cuts the α-factor in half,
so that a cells can recover
from being exposed to α-factor
if they don't mate successfully.
So what we want to do
is ask, in detail,
how cells respond to pheromone gradients,
and to do that we have to make gradients
that are reproducible,
whose structure we know about,
and we used to do that using something called microfluidics,
and so I'm going to show you the top view
and a cross section
of a microfluidic chamber.
So, this is looking from the top.
The bottom, behind the screen,
would be a microscope slide.
The top, closer to you,
would be a coverslip,
cells would be glued on top,
and what you can see is when you look down,
fluid comes in in two streams.
This one has α-factor
and this one, here,
which goes up in this way doesn't have α-factor.
Because of the dimensions of the device
-- this is only about a millimeter across --
and the speed of the flow,
there's no turbulent mixing.
Anything that's going to move from one side of the channel
to the other
has to do it by diffusion.
And what that means is that this blue wash,
which is the pheromone,
gradually gets more and more spread out across the gradient,
as you go down,
and at each point in the gradient
we can draw a cross section,
and we can plot the concentration of the pheromone
as you go across that cross section,
and you can see as you go from the top of the device
and you flow down,
the gradient just gets shallower and shallower.
So that's looking from the top.
If we cut it like this
and look at a cross section,
here's our microscope looking down at the cells inside.
Here are the cells that are glued to the coverslip,
and this is the liquid that's flowing.
It would be flowing right out of the screen
towards you in this image.
So, what I'm going to show you is what happens
to cells in such a chamber.
So, in exactly the same was as the one on the left side,
the flow is going to be in this direction here.
This is the high concentration of pheromone
on this side of the channel.
This is the low concentration.
When I run the movie,
what you're going to see
is each time the movie loops around,
these white circles will draw your attention
to a different bunch of cells.
First, you'll see cells that don't shmoo,
they make buds,
but they make the buds
towards higher pheromone concentrations.
Then you'll see a group of cells over here
that just get bigger and bigger like basketballs,
so they're still growing symmetrically.
The cells around here
are going to make shmoos;
they're going to head straight up the gradient.
Then there are going to be some cells here
which start in the wrong direction and then turn.
And the guys at the top,
at the highest pheromone concentration,
are going to be hopelessly confused,
They're not going to know which direction to go in.
Okay, so here we go.
So, here you see the guys bud
straight up the gradient.
Here are the beach balls.
Here's a cell that's going to shmoo
straight up the gradient.
Here's a cell that's going to
turn up the gradient.
And these are the hopelessly confused.
So, I'll let it loop through one more time,
and what you're going to see
is these guys are going to bud straight up the gradient.
These are the beach balls, right here.
Cells shmooing straight up the gradient
towards an imaginary partner.
And here are cells turning up the gradient.
And these guys are the hopelessly confused.
So, I'm going to leave it there.
There's another slightly longer talk
that goes into much more detail,
that goes into trying to understand how it is that cells break symmetry,
and how it is that yeast cells can mate successfully
that vary under conditions,
which in a human analogy,
would be a man and a woman
on a desert island,
but no one else,
and the crowdiest, sweatiest college mixer
you've ever been in,
with thousands of people of two opposite sexes,
and at the end of that,
yeast cells are so good at finding partners
that almost no one goes home
at the end of the day
broken hearted and crying.
And if you want to see that, keep watching.