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OK.  I think we'll get started.
We're moving into another phase of

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the course today talking about cell
biology.  There are going to be

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three lectures on cell biology
covering a variety of topics.

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So we've been talking about cells in

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various contexts over the course of
several lectures,

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actually, but more or less we've
talked about them as static objects

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that carry out replication and
transcription and translation and so

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on and so forth.
But, in fact, as I'm sure you are

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aware, cells are highly dynamic
structures which do many things at

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all times.  And this is a movie from
which shows the movement of cells.

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Cells will move randomly when you
place them in a dish,

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but they'll also move in a directed
fashion in response to factors in

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their environment that attract them
or they'll move away from factors

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that repel them.
So cell movement is an example of a

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dynamic process.
Cells in the immune system,

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for example, are cells that do a lot
of very critical movement to get to

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the site of a wound,
to get to the site of an infection,

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and then to elicit the proper
response to that infection.

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It's a late arriving crowd today.
Another aspect of the dynamic

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nature of cells is the fact that
they divide.  And I've actually

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shown you this movie previously.
From early embryogenesis through the

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formation of the fully formed
organism and then throughout adult

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life, cells are receiving signals to
divide at the appropriate times and

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to stop dividing when it's not
longer necessary.

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This is a process of signal
transduction, receipt of a signal

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which tells the cell it's time to do
something, and then to process that

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signal in a way to leads to the
proper cellular response.

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Maybe one of the most well
understood or familiar examples of

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this process of signal transduction
is the neuron depicted here in its

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actual state filled with a dye so
you can see its various processes

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and diagramed here.
Neurons, of course,

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are part of the information transfer
in the nervous system where signals

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are received from these structures
here called dendrites.

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That signal is then propagated
through the body of the neuron

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through these long structures called
axons and then released.

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The signal finally acts upon the
structures at the very end of these

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nerve terminals leading to the
release, for example,

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of neurotransmitters.  These are
impressive cells for a variety of

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reasons.  And Professor Sive will
actually give you some lectures

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which deal more specifically about
signaling in the nervous system.

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One of the ways that they're
impressive is their sheer length.

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You have neurons in your body that
are more than a meter in length,

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and whales have neurons in their
body that are tens of

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meters in length.
So these are remarkable cells which

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carry out very specific functions.
So basically what we want to talk

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about over the course of today's
lecture, we'll get there eventually,

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we have to do some other stuff first,
is the process by which cells

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respond to a stimulus.

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And then, much as a computer would
do, to process that signal in very

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sophisticated ways,
which I'll introduce to you this

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time and talk about in greater
detail next time as well,

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to lead to a particular cellular
response.  And I've given you three

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examples of cellular responses in
the movies and images this morning.

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For example, the cells might respond
by moving in a particular directed

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fashion.  They might divide because
it's time for them to divide.

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Or they might, in response to this
signal, release a neurotransmitter

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--

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In the peripheral nervous system or
in the brain.  So what we want to

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understand is what does this really
mean?  What are the mechanisms that

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cells use to process signals?
And you'll actually see analogies

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to integrated circuits as we go
through some examples.

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Before we get there,
we need to understand a little bit

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about how cells are built.
Largely, but not entirely,

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the structure of a cell is related
to its function.

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So neurons are an example.
Their particular structures allow

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them to do the things
that they do.

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Erythrocytes are another example.
Small red blood cells, which are

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oxygen-carrying cells,
are built the way they're built to

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do what they need to do.
And largely, but not entirely,

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a cell's structure is determined by
what proteins are expressed.

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And also where those proteins are

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found inside the cell.
Where those proteins are localized

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inside the cell.
So I want to spend a little bit of

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time talking about protein
localization.

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So I want to spend a little bit of
time talking about protein

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localization, how is it that
proteins within your cells get to

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where they're going,
in a sense know where they're

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supposed to go.

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And a critical tool for
understanding this process of

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protein localization --

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-- is fluorescence imaging.

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And so I think you're all aware of
standard light microscopy where you

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pass white light through a sample,
let's say a cell or a collection of

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cells, and based on the absorbance
of the light through the different

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structures within those cells you're
actually able to visualize what the

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cells look like.
With fluorescence microscopy,

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what you do is shine UV light onto
the cell, which brings energy to the

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cell and allows molecules that will
fluoresce in response to that

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wavelength of UV to be visualized.
So if you have a fluorescent signal

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within the cell,
you can see where that signal is

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coming from using fluorescence
microscopy or fluorescence

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imaging.
So how is it that we get

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fluorescence from the particular
structures, proteins or other things

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that we want to see inside the cell?
Well, there are two general ways of

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doing that.  The first is
fluorescent antibodies.

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So let's imagine we have a protein

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of interest.  We want to know where
it is inside the cell.

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And we're able to make that protein,
say, in bacteria.

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We can inject that protein into a
bunny, that's a bunny,

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or into a mouse, that's a mouse.
OK.  These are the common organisms

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used for production of antibodies.
We'll actually talk about how

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antibodies are made and how they
recognize the things they recognize

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in subsequent lectures.
But I think you all know that

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antibodies are made by the body to
recognize foreign stuff.

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In this case, this protein that
we've produced is foreign to the

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bunny or to the mouse.
And so in response to that it makes

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an antibody, or a series of
antibodies.  These are proteins

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which have a particular structure
that we often diagram like this.

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They have four chains, two big ones
and two little ones.  So

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these are antibodies.
And importantly the antibodies are

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specific.  That is they will bind to
this protein but they won't bind to

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other proteins.
So they specifically recognize this

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protein which is called the antigen
in the context of antibodies.

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We can purify these antibodies and
then chemically modify them to add a

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fluorochrome.

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A chemical moiety that will
fluoresce in response to a

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particular UV wavelength of light.
We can then apply those

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fluorescently labeled antibodies to
cells that we've put onto a glass

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slide and actually poked holes in to
allow the antibody to access the

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inside of the cell.
And then we can visualize where the

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fluorescence comes from within the
cell using fluorescence

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imaging microscopy.
So, for example,

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if the protein of interest is in the
nucleus, and we have labeled our

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antibody with a fluorochrome that
fluoresces in the green wavelength,

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now when we visualize this cell by
fluorescence microscopy the nucleus,

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if the protein is nuclear, will look
green.  OK?  And this has been used

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in cell biology for a very long time,
and it's still used today.

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It's a very powerful method for
determining protein localization.

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This method is called
immunofluorescence.

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Immuno for the antibody and

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fluorescence.  And you can actually
use multiple antibodies labeled with

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different colored fluorochromes and
find multiple proteins in relation

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to one another.
So that's one method.

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To an extent, this method has been
replaced by a new method which

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relies on the fact that there are
naturally fluorescent molecules.

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Naturally fluorescent proteins,

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I should say.  You've actually all
seen these.  If you go to the beach,

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you might have encountered jelly
fish which fluoresce.

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And they fluoresce because they
make a protein which itself

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fluoresces when activated by the
proper UV wavelength.

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These proteins have been isolated
from these species,

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cloned into plasmids,
and then used in recombinant DNA

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techniques, like I told you about
before, to visualize where other

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proteins go inside the cell.
And I'll give you some examples of

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that in a minute.
But here's the jelly fish naturally.

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Here are these structures.
The jelly fish actually makes a

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light which then causes these
protein bundles to fluoresce green.

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The gene was cloned,
as I said.  It was given the name

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GFP for green fluorescence protein.

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And a cDNA copy of the mRNA is used
in molecular biology all over the

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place to do an experiment like this.
And I'll take you through an

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example of another one like this in
a moment where you can actually fuse

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your gene of interest,
gene X, cDNA corresponding to that

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gene with the GFP cDNA to make a
fusion protein that has your gene of

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interest, plus GFP as one
long polypeptide.

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And now this protein will fluoresce.
And you'll be able to visualize it.

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And, again, I'll give you an
example of how that's done in a

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moment.  We've also used this,
we, the field has used this to make

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organisms, other organisms
which fluoresce.

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In my lab, for example,
we've mad green mice which express

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the GFP protein in the cells of the
skin of the mouse.

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So if you shine UV light on the
mouse it turns green.

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It's kind of cool.  And others have
made pets that fluoresce.

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Surprising, but true, you can
actually buy a fluorescent rabbit.

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I'm not sure who sells them, but
they are available commercially.

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And I decided to show you a picture
of one, of a rabbit called Alba the

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fluorescent bunny,
which should be showing here.

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But Alba is actually a little bit
shy so she doesn't always come.

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Alba the fluorescent bunny.  Oh,
here she comes.  So this is actually

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a fluorescent rabbit which was made
by introducing this GFP gene into

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the cells of the skin,
such that it expressed in the cells

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of the skin of the rabbit.
So it's a very powerful technique,

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but let me give you a few more
examples of how it might be useful.

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I know.  Everybody now wants one.
So if you have a cDNA for GFP,

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and actually there are clever
molecular engineers who have

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modified this gene GFP to produce
fluorescent proteins that fluoresce

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in the red wavelength.
So they're RFP,

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red fluorescent proteins.
There are yellow fluorescent

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proteins.  There are blue called
scion CFP fluorescent proteins.

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So there's a whole kit, a whole
coloring box, if you will,

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of different color fluorescent
proteins that you can play with.

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You can then add that, in
techniques that you are familiar

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with, to a cDNA of your
gene of interest.

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So this is a cDNA of your gene of

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interest.  If you now combine those
using recombinant DNA techniques

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that we told you about in previous
lectures, you could make a plasmid

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which has GFP fused directly in
frame, to make a long open reading

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frame with your gene of interest.
OK?  And in that plasmid you would

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also include a promoter to allow the
expression of this fusion.

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And it's also necessary to have,
at the other end, a polyadenylation

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sequence, again,
to insure proper expression of this

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fusion protein in the cells.
And this would then be housed

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inside of a plasmid vector.  OK?
We could then transfect,

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introduce, using methods similar to
transformation,

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but now not into bacteria.
Instead into mammalian cells.

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We could introduce this plasmid
into cells in culture so that in the

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nucleus of these cells that I've
modified, I now have this new gene

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present which can express a green
fusion protein with my

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gene of interest.
Now, importantly,

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the goal of this experiment is to
understand where the protein that's

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encoded by my gene of interest is
localized.  And the way that I'm

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going to do that is to follow where
the green color goes.

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If it's a cytoplasmic protein the
green will be in the cytoplasm.

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If it's a nuclear protein the green
will be in the nucleus and so on.

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So if I now look at this cell by
fluorescence microscopy,

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I will see the green signal in the
nucleus.

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OK?  It's a very,
very powerful technique that allows

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us to follow the products of genes
of interest wherever they might go

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inside of cells.
And I just made a collection of

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images from the literature.
Here you can see, actually,

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two different proteins being
visualized simultaneously.

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The blue is actually a DNA stain.
That shows you were the nuclei are.

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The green is one particular protein.
And you can see that it's largely

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cytoplasmic.  And the red is another
protein rimming the plasma membrane

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of these cells.
There are a whole bunch of cells

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here.  You can see their shape by
where the red color is.

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And the green is then marking the
other protein which is largely

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cytoplasmic.  In this example,
similar to what I drew on the board,

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the fusion protein is in the nucleus.
So the fusion protein is a nuclear

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protein.  It drags the GFP with it
into the nucleus.

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This is a structure called the
endoplasmic reticulum,

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the ER.  We'll come back to it later
in the lecture.

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It's involved in the sorting of
proteins to different parts of the

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cell.  And here's an ER protein
that's been fussed to GFP.

225
00:18:11,000 --> 00:18:14,000
So now the signal is coming from
this structure inside the cell,

226
00:18:14,000 --> 00:18:17,000
the endoplasmic reticulum.  And here
is a protein which resides in the

227
00:18:17,000 --> 00:18:21,000
mitochondria, that energy-producing
organelle inside the cell.

228
00:18:21,000 --> 00:18:24,000
You can drag GFP into the
mitochondria to highlight

229
00:18:24,000 --> 00:18:28,000
those structures.
Here's a red example,

230
00:18:28,000 --> 00:18:32,000
RFP fused to a cytoskeletal protein,
so you now see these long filaments

231
00:18:32,000 --> 00:18:36,000
inside the cell now fluorescing red.
And here's another kind of neat

232
00:18:36,000 --> 00:18:40,000
example.  This is a protein that
moves.  It's normally present in the

233
00:18:40,000 --> 00:18:44,000
cytoplasm.  As you can see here it's
filling the cytoplasm of the cell

234
00:18:44,000 --> 00:18:48,000
and it's not in the nucleus which is
labeled green.

235
00:18:48,000 --> 00:18:52,000
However, in response to a signal
the protein moves.

236
00:18:52,000 --> 00:18:56,000
It goes from the cytoplasm into the
nucleus.  It might be,

237
00:18:56,000 --> 00:19:00,000
for example, a transcription factor,
which is kept in the cytoplasm so

238
00:19:00,000 --> 00:19:04,000
that it won't turn on the expression
of its target genes.

239
00:19:04,000 --> 00:19:08,000
But in response to the signal it now
moves from the cytoplasm into the

240
00:19:08,000 --> 00:19:12,000
nucleus such that it can access the
DNA and begin to turn on the

241
00:19:12,000 --> 00:19:17,000
expression of those genes.
OK?  So proteins can be followed

242
00:19:17,000 --> 00:19:21,000
dynamically.  And you can actually
do movies of these things

243
00:19:21,000 --> 00:19:30,000
over time.  OK.

244
00:19:30,000 --> 00:19:36,000
So proteins can be moved from place
to place.  How do they get there?

245
00:19:36,000 --> 00:19:42,000
What is the mechanism by which
proteins move from one compartment

246
00:19:42,000 --> 00:19:48,000
of the cell to another?
How do they know where to go?

247
00:19:48,000 --> 00:19:54,000
There are two answers to this
question.  One is signals

248
00:19:54,000 --> 00:20:02,000
in the polypeptide.

249
00:20:02,000 --> 00:20:06,000
Signals within the polypeptide tell
the protein where to go.

250
00:20:06,000 --> 00:20:11,000
These signals can be short,
a few amino acids, four, five amino

251
00:20:11,000 --> 00:20:15,000
acids, they can be a little longer,
15 or 20 amino acids, but they

252
00:20:15,000 --> 00:20:20,000
mostly function as a molecular zip
code.  They can be read by the

253
00:20:20,000 --> 00:20:25,000
cell's protein sorting machinery.
Like a post office reads a zip code,

254
00:20:25,000 --> 00:20:30,000
it knows to send certain letters to
certain places.

255
00:20:30,000 --> 00:20:34,000
This machinery reads these signals
and sends proteins to different

256
00:20:34,000 --> 00:20:38,000
places within the cell.
There's also a separate and

257
00:20:38,000 --> 00:20:45,000
sometimes related mechanism --

258
00:20:45,000 --> 00:20:48,000
-- known as post-translational
modification.

259
00:20:48,000 --> 00:20:53,000
Here it's not a sequence within the

260
00:20:53,000 --> 00:20:56,000
protein amino acid sequence.
It's not a signal within the amino

261
00:20:56,000 --> 00:20:59,000
acid sequence,
but rather it's something that's

262
00:20:59,000 --> 00:21:02,000
added onto the protein after it's
been made.  That's why it's called

263
00:21:02,000 --> 00:21:06,000
post-translational.
And, again, this can influence where

264
00:21:06,000 --> 00:21:11,000
the protein ends up inside the cell.
So let me give you some examples of

265
00:21:11,000 --> 00:21:16,000
what I'm talking about here.
With respect to signals that are

266
00:21:16,000 --> 00:21:22,000
contained within the polypeptide,
proteins that are synthesized in the

267
00:21:22,000 --> 00:21:27,000
cytoplasm on ribosomes,
this is a ribosome, this is a mRNA,

268
00:21:27,000 --> 00:21:32,000
5 prime end, 3 prime end, will give
rise, through the process of

269
00:21:32,000 --> 00:21:36,000
translation, to proteins.
And when this process is completed,

270
00:21:36,000 --> 00:21:40,000
those proteins will be released into
the cytoplasm of the cell.

271
00:21:40,000 --> 00:21:44,000
OK?  Most translation occurs freely
inside the cytoplasm of the cell.

272
00:21:44,000 --> 00:21:48,000
There are exceptions to that, as
I'll show you in a moment.

273
00:21:48,000 --> 00:21:52,000
Most of it takes place freely in
the cytoplasm of the cell,

274
00:21:52,000 --> 00:21:56,000
so when the process is done protein
winds up in the cytoplasm.

275
00:21:56,000 --> 00:22:00,000
These proteins have two fates
generally speaking.

276
00:22:00,000 --> 00:22:04,000
One is that they remain cytoplasmic.
And I gave you some examples of

277
00:22:04,000 --> 00:22:08,000
cytoplasmic proteins earlier.
Lots of the enzymes that are

278
00:22:08,000 --> 00:22:12,000
present inside your cells just hang
out inside the cytoplasm and do

279
00:22:12,000 --> 00:22:17,000
their job.  Other proteins are
synthesized first in the cytoplasm

280
00:22:17,000 --> 00:22:21,000
will end up going to the nucleus,
and these proteins are

281
00:22:21,000 --> 00:22:25,000
distinguishable by virtue of the
fact that the ones that end up in

282
00:22:25,000 --> 00:22:30,000
the nucleus have a little
tag on them.

283
00:22:30,000 --> 00:22:37,000
And this little tag has a name
called the nuclear localization

284
00:22:37,000 --> 00:22:47,000
sequence.

285
00:22:47,000 --> 00:22:52,000
This is one of the short tags.
It can be just four or five amino

286
00:22:52,000 --> 00:22:58,000
acids, usually positively charged.
And inside the cytoplasm of this

287
00:22:58,000 --> 00:23:04,000
cell there are proteins which will
recognize that little tag.

288
00:23:04,000 --> 00:23:10,000
Attached to it these proteins
belong to a class known as importins.

289
00:23:10,000 --> 00:23:16,000
And what the importins do is drag
the protein that has an NLS to the

290
00:23:16,000 --> 00:23:22,000
nucleus where they interact with a
structure within the membrane of the

291
00:23:22,000 --> 00:23:28,000
nucleus known as the nuclear
pore complex.

292
00:23:28,000 --> 00:23:37,000
And importin then drags the protein

293
00:23:37,000 --> 00:23:41,000
into the nucleus.
It imports it into the nucleus.

294
00:23:41,000 --> 00:23:45,000
So nuclear proteins get into the
nucleus by virtue of the fact that

295
00:23:45,000 --> 00:23:49,000
they have a nuclear localization
sequence on them.

296
00:23:49,000 --> 00:23:53,000
OK?  And you might imagine,
and probably you will think hard

297
00:23:53,000 --> 00:23:57,000
about how you could visualize this
process of nuclear importation,

298
00:23:57,000 --> 00:24:01,000
for example, in a problem set.
So that's how cytoplasmic resident

299
00:24:01,000 --> 00:24:05,000
proteins are distinguished from
nuclear proteins.

300
00:24:05,000 --> 00:24:08,000
What about the proteins that fully
leave the cell?

301
00:24:08,000 --> 00:24:12,000
Many proteins that you make
actually get secreted.

302
00:24:12,000 --> 00:24:15,000
They leave the cell entirely.
Still other proteins wind up on the

303
00:24:15,000 --> 00:24:19,000
membrane itself.
We're going to talk later in the

304
00:24:19,000 --> 00:24:22,000
lecture and next time about
receptors that sit on the outside of

305
00:24:22,000 --> 00:24:26,000
your cell waiting for signals to be
sent.  Those proteins have to get

306
00:24:26,000 --> 00:24:30,000
there, and there's a special
mechanism that insures that.

307
00:24:30,000 --> 00:24:43,000
So this applies to both secreted,

308
00:24:43,000 --> 00:24:49,000
as well as what we refer to as
transmembrane proteins.

309
00:24:49,000 --> 00:24:57,000
And here the situation is a little

310
00:24:57,000 --> 00:25:01,000
bit different.
In this case, when the ribosomes

311
00:25:01,000 --> 00:25:05,000
synthesize the very first few amino
acids, the very first polypeptide

312
00:25:05,000 --> 00:25:09,000
chain, at the end,
the end terminal end of these

313
00:25:09,000 --> 00:25:13,000
polypeptides, as they're emerging
from the ribosome there is a longer,

314
00:25:13,000 --> 00:25:17,000
more like 15 or 20 amino acid
sequence known as the

315
00:25:17,000 --> 00:25:25,000
signal sequence.

316
00:25:25,000 --> 00:25:28,000
If a protein has a signal sequence
on its end you know it's going to

317
00:25:28,000 --> 00:25:31,000
end up as a secreted protein or a
transmembrane protein.

318
00:25:31,000 --> 00:25:34,000
And when it emerges from the bottom
of the ribosome,

319
00:25:34,000 --> 00:25:37,000
as depicted here,
there's a specialized protein that

320
00:25:37,000 --> 00:25:40,000
will bind to it,
it's actually a complex of proteins

321
00:25:40,000 --> 00:25:44,000
called the signal recognition
particle.

322
00:25:44,000 --> 00:25:55,000
SRP has a very important function,

323
00:25:55,000 --> 00:25:59,000
actually two important functions.
Firstly, when it binds to the

324
00:25:59,000 --> 00:26:03,000
ribosome, when it binds to the
signal sequence like this it leads

325
00:26:03,000 --> 00:26:11,000
to translational arrest.

326
00:26:11,000 --> 00:26:17,000
It stops translation.
It blocks the process of

327
00:26:17,000 --> 00:26:24,000
translation right at this point.
OK?  The next thing that happens is

328
00:26:24,000 --> 00:26:30,000
that it drags this complex of the
ribosome with its little nascent

329
00:26:30,000 --> 00:26:37,000
polypeptide to a structure known as
the endoplasmic reticulum.

330
00:26:37,000 --> 00:26:51,000
In the membrane of the endoplasmic

331
00:26:51,000 --> 00:26:57,000
reticulum.  That was really loud.
Is it that phone?  I got to do this

332
00:26:57,000 --> 00:27:02,000
the other day in a class.
Hello.  Oh, they hung up.

333
00:27:02,000 --> 00:27:06,000
Last time I was teaching a class
and the computer guy left his cell

334
00:27:06,000 --> 00:27:11,000
phone, and it rang.
And the name of the person calling

335
00:27:11,000 --> 00:27:16,000
actually showed up on the screen,
so I picked it up and I said Herb.

336
00:27:16,000 --> 00:27:20,000
And we actually talked for about
five minutes and I told him I had to

337
00:27:20,000 --> 00:27:25,000
go and keep teaching a class.
Anyway, inside the endoplasmic

338
00:27:25,000 --> 00:27:30,000
reticulum there's another receptor,
not unlike the nuclear pore complex.

339
00:27:30,000 --> 00:27:35,000
This is called the SRP receptor.
It binds to this thing SRP, signal

340
00:27:35,000 --> 00:27:41,000
recognition particle.
And this then allows SRP to go away

341
00:27:41,000 --> 00:27:47,000
and allows translation to continue
allowing the protein to be

342
00:27:47,000 --> 00:27:53,000
synthesized through this membrane
and end up inside the endoplasmic

343
00:27:53,000 --> 00:27:59,000
reticulum.  So the end result of
this process is that the protein

344
00:27:59,000 --> 00:28:05,000
will now reside within this
structure, this membrane-bound

345
00:28:05,000 --> 00:28:12,000
structure.
Subsequently, the protein leaves

346
00:28:12,000 --> 00:28:24,000
this structure in a vesicle.

347
00:28:24,000 --> 00:28:29,000
It buds off the membrane,
gets carried like cargo in a little

348
00:28:29,000 --> 00:28:34,000
pod coming off the mother ship here.
It goes to another sorting

349
00:28:34,000 --> 00:28:39,000
structure called the Golgi apparatus.
And it eventually goes to the

350
00:28:39,000 --> 00:28:45,000
plasma membrane where it fuses to
the plasma membrane allowing this

351
00:28:45,000 --> 00:28:50,000
secreted protein to leave the cell.
I realize that this is a little bit

352
00:28:50,000 --> 00:28:56,000
messy, but there are comparable
pictures in your book.

353
00:28:56,000 --> 00:28:59,000
The point being that these proteins
have a signal which drags them to

354
00:28:59,000 --> 00:29:03,000
the ER.  There's then a sorting
process that takes them out of the

355
00:29:03,000 --> 00:29:07,000
cell, takes them to the plasma
membrane and then out of the cell.

356
00:29:07,000 --> 00:29:11,000
So that's how you deal with
secreted proteins.

357
00:29:11,000 --> 00:29:14,000
How do you deal with proteins that
end up in the membrane?

358
00:29:14,000 --> 00:29:18,000
If they don't get secreted they
wind up resident in the plasma

359
00:29:18,000 --> 00:29:22,000
membrane.  This happens by virtue of
another signal.

360
00:29:22,000 --> 00:29:25,000
And I'm going to pick up this
process, the beginning of the

361
00:29:25,000 --> 00:29:29,000
process is exactly the same.
I'm going to pick up this process

362
00:29:29,000 --> 00:29:33,000
at a slightly later point where we
have the SRP receptor

363
00:29:33,000 --> 00:29:37,000
bound by a ribosome.
It's actually partway through the

364
00:29:37,000 --> 00:29:42,000
process of producing a polypeptide
that has wound up inside the ER.

365
00:29:42,000 --> 00:29:47,000
For proteins that wind up
ultimately in the membrane there's

366
00:29:47,000 --> 00:29:53,000
another sequence called a
stop transfer sequence.

367
00:29:53,000 --> 00:30:02,000
And when a stop transfer sequence is

368
00:30:02,000 --> 00:30:06,000
hit and recognized by this complex
it causes the ribosome to

369
00:30:06,000 --> 00:30:11,000
disassociate from the mRNA,
or rather allows the synthesis to

370
00:30:11,000 --> 00:30:16,000
occur without continuous transfer
through this complex.

371
00:30:16,000 --> 00:30:20,000
So the protein just gets built on
this side of the membrane.

372
00:30:20,000 --> 00:30:25,000
And the result of that then is a
protein that ends up halfway in and

373
00:30:25,000 --> 00:30:30,000
halfway out of the endoplasmic
reticulum.

374
00:30:30,000 --> 00:30:35,000
If you then take this through the
sort of sorting steps that I just

375
00:30:35,000 --> 00:30:40,000
outlined for secreted proteins,
this protein will end up with part

376
00:30:40,000 --> 00:30:46,000
of it on the outside and part of it
on the inside of the cell.

377
00:30:46,000 --> 00:30:53,000
OK?  So that's how we deal with

378
00:30:53,000 --> 00:30:57,000
proteins that are transmembrane.
And you'll actually see proteins

379
00:30:57,000 --> 00:31:01,000
that go through the membrane
multiple times.

380
00:31:01,000 --> 00:31:05,000
That's because they have signal
sequences and stop transfer sequence,

381
00:31:05,000 --> 00:31:10,000
signal sequences and stop transfer
sequences that allow them to

382
00:31:10,000 --> 00:31:15,000
transition through the membrane more
than once.  Many of the proteins

383
00:31:15,000 --> 00:31:20,000
just once but other proteins
multiple times.

384
00:31:20,000 --> 00:31:25,000
OK.  So that's the way it's done in
terms of signal recognition.

385
00:31:25,000 --> 00:31:32,000
I also mentioned that there is this

386
00:31:32,000 --> 00:31:38,000
class of posttranslational
modification.  Proteins get modified

387
00:31:38,000 --> 00:31:45,000
after they're made by translation.
And the examples include

388
00:31:45,000 --> 00:31:51,000
proteolysis.  The protein might
actually get cleaved,

389
00:31:51,000 --> 00:31:58,000
cut.  Half of it goes one place.
The other half goes somewhere else.

390
00:31:58,000 --> 00:32:07,000
Phosphorylation.

391
00:32:07,000 --> 00:32:13,000
Addition of a phosphate can
determine the localization of a

392
00:32:13,000 --> 00:32:19,000
protein.  Glycosylation.
Addition of sugar groups,

393
00:32:19,000 --> 00:32:25,000
carbohydrates onto the protein can
determine where the protein goes.

394
00:32:25,000 --> 00:32:33,000
And finally lipid addition.

395
00:32:33,000 --> 00:32:37,000
Addition of a lipid molecule can
determine where the protein goes.

396
00:32:37,000 --> 00:32:41,000
And I'll give you one example of
that last one.

397
00:32:41,000 --> 00:32:46,000
A protein that we work on in my lab,
the protein called RAS,

398
00:32:46,000 --> 00:32:50,000
which will actually come up in the
next lecture.  It's a signaling

399
00:32:50,000 --> 00:32:55,000
protein, which actually turns out to
be very important in cancer.

400
00:32:55,000 --> 00:32:59,000
In its unmodified state the protein
is cytoplasmic.

401
00:32:59,000 --> 00:33:04,000
However, it gets modified by an
enzyme called farnesyltransferase.

402
00:33:04,000 --> 00:33:12,000
Which modifies the very end of the

403
00:33:12,000 --> 00:33:18,000
protein, the C terminal end of the
protein, and it sticks onto it a

404
00:33:18,000 --> 00:33:24,000
lipid group, about a 20 carbon lipid
group, which is very much like the

405
00:33:24,000 --> 00:33:30,000
fatty acid side chains in
the plasma membrane.

406
00:33:30,000 --> 00:33:33,000
And, in fact, this is a very
hydrophobic structure.

407
00:33:33,000 --> 00:33:37,000
And it ends up dragging the protein
to the plasma membrane.

408
00:33:37,000 --> 00:33:41,000
Here's the plasma membrane.
I'm drawing it now as a lipid

409
00:33:41,000 --> 00:33:45,000
bilayer, whereas I drew it as a
single line previously.

410
00:33:45,000 --> 00:33:48,000
And that lipid tail, which has been
stuck on the RAS protein,

411
00:33:48,000 --> 00:33:52,000
inserts into the lipid bilayer and
localizes RAS then to the plasma

412
00:33:52,000 --> 00:33:56,000
membrane.  OK?
So here are examples of

413
00:33:56,000 --> 00:34:00,000
post-translational modifications
that effect where the protein

414
00:34:00,000 --> 00:34:04,000
ends up going.
So we can determine where the

415
00:34:04,000 --> 00:34:08,000
protein goes, that's very important.
I also want to make the point

416
00:34:08,000 --> 00:34:12,000
before we move onto the next topic
that protein-protein interactions

417
00:34:12,000 --> 00:34:16,000
are very important in determining
function.  These proteins that we've

418
00:34:16,000 --> 00:34:20,000
been talking about almost never,
probably never do their functions by

419
00:34:20,000 --> 00:34:24,000
themselves.  They interact with
other proteins.

420
00:34:24,000 --> 00:34:28,000
And these are determined,
again, by sequences that bind to one

421
00:34:28,000 --> 00:34:31,000
another between the two proteins.
So this is actually a protein

422
00:34:31,000 --> 00:34:35,000
structure from a colleague here at
MIT, Thomas Schwartz,

423
00:34:35,000 --> 00:34:39,000
solved by him.  What you're going to
look at hopefully,

424
00:34:39,000 --> 00:34:42,000
this is a ribbon diagram of two
proteins, the one in blue and the

425
00:34:42,000 --> 00:34:46,000
one in pink.  And you can see that
these two proteins bind to each

426
00:34:46,000 --> 00:34:49,000
other based on amino acid residues
at this interface between the two

427
00:34:49,000 --> 00:34:53,000
proteins.  This ribbon diagram will
be replaced by a space-filling model.

428
00:34:53,000 --> 00:34:57,000
This is what the protein really
would like inside the cell.

429
00:34:57,000 --> 00:35:00,000
And here's that interface more
clearly visualized with residues in

430
00:35:00,000 --> 00:35:04,000
light blue on one side,
pink on the other that allows the

431
00:35:04,000 --> 00:35:07,000
two proteins to interact.
So protein-protein interaction is

432
00:35:07,000 --> 00:35:11,000
extremely important,
allowing proteins to interact either

433
00:35:11,000 --> 00:35:15,000
for a long time because they carry
out some essential function together

434
00:35:15,000 --> 00:35:19,000
or very transiently.
Protein-protein interactions can be

435
00:35:19,000 --> 00:35:23,000
long-lasting or very transient.
And to just give you a visual

436
00:35:23,000 --> 00:35:27,000
reminder of that,
the transient interaction,

437
00:35:27,000 --> 00:35:31,000
this I took a couple of years ago,
Johnny Damon getting run into by

438
00:35:31,000 --> 00:35:35,000
Damian Jackson.
A very transient but actually quite

439
00:35:35,000 --> 00:35:39,000
important interaction between these
two baseball players.

440
00:35:39,000 --> 00:35:43,000
And then there can be longer
lasting interactions.

441
00:35:43,000 --> 00:35:48,000
Here's Brad Pitt, well,
it's not that long-lasting,

442
00:35:48,000 --> 00:35:52,000
I guess, but the point is that
proteins do interact either very

443
00:35:52,000 --> 00:35:56,000
transiently or in a long-lasting way.
Jennifer actually did the cutting

444
00:35:56,000 --> 00:36:01,000
out herself of this picture
that she sent me.

445
00:36:01,000 --> 00:36:08,000
OK.  Finally, proteins can change

446
00:36:08,000 --> 00:36:12,000
their shape.  Proteins can change
their shape in response to binding

447
00:36:12,000 --> 00:36:16,000
of other proteins or binding of
cofactors that they interact with.

448
00:36:16,000 --> 00:36:21,000
And here's another movie from
Thomas Schwartz.

449
00:36:21,000 --> 00:36:25,000
This is a signaling protein,
much like the RAS protein actually.

450
00:36:25,000 --> 00:36:29,000
And you'll see that upon binding to
a nucleotide here,

451
00:36:29,000 --> 00:36:34,000
the protein dramatically changes its
shape.

452
00:36:34,000 --> 00:36:38,000
It actually unwinds a little bit and
exposes a new structure for binding.

453
00:36:38,000 --> 00:36:42,000
And this is one of the critical
ways that proteins participate in

454
00:36:42,000 --> 00:36:46,000
this signal transduction,
signal processing function.

455
00:36:46,000 --> 00:36:50,000
They receive signals, change their
shape, and this allows them to bind

456
00:36:50,000 --> 00:36:54,000
to other proteins or carry out other
functions.  OK.

457
00:36:54,000 --> 00:36:58,000
So I want to move now to the
signaling introduction for the last

458
00:36:58,000 --> 00:37:02,000
12 minutes or so.
I started the day with this slide of

459
00:37:02,000 --> 00:37:06,000
cells moving around.
They're receiving signals from one

460
00:37:06,000 --> 00:37:10,000
another or from the medium and
they're responding to it.

461
00:37:10,000 --> 00:37:14,000
I also gave you examples of cells
dividing or not dividing,

462
00:37:14,000 --> 00:37:18,000
cells differentiating, turning into
one type of end-stage cell or

463
00:37:18,000 --> 00:37:22,000
another, changing their function.
We'll talk later about cells that

464
00:37:22,000 --> 00:37:26,000
receive signals and kill other cells,
cells that receive signals and

465
00:37:26,000 --> 00:37:30,000
commit suicide in a couple
of more lectures.

466
00:37:30,000 --> 00:37:36,000
So cells will do very complicated
things in receipt of various signals.

467
00:37:36,000 --> 00:37:42,000
And this general process is known
as signal transduction.

468
00:37:42,000 --> 00:37:49,000
And basically it's the ability of
cells to respond to things in their

469
00:37:49,000 --> 00:37:55,000
extracellular environment,
stimuli in their extracellular

470
00:37:55,000 --> 00:38:01,000
environment.
These could be physical stimuli,

471
00:38:01,000 --> 00:38:06,000
light, odorants, or they could be
biological stimuli,

472
00:38:06,000 --> 00:38:12,000
peptides, proteins, hormones,
which then get sent first from the

473
00:38:12,000 --> 00:38:17,000
outside of the cell and then to the
inside of the cell leading to either

474
00:38:17,000 --> 00:38:29,000
short-term responses --

475
00:38:29,000 --> 00:38:33,000
I'll give you an example of glucose
secretion in response to a hormone

476
00:38:33,000 --> 00:38:37,000
epinephrine or adrenalin.
This happens exclusively inside the

477
00:38:37,000 --> 00:38:42,000
cytoplasm of the cell.
The signal is received,

478
00:38:42,000 --> 00:38:46,000
processed, and the result is the
secretion of glucose.

479
00:38:46,000 --> 00:38:51,000
Other signals will make their way
into the nucleus of the cell,

480
00:38:51,000 --> 00:38:55,000
and this might lead to changes in
gene expression.

481
00:38:55,000 --> 00:39:00,000
New genes are turned.
Others might be turned off.

482
00:39:00,000 --> 00:39:04,000
Changes in gene expression.
And this might lead to a longer

483
00:39:04,000 --> 00:39:09,000
term response.
And we'll give you an example of

484
00:39:09,000 --> 00:39:14,000
this in the next lecture.
So how does this work?  Well,

485
00:39:14,000 --> 00:39:19,000
it's different in every way.  Each
example is slightly different.

486
00:39:19,000 --> 00:39:24,000
I show you this slide to give you
sort of a visual picture of what

487
00:39:24,000 --> 00:39:28,000
it's like, we think.
Like one of these,

488
00:39:28,000 --> 00:39:32,000
what do they call them,
Kineto-something or rather

489
00:39:32,000 --> 00:39:35,000
sculptures that you see at Logan
Airport, for example,

490
00:39:35,000 --> 00:39:39,000
where a small perturbation at one
end is propagated through a series

491
00:39:39,000 --> 00:39:42,000
of devices and leads to sounds and
whistles and movements taking place

492
00:39:42,000 --> 00:39:46,000
within the structure.
It's not unlike dominos.

493
00:39:46,000 --> 00:39:49,000
When you flip a domino at one end
it can spread,

494
00:39:49,000 --> 00:39:53,000
increasing its effect because of the
involvement of additional dominoes

495
00:39:53,000 --> 00:39:57,000
as it goes along a complex
structure.

496
00:39:57,000 --> 00:40:01,000
We actually found,
as we were looking at the Web for

497
00:40:01,000 --> 00:40:06,000
pictures of this movie of Mr.
Domino, does anybody know what Mr.

498
00:40:06,000 --> 00:40:10,000
Domino is?  I had no idea.  It's a
computer game,

499
00:40:10,000 --> 00:40:15,000
and they have a cool little
animation which I thought I would

500
00:40:15,000 --> 00:40:19,000
show you.  Here's Mr.
Domino.  And it's actually kind of

501
00:40:19,000 --> 00:40:24,000
relevant because he'll go and his
signal is being transmitted along a

502
00:40:24,000 --> 00:40:29,000
linear path involving other types of
molecules spreading out.

503
00:40:29,000 --> 00:40:34,000
Again, other types of molecules
being involved,

504
00:40:34,000 --> 00:40:39,000
and so on and so forth.
It's not a great example but it is

505
00:40:39,000 --> 00:40:44,000
kind of fun.  OK.
So before I give you a specific

506
00:40:44,000 --> 00:40:49,000
example, I want to give you some
principles about signal transduction.

507
00:40:49,000 --> 00:40:55,000
And, again, the principles that are
relevant here in biology are not

508
00:40:55,000 --> 00:41:00,000
dissimilar to signal propagation in
electrical engineering,

509
00:41:00,000 --> 00:41:05,000
for example.
Signal transduction in its simplest

510
00:41:05,000 --> 00:41:11,000
form means that something which
takes some form,

511
00:41:11,000 --> 00:41:16,000
like a ligand binding to a receptor
on the surface of the cell,

512
00:41:16,000 --> 00:41:22,000
changes its form inside the cell.
It causes a change at the membrane

513
00:41:22,000 --> 00:41:28,000
to produce a different type of
response inside the cell.

514
00:41:28,000 --> 00:41:32,000
Importantly, for almost all of these
signal transduction pathways,

515
00:41:32,000 --> 00:41:37,000
what happens at the membrane is
usually inadequate to change

516
00:41:37,000 --> 00:41:42,000
whatever process is to be changed
inside the cell such that it is

517
00:41:42,000 --> 00:41:47,000
necessary for signal amplification
to take place.

518
00:41:47,000 --> 00:41:52,000
And we'll give you examples of how
signal amplification occurs in

519
00:41:52,000 --> 00:41:57,000
biology.  But rather than making one
square, in fact, you might

520
00:41:57,000 --> 00:42:02,000
make multiple squares.
There's also the opportunity,

521
00:42:02,000 --> 00:42:07,000
and it's often the case for signal
diversification,

522
00:42:07,000 --> 00:42:12,000
so that in response to the same
signal, in fact,

523
00:42:12,000 --> 00:42:16,000
processed slightly different ways
temporally or spatially with inside

524
00:42:16,000 --> 00:42:21,000
the cell you might make,
sorry, let's make this a circle.

525
00:42:21,000 --> 00:42:26,000
You might make two different
responses that coordinate the

526
00:42:26,000 --> 00:43:00,000
overall cellular response.

527
00:43:00,000 --> 00:43:03,000
There's the opportunity for what we
call signal integration.

528
00:43:03,000 --> 00:43:11,000
So with respect to signal

529
00:43:11,000 --> 00:43:16,000
integration, you might imagine one
signal which produces a particular

530
00:43:16,000 --> 00:43:21,000
response.  A separate signal which
produces a distinct response.

531
00:43:21,000 --> 00:43:26,000
But if you add the two together,
that is if both signals are received

532
00:43:26,000 --> 00:43:32,000
simultaneously, you get
some third response.

533
00:43:32,000 --> 00:43:35,000
So signal integration,
again, not unlike electrical

534
00:43:35,000 --> 00:43:39,000
engineering.  And finally
signal modulation.

535
00:43:39,000 --> 00:43:47,000
So let's imagine again that we have,

536
00:43:47,000 --> 00:43:52,000
in some circumstances, a particular
signal giving a particular response.

537
00:43:52,000 --> 00:43:57,000
If, in a different context with a
different signal being received by

538
00:43:57,000 --> 00:44:02,000
the cell at the same time,
instead of producing this response,

539
00:44:02,000 --> 00:44:08,000
there's an inhibitory effect so that
nothing is produced.

540
00:44:08,000 --> 00:44:12,000
This signal inhibits the processing
of this signal,

541
00:44:12,000 --> 00:44:17,000
so this is inhibitory.
And you could imagine,

542
00:44:17,000 --> 00:44:21,000
and we know exists, the opposite
such that in the presence of this

543
00:44:21,000 --> 00:44:26,000
signal plus now a distinct signal
there's an augmentation of the

544
00:44:26,000 --> 00:44:31,000
response so that now you make much
more of whatever you were making.

545
00:44:31,000 --> 00:44:36,000
So this is augmentation.
So all of these things happen in

546
00:44:36,000 --> 00:44:42,000
the process of cellular signal
transduction, cellular signal

547
00:44:42,000 --> 00:44:47,000
processing.  At the beginning of
this process, for at least the

548
00:44:47,000 --> 00:44:53,000
examples we're going to talk about,
things happen at the plasma membrane.

549
00:44:53,000 --> 00:45:05,000
So here is the plasma membrane again.

550
00:45:05,000 --> 00:45:09,000
You can think of the plasma
membrane as sort of the outside of

551
00:45:09,000 --> 00:45:13,000
your house with the antennas
sticking up into the air waiting for

552
00:45:13,000 --> 00:45:18,000
signals to be received.
Well, in biological circumstances,

553
00:45:18,000 --> 00:45:22,000
those antennas are polypeptide
receptors.  These are in this

554
00:45:22,000 --> 00:45:26,000
category.  Or proteins that have
transmembrane domains,

555
00:45:26,000 --> 00:45:31,000
get stuck in the membrane,
and end up on the plasma membrane.

556
00:45:31,000 --> 00:45:35,000
These polypeptide receptors are
sitting out there.

557
00:45:35,000 --> 00:45:40,000
There are often thousands of a
given polypeptide receptor type on

558
00:45:40,000 --> 00:45:45,000
the outside of a cell.
And these bind to ligands that fit

559
00:45:45,000 --> 00:45:49,000
inside them.  There's
complementarity between the shape of

560
00:45:49,000 --> 00:45:54,000
the ligand and the receptor such
that you get specific binding of the

561
00:45:54,000 --> 00:45:59,000
ligand to the receptor.
The consequences of binding of

562
00:45:59,000 --> 00:46:03,000
ligand to receptor we generally
think of now as changing the shape

563
00:46:03,000 --> 00:46:08,000
of the receptor.
Something that happens on the

564
00:46:08,000 --> 00:46:13,000
outside through binding of the
ligand to the receptor is propagated

565
00:46:13,000 --> 00:46:17,000
through this polypeptide chain to a
change in the structure at the

566
00:46:17,000 --> 00:46:22,000
inside.  OK?  It's as though I took
this guy's head and I twisted it.

567
00:46:22,000 --> 00:46:27,000
Eventually his feet are going to
move.  The same thing here.

568
00:46:27,000 --> 00:46:31,000
If I make a change out here,
I can propagate that change through

569
00:46:31,000 --> 00:46:36,000
the polypeptide chain and change the
inside structure.

570
00:46:36,000 --> 00:46:41,000
So if you imagine a ligand which
has a particular structure,

571
00:46:41,000 --> 00:46:45,000
I'm sorry a receptor that has a
particular structure,

572
00:46:45,000 --> 00:46:50,000
when you add the ligand you now
change the structure.

573
00:46:50,000 --> 00:46:55,000
And take a protein that might be
inactive, this might have some

574
00:46:55,000 --> 00:47:00,000
enzymatic activity,
from inactive to one that's active.

575
00:47:00,000 --> 00:47:05,000
Or another example,
if I take this receptor and I bind

576
00:47:05,000 --> 00:47:11,000
the ligand, change its structure,
I might now allow, based on this

577
00:47:11,000 --> 00:47:16,000
change in its structure,
an interaction between this protein

578
00:47:16,000 --> 00:47:22,000
and some other signaling molecule,
some other signaling protein.  And,

579
00:47:22,000 --> 00:47:28,000
again, we know examples of all of
those things.

580
00:47:28,000 --> 00:47:44,000
Usually, maybe always,

581
00:47:44,000 --> 00:47:48,000
the change that takes place at the
plasma membrane,

582
00:47:48,000 --> 00:47:52,000
like the first domino that you flip,
doesn't have enough energy

583
00:47:52,000 --> 00:47:56,000
associated with it to change the
biological response.

584
00:47:56,000 --> 00:48:00,000
It's necessary to propagate that
signal and to amplify that signal

585
00:48:00,000 --> 00:48:05,000
through various stages.
This process of signal amplification

586
00:48:05,000 --> 00:48:11,000
is very important.
And there are two general classes

587
00:48:11,000 --> 00:48:17,000
of signal amplification.
One is the production of secondary

588
00:48:17,000 --> 00:48:23,000
signaling molecules called second
messengers.  And the example that

589
00:48:23,000 --> 00:48:30,000
we'll give you next time is the
second messenger cyclic AMP --

590
00:48:30,000 --> 00:48:34,000
-- which can be produced in large
amounts following the binding of a

591
00:48:34,000 --> 00:48:39,000
ligand to a receptor,
thereby amplifying the signal.

592
00:48:39,000 --> 00:48:43,000
And the other, which we'll give you
as two examples next time,

593
00:48:43,000 --> 00:48:48,000
is enzymatic, very often kinase
cascades.  Enzymatic cascades where

594
00:48:48,000 --> 00:48:52,000
you turn on the activity of one
enzyme, which turns on the activity

595
00:48:52,000 --> 00:48:57,000
of more other enzymes,
which turn on the activity of still

596
00:48:57,000 --> 00:49:01,000
more other enzymes and so on.
So, we'll talk about specifics

597
00:49:01,000 --> 00:49:04,000
related to these next time.