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I think a lot of you know me already.
I just want a show of hands-- how many of you are new to the
Bio Core sequence and are taking
Bio42 out of sequence?
It's OK.
You will be in good company.
So there are a few of you.
For this reason, we are going to go over a few details from
the syllabus.
But before I do that, I'm going to introduce you to the
professors who are teaching the course.
Professor Kang Shen, he's going to be teaching the cell
bio part, the first part, and development.
And then Professor Pat Jones is going to be teaching you
immune system, immunology, and Professor Craig Heller, who's
going to be doing the bulk of the physiology.
Professor Robert Sapolsky is going to be teaching you
neurobiology.
He's not here today, but one thing I can tell you is that
by the end of Professor Sapolsky's section, you'll
know that the brain controls everything.
Including the systems that Professor Heller talks about.
So going back to the syllabus, however, I just
wanted to let you know about my office
hours for this quarter.
They're a little bit different than they were last quarter,
so pay attention to that.
One of the kinds of things I'm useful for is
pretty much everything that doesn't have to
do with your material.
So if you have questions on your material, your best bet
is the professors and the TAs for the course.
If you have questions that don't fit into the category of
material, then I'm probably your person.
So definitely email me or come see me during office hours.
I'm also happy to talk to you about the material, but if
we do have students who have questions about course
logistics or want to discuss their grades, or how they're doing in the course
then they can get priority over students with questions on material.
A few quick things about the syllabus-- there are two
textbooks you have to buy.
It's fine if you buy the penultimate edition.
We've provided you readings from both the latest edition
as well as one that's older.
You will find the readings on the last two pages of the
syllabus, and we highly recommend that you do get these
before you come to class.
Exams, there are two midterms and a final.
Please make note of the dates and make sure that you can
attend them.
If you have a conflict, a legitimate conflict--
and for the definition of a legitimate conflict, please
look at the syllabus--
please contact me ASAP so we can make arrangements for you. Okay?
So take a look at the dates and make sure
that you can be there.
Problem sets, there's going to be eight of them.
They're taken on coursework on your own time.
There's PDF versions that are going to be available on the
class website so you can work off one if you want.
Make sure you submit them on time so that you can get
credit for it.
We don't accept late problem sets, because it's really hard
to manage and determine how late is late,
and things like that.
So we give you a sufficient amount of time.
For each problem set, the problem set will be released
on Monday and it will be due the next Friday,
not the same Friday.
So you have a lot of time.
Sections we're going to start next week.
And sections are handled by our very able TAs, and I'd
like to quickly introduce them to you.
So Nathan Barnett--
Nathan, many of you will recognize, is a very familiar
face because he basically TAed Bio41 also, and he was an
undergrad here.
Diana Li.
She's--
So that's
Diana, Brad Turnwall, Ben Saligman, and Elan Coutner.
So these are your-- actually, I'm missing--
I always do this.
Meg, Meg Tabitha is also a returning TA from Bio41.
You can check out their bios on our class website.
You can also sign up for discussion sections with them.
We highly recommend that you go to section.
There's a lot of material in this course, and it's always
nice to be able to revisit the material, really absorb it
well, and get a different perspective on the material
from these TAs.
So definitely attend sections.
One other thing is before you go to section, just make sure
that you double check the location of sections, because
sometimes we change them at the last minute because of
registrar events.
OK, as far as navigating all the stuff that is available to
you online, there are two sites.
There's the coursework site, and then there's the reading
website, the class website, where a lot of stuff that
doesn't fit on coursework, we end up putting it
on the class website.
So definitely check out both sites
and see what's available.
We also have this piazza, discussion section--
discussion forum, sorry-- and it's a great place to ask
questions on the material.
As you can see, we are videotaping these sessions,
but please do not rely on them.
There are times when we may not be able to post the video
on time, or for technical reasons, the
video may not be available.
So definitely come to class.
We are expecting that you're going to be using these videos
more as a study tool to revisit material that kind of
went by quickly and you just want to have
another stab at it.
So the class videos are not going to be on coursework.
They'll be on a different site, and we'll send you the
link for that.
Before I end, I just want to say, there are a lot
resources for you.
The professors are your biggest resource.
They have office hours and they'd love to see you.
So definitely go see them.
Sections are a great resources.
Center for Teaching and Learning, there are
undergraduates who were in your same situation last year,
they did a fabulous job, and they want to help you.
They have drop-in hours and we'll be
posting a link for that.
There are a lot of resources, so don't struggle on your own.
Don't spend five hours when you can just talk to somebody
and get your question cleared right away.
So try to spend your time very strategically and wisely, and
we all want to help you succeed in this course.
Finally, there will be pizza socials.
If you look at the last two pages of the syllabus, you'll
notice that the professors have indicated when they're
having their socials, and we will announce to you how to
sign up for those, OK?
Any questions about logistics before I turn it over to the
professors?
All right, great, thank you.
So Professor Shen?
So, welcome everyone to Bio42.
Every year I come up here and I say hi, my name's Kang Shen,
and I've been in the department for x
number of years now.
I can say it's over 10 years, so it's a scary thought.
So this is a new thing that we're going to do for the
first five minutes, so each of us is going to come up and
then we're going to give you an overview of what this whole
Bio42 lectures are going to be about.
And we didn't do this last year, and from the student
feedback, there seems to be the need for doing that before
we delve into each of our sections and get all the
details of how cells work and systems work.
So I'm just going to get this process started, and then Pat
and Craig will come up and just say a few things about
the overview about their lectures.
So in Bio41, in the last semester, you guys learned
about the building blocks, the biochemical reactions, that is
the basis of what's happening--
to biology that occurs in our body.
And so here in the first part of the lectures, we're going
to now move on to cells.
It's all about cells the first month or so, and we know that
there are many cell types that makes our bodies.
And these cells are critical, are important, essentially the
unit that makes the individual organism.
And the cells make tissues, and in our bodies there are
four different types of major tissues--
these are epithelial tissues, nervous tissues, muscles, and
connective tissues.
And these tissues then make organs, and these organs are
of course are literally larger, and they've been
recognized by early biologists when you do a dissection.
When you cut open a dead animal you see their organs--
their hearts, their kidney and brain.
And each individual organ is made up of these four tissues.
And then the organs perform a particular function.
The heart is pumping the blood, and the kidney is
making urine, it's filtering the toxic material, and
keeping the valuable solvent.
And the organs then makes systems, and each of the
systems include multiple organs, and these systems can
function independently to achieve a function.
So we're going to get into all these details
in individual lectures.
And of course the systems don't function independently,
and it's the coordination between the systems that give
rise to the variety of complicated behavior that
individual organisms possess.
So here are the layouts, and in the first eight lectures I
will discuss with you about how cells work.
What are the fundamental features of the cells?
What are the things that build cells?
What are the molecular mechanisms that determines the
cells to be the type that they are?
And then after that we'll spend just two lectures very
briefly talk about a very important question of
developmental biology--
this is how a single cell, the fertilized egg, gives rise to
an organism through a series of differentiation processes.
And then after I'm done with my parts, you'll hear from Pat
about the immune system, and then Robert will come in and
teach you about the nervous system and
the endocrine system.
And this is followed by Craig--
Professor Heller's lectures on how physiology works as a
whole organism.
So I'll give the microphone to Pat for the immune system.
Hi everybody.
Happy you're here, and welcome back to campus.
I'm the resident immunologist in the biology department, and
I'm going to be talking about this-- it's really the first
of the physiological systems in the body that we're going
to be covering in the course.
That's particular systems that have important functions that
are necessary for survival.
So I think probably everybody has a pretty good sense of
what their immune system does.
It protects us against invasion by various bad
things, mostly germs--
bacteria, viruses, parasites, fungi, also various toxins.
And the fact that you're pretty much healthy-- so the
fact that you're all here, I don't hear too much sneezing,
as the flu is just arriving in this part of the country--
means that our immune systems are all working pretty well.
So it's a good physiological system to start with, because
it's a system that is carried out by particular cells, but
these cells are really a cellular system rather than an
organ system-- that they're not hard wired into place,
like the nervous system is, or the digestive system.
Rather, the cells of the immune system are white blood
cells that function by circulating around in the body
in a surveillance mode, screening the body for
infection by various pathogens.
So again, the fact they we're healthy means that the immune
system is working.
It's also very good system to start with because it really
integrates and covers, encompasses, a lot of material
you had in Bio41, as well as material that you'll hear from
Professor Shen on cells in developmental biology, in that
it illustrates how there's integration of various unusual
biochemical and molecular functions and properties so
that the cells can respond to virtually anything foreign
that enters the body.
So it's not pre-programmed to respond to particular
pathogens--
rather, the immune system has evolved to be able to protect
us against anything foreign that enters the body through
receptors that are generated by very unusual molecular
genetic phenomena.
So shown on the right are some examples of mechanisms of
immune responses.
So in response to various infections, we make
antibodies,
proteins, that will attack cells.
So shown on the right is antibodies
killing E. coli bacteria.
If we have cells in the body that have become altered and
harmful, such as tumor cells, or virus infected cells, some
of the cells of the immune system-- the T lymphocytes--
directly recognize them as being harmful, attack them,
and kill them, as shown on the bottom, where we see T cells
killing tumor cells.
So one of the great things about the immune system is
that it manages responses that are tailored for the
particular kind of invasion.
So I look forward to talking with you about these really
fascinating phenomena, and again, very unusual molecular
and cellular processes that allows us to protect ourselves
from invasions and to stay healthy.
All right, so I'll now pass it on to Craig, and I'll see you
in a few weeks.
Hello everyone.
It's great to see you here, and I hope that by the time we
get to my lectures at the end of the quarter, you'll still
all be here, and will not be depending on those videos.
The videos are going to be great, but there's no
substitute for actually coming, interacting with your
colleagues, interacting with the faculty, and actually
participating intellectually in the subject
matter of the course.
So Kang just mentioned to you the basic
organization of course.
You learned all about the basics of how you build a
cell, now you're going to see how the cells operate.
And through the course, you're going to see how these cells
are organized into tissues, organs, organ systems, and
eventually organisms.
So we don't have to go back over that again.
But what I wanted to tell you is the approach that we're
going to take when we get to my portion of course, and I'll
also talk about Robert's portion.
Robert's going to tell you about the nervous system and
the endocrine system.
And along with the immune system, these are the three
systems of information in the body.
Now information is just as important in biology as it is
across the street in computer science.
And it's these three systems in the body that enable us to
store information, to use information, process
information, and so forth.
So Robert's going to tell you about the endocrine and the
nervous systems, and then I'm going to put them to work in
telling you how these systems control our internal
environments and everything that we do to maintain healthy
status of the body.
So you might think of Robert's part as being everything above
the neck, and my part as being everything below the neck.
But indeed, there be quite a bit of
interaction between the two.
When we talk about physiology, what we're going to do is
first of all identify what parameters
we're concerned with.
Whether it's the pH of the body fluids, whether it's the
oxygen concentration, the CO2, or whatever it is.
And then we're going to identify the physical and
chemical and biological challenges to these
parameters.
What could possibly cause you to deviate from what's normal,
what's healthy.
And then we're going to ask how the various organs and
organ systems regulate these parameters of our internal
environment, and different evolutionary strategies.
We are biologists, we're not just human biologists, we're
concerned about how evolution has created various solutions
to the problems that we're going to be dealing with.
And then, of course, the most important topic in physiology
today is questions of control and regulation.
How are these organ systems controlled, regulated, and how
are they integrated?
Now advice for getting through this course successfully, for
getting over the bar.
Do the reading.
We are limited to three days a week for an hour and a half to
cover or a very large portion of biology.
You're going to have to know a lot more than what we can
possibly teach you in these three hours per week.
So do the reading, because the reading is going to take you
into areas that we won't have time to cover.
And quite frankly, it's advantageous to us and to you
that we don't have to cover everything, because we can
therefore spend more time on the things that are really
interesting, and also have some fun.
So do the reading, and do it before you come to class so
you can ask questions.
If there's something we didn't cover, you can ask a
question about it.
Study actively.
Don't just read the book with your yellow marker and cover
all sorts of things with red underlines, and so forth.
Use the lecture material, use the book to formulate
questions in your own mind.
If you see a topic heading in the book or in the lecture
syllabus, turn it into a question.
See if you can answer that question.
And when you can start answering all the questions
that you can formulate by covering up the paragraph and
just reading the title in the book or in the lecture note,
you will have mastered the course material.
And study regularly.
Don't wait until the night before the midterm, or the
night before the final.
Keep up, because there's a huge amount of information
that you're going to learn this quarter, and we want you
to learn it successfully.
OK?
Kang.
All right.
So before I get started about today's lectures, I just want
to use one minute to tell you a little bit about what I do
when I don't teach Bio42 here.
I spent the majority of my time running a research lab
across the street, and that's my primary job, I would say.
So in my lab, students and post-docs are interested in
studying the molecular mechanisms of a bunch of
interesting things about nerve cells.
We're interested in cells, but we're interested in a specific
type of cells, and the neurons.
So we're focusing on how neurons make specific
connections, and how when the axons is really long, how do
you actually make sure the materials are getting down the
axon and getting off at the right place?
And we're interested in how to build a cell with the
incredibly complex morphology.
And these are all very interesting questions, and you
can read some of that on our website.
And although we're trying to push the envelope on our
understanding of these very specific questions, what I
realized when I was preparing for these series of lectures
is that a lot of what we work on has intimate relationship
to what we will be talking about in the class.
For example, we're going to be talking about the very cool
molecular motors--
the cars, the vehicles that drives little tiny organelles
and put them in different places of the cell.
So we are actually going to talk about things like that in
this lecture series.
But of course, in the lab, we're trying to learn what's
unknown, trying to be on the frontiers of these
discoveries.
Just give you a very simple example, this is a purkinje
cell, is one type of neurons in our cerebellum, the part of
the brain that is very important for our movement.
And that's purkinje cell is famous because it has
extremely elaborate processes, and this is really the peacock
of cells, in terms of morphology.
And you can see that these are all dendrites.
As Craig told you about neurons, the nervous system is
a processed information.
And in order to process information, you have to sense
the information and then have output.
So all these very much branched processes, the
dendrite is the antenna of the nerve cell that receives
information.
And then there's a single axon that comes out the cell.
So this cell is incredibly complicated because it has
this very elaborate sets of antenna.
So now the question becomes, how can a cell build such
complex dendritic morphology.
And we don't study this in human--
it's very difficult to do experiments in human--
but we look for other cell types that are analogous to
this highly elaborate dendritic
cells in other organisms.
For example, in very tiny nematodes--
round nematodes, C. elegans,
and there are cells--
this is another neuron--
with very elaborate dendrite.
They're not quite as elaborate as this, but you can see that
dendrite branches very regularly to make these, again
receiving apparatus.
So we take an organism like this, and then we do genetic
experiments.
And for example, we found that if you just screw up one gene
with a single permutation in the particular gene, it
completely disrupts the way that the dendrite forms.
And then through understanding what is this gene and how this
gene functions, we are making progress in understanding how
a complex cell like this acquired its morphology.
So this is the kind of thing that we do in the lab.
And if you're interested, go read our websites and we do
provide opportunities for undergrads to work in the lab,
but I ask for a certain commitment.
If you're interested in that, come talk to me.
So my office hours are going to be from 9:30 to 10:30 on
Thursdays while I teach, so you're welcome
to come to the lectures.
And so I think Waheeda has covered most of this, and I
just wanted to tell you guys about another resource.
This is going to be posted, so you don't
have to write it down.
And this is a video collections that a bunch of
students in Harvard really made to give you a visual
impression of a lot of the processes that we'll be
talking about.
It's probably not 100% scientifically correct, but
mostly it is.
But it gives you really a visual animation
impression of it.
It helps to remember the process, it helps to put what
we're talking about-- the more abstract concept in the class
into a more visually pleasing media.
So you can check this out.
So now we're going to start today's lecture.
So as we said, the cells can do amazing things.
What are the cool things that cells can do?
Can anyone give me an example of what is an interesting
thing that a cell can do, let's say a biochemical
reaction cannot achieve?
You all know that you can add an enzyme and it can convert a
substrate into a product in a test tube, right?
That's what you guys have learned in Bio41.
But what is a cool thing that a cell can do that's beyond
these biochemical reactions?
Any examples?
Selective transport through a membrane,
for instance?
I mean, certain, what you'd call
cellular structure, a series of different molecules arranged
in a certain way to do that?
Absolutely, absolutely.
You need many molecules in order to transport a
particular organelle from one side of the
cell to another, yes.
But closer to, let's say, what organisms can't do.
For example for neurons, like what can a neuron do that you
can appreciate?
What can a neuron do?
Any examples of--
yes, go ahead.
Take in a stimulus and generate a response?
Excellent.
So it takes a stimulus and generates a response.
So in this particular experiment, this is the
genetically modified fly.
It's a fruit fly, it's a very small one, and here we're
viewing it on a microscope.
And here what you will find is that this fly, every time that
there's a light flashed, this fly jumps.
And this fly is not a normal fly, but it's a genetically
engineered fly, and two of these hundreds of thousands of
neurons in this fly, but only two of them express a channel
protein that responds to light.
And these invertebrates have these escape neurons.
They're huge neurons, and they're there to escape from,
let's say, predators.
These researchers, what they did is that they forced the
expression of this exogenous channel protein.
Normal flies don't have them, and these channel proteins
upon the light stimulus will activate this neuron.
So therefore, as you can see, when there's a flash, these
neurons get excited and no matter what else happens, the
flies will jump.
So generating behavior--
sensing a stimulus and generating a behavior.
That's an amazing thing cells can do.
There's many other things that cells can do
that's quite amazing.
For example, a fertilized egg.
A fertilized egg contains all the information that is
required to make a whole organism.
We're going to talk about this in a development lectures.
But all you need is to have that single cell, and then
it'll undergo a series of divisions, some cells will
undergo this type of differentiation, other cells
will differentiate into other tissues, and then these
tissues actually interact to make a intact organism.
So that's pretty amazing, the fact that the single cell
contains all the information that is
required to make an organism.
That's remarkable.
And then the immune cells that Pat just talked about has the
capability of recognizing things that it has not seen
ever, and the type of things that it can
recognize is infinite.
So that's pretty remarkable.
The immune system has evolved to recognize pathogens, and
the passages are number one, hasn't seen by the individual
organism, number two, the pathogens are also evolving,
so they're are changing all the time.
So the immune system has evolved ways to create a
recognition system that essentially can recognize just
about an infinite number of things.
So how that occurs, that's pretty amazing.
I give you three examples of very specialized cells.
These cells either have potency, or has a
very fast acting response, or you can generate so many
varieties of receptors that can recognize just about
everything.
So these are very specialized cells.
There are many different cell types in our bodies that can
do a particular sophisticated task.
But before we really talk about these, we want to focus
our energy in the first eight or so lectures on the
commonality of cells.
What do all the cells have in common?
What are the basic building blocks of cells, and what are
the basic principles of building a cell from scratch--
from DNA, from protein?
So there are many questions involved in building a common
cell, but the question that we're specifically going to
focus on is how to target the proteins to the right place.
So you've talked about it in Bio41, you've learned a lot
about biochemical reactions, and those things happens in
test tubes, right?
So you add an enzyme, add a substrate, it'll be converted
into products.
And in cells, there's many biochemical reactions.
All the reactions that you've learned in the last semester
occurs in cells.
But they don't occur in just in a mixed bag of solutions.
So they have to be at particular places.
So we're going to focus our energy on how proteins are
targeted to the right place.
Can anyone think of why we want to do that?
What is the advantage of having this biochemical
reaction occurring in one place, and the other one
occurring in another place?
What is the advantage of that, instead of just mixing
everything in a test tube and let's let them all do their
thing randomly?
What was the advantage of some kind of organization?
[INAUDIBLE]
faster.
Faster.
Can you elaborate that?
[INAUDIBLE]
closer to each other, so they're
more likely to interact.
Great, great, that's
absolutely a very big advantage.
So you've learned about metabolic processes that often
contain more than one set of biochemical reactions.
You need to take the substrate and convert to the first
product, and then immediately there's another enzyme that
will convert that into the next the set of intermediate.
So having these enzymes that catalyzes the same series of
biochemical reactions together will increase the efficiency
of the reactions.
Great, OK, so that's one advantage.
Any other advantages?
[INAUDIBLE].
Absolutely.
So once you have them all together, you can potentially
regulate so that the conditions that favors that
interactions.
Great, so increasing efficiency.
Any other advantages?
So cell--
yes, go ahead, sorry.
It's OK.
I don't know if it's ever happened [INAUDIBLE], I've
never heard of an example of this happening, but if I had
two reactions, and [INAUDIBLE] for some reason--
Yes.
--but they'll screw each other up--
Yes.
--then I've got to put them in different rooms.
Yes.
Great points.
That's exactly the second point that I was
going to show you guys.
So any biological reaction is reversible.
So on one hand, you need to synthesize proteins.
On the other hand, you need to degrade proteins.
Any protein in our body has a half life, so it lives for a
certain amount of time, and then it gets degraded.
So imagine the mechanism to degrade proteins and the
mechanisms to synthesize proteins.
They have to be put in different places, right?
So indeed we have very potent ways to degrade proteins.
So those things are toxic if you let them just go
everywhere.
So these are the two advantages of segregating
compartmentalized proteins.
And one is to increase efficiency, and second is to
minimize damage to the cells.
This is especially important for biochemical reactions that
are hazardous, that are involved in degradation,
involved in turning down--
[INAUDIBLE].
Already?
[INAUDIBLE].
Did I even turn it on?
[INAUDIBLE].
OK, so now I have another challenge for you guys.
So now we know that it's important to keep certain
proteins in one place or confine them to a particular
sub-cellular compartment.
What are the ways of doing that?
Can you think of any ways to achieve that?
[INAUDIBLE].
You can localize specific reactions to organelles.
Of course.
Can you be slightly more specific?
Like what are the ways of--
For example you can import certain molecules into the
mitochondria so they react to the mitochondria, and then
export other molecules out of the mitochondria.
Great, so mitochondria is a membrane organelle.
So that means inside the cell, there are lipids that can be
utilized to compartmentalize solutions.
So lipids are hydrophobic, so that that most of the proteins
are hydrophilic.
So once you have a layer of lipids, it's sort of like
putting a bunch of things in a plastic bag so that they can
be contained on their own.
Using lipid to create membrane organelles is a great way of
compartmentalizing molecules.
Anything else?
Any other ways that can achieve--
so a lot of it is done through exactly what she said.
Sending things into particularly lipid-defined
organelles.
But there's another way of doing it.
This one's a little harder.
So the other one is using scaffolding molecules.
It's basically even if you're in the same swimming pool, if
certain proteins are just holding on to the same, let's
say, beacon, then they would still be very
close to each other.
So there are scaffolding molecules whose role is just a
grab onto various proteins that are doing this common
thing, and this represents another way of concentrating
proteins to the same sub-cellular compartment.
So what I just told you this is some very basic concept of
compartmentalizing cells.
And just to put it in a more concrete words, cells are--
we have to study them, because they're the smallest unit of
independent life.
And if you look on our planet, there's two type of cell.
The prokaryotic cells--
these are very simple cells, bacterias and cyanobacterias.
And these cells are simple in the sense that they have a
surrounding lipid membrane that separates them from the
external world, and within the membrane there are DNA, of
course, these are genetic material, DNA and RNA.
And then they're primitive, they're very simple.
They're a little compartment within the [INAUDIBLE]
of these prokaryotic cells.
And of course most of the other animal kingdom, the
planet is populated with fungi, plants, and animal
cells, and these are eukaryotic, and eukaryotic
cells are much larger.
And of course they vary in size as well.
The fertilized egg could be as big as one millimeter--
it can be seen by naked eye.
And these cells are much more sophisticated in several ways.
And one is that they have lots of membrane organelles, like
the organelles that we talked about a little bit already.
And these organelles inside the cells, these organelles
are where the compartments are built and where different
biochemical reactions took place.
And to see these organelles, there has be some very high
resolution microscopy techniques, such as the
electron microscopy, because they're so small.
So the plasma membrane has the prokaryotic cells, the
eukaryotic cells has a lipid membrane that separates the
cells from the outside world.
And then within the lipids, there's cytosol, and that's
basically the solution between the organelles, and it's full
of proteins.
And its gelatinous.
It's kind of like the egg white.
So when you crack an egg, and you see
this not quite liquid--
they're liquid, but they're very, very gelatinous.
And that's what the cytosol feels like.
And between the cytosols there are intracellular membranes,
and that forms distinct compartments.
And you've heard mitochondria, which houses a lot of the
reactions that generate ATP, and you've probably heard of
endoplasmic reticulum, which is the site of membrane
protein synthesis, and then there are lysosomes, and this
is the degradation factory.
The house lots of proteases which chop up proteins and
recycles amino acids from there,
So here is a layman's analogy--
so different compartments are like
different rooms in a house.
You've got a kitchen where you do the cooking, and bedrooms
where you rest.
Is that funny?
So so of course that the compartments are defined by
the proteins that's in them.
The kitchen is defined by things that you need to
cook, and so on.
So now what I want to do is to take a slight detour, and then
tell you about two of the technologies that have been
commonly used to study where proteins are within the cell.
So since I said that of the key question that we'll be
talking about is how to send proteins into these different
rooms within the cell, then we have to have a way to
recognize where these proteins are.
So the first technologies got indirect immunofluorescence.
And essentially, the goal here is to understand where this
particular protein, which is drawn as these pink dots, are
within the cell.
So in order to do that, you have to have
two things in hand.
One thing is that you have to have a reagent
recognize this protein.
And second is that this reagent has to glow in some
way so that you can see it by microscope.
So this is achieved as following--
utilizing the immune system's unique ability to generate
many reagents that specifically recognize
proteins, you can purify the protein that you're interested
in, which are these dots, right?
And then you inject them into rabbits.
So a rabbit has never seen them before, so once he sees a
exogenous protein, it'll generate
antibodies against it.
So the antibodies can bind very
specifically to this protein.
You purify the antibody--
you could actually essentially just tag the antibody with a
fluorescent dye.
So you can conjugate this antibody with a fluorescent
dye, so that now you can see this antibody.
And then you can introduce the antibody into the cells by
fixing the cells, by putting holes on the cells with the
detergent so that the antibody will get in.
And then if the antibody's tagged with the fluorescent
dye, then you can see it with fluorescent microscopy.
This is not usually how people do it, because this step of
labeling a primary antibody with the florescent dye is a
finicky reaction that doesn't go very well.
So you have to kind of troubleshoot it a lot to get a
good label.
So that's why people don't really do it, and so instead
what happens is that there are these things called secondary
antibodies.
Essentially these secondary antibodies are antibodies that
recognize these rabbit's antibodies.
So you're [INAUDIBLE]
the way that you make it is that you take a rabbit
antibody and inject it into, let's say, donkey.
And then the donkey has never seen rabbit [INAUDIBLE]
protein.
So once he sees the rabbit proteins, he says hey that's
[INAUDIBLE].
I've gotta make some antibodies to
recognize these things.
So they make it a set of donkey antibodies that
recognize the rabbit antibody.
So then you basically fluorescence tag these, and
all you have to do is to tag--
so the company has done this for us.
So in the lab, what we have is we have a series of secondary
antibodies that are florescence labelled.
So for example, you can have florescence labeled secondary
antibodies against rabbit antibodies.
So that means it can recognize all the rabbit antibodies.
So within the rabbit antibodies, there's this
common part that essentially is the same between all the
rabbit antibodies.
So that way all you need is to have a few secondary
antibodies that the companies have already
made, it's on the shelf.
So what you do is you make a primary antibody, and then you
add a secondary antibody with the florescence tag, and then
you can see this indirect immunofluorescence.
So it might be a little bit mind boggling for now, but if
you go through this again on your own after
class, you'll get it.
So essentially you can visualize the fluorescence by
fluorescent microscopy here--
I apologize for the lighting.
It's not so ideal, but you can see that this is to show you
that indeed you can see there are different fluorescence
patterns you can observe when you use different antibodies.
So for example, here the staining is obtained it from
an antibody recognized the nuclear protein.
It's in the center of the cell, and here is recognizing
of major cytoskeletal component called actin.
The lysosomes are punctated structures that are
distributed all across the cytosol.
The mitochondria look very different.
So if you use different colored dyes, you can actually
do a quadrupled or quintupled stainings, and you can
visualize different components of the cells simultaneously.
So that you really gives you the power of localizing
different proteins at the same time, and understanding the
relationship with each other.
So that's one way, that's indirect immunofluorescence--
this [? is involved in ?]
purifying your proteins and then injecting them into a
antibody generating organism, and then purifying these
antibodies, taking advantage of this very specific
recognition, and coupling the antibody with a dye, and then
visualizing them in cells.
And a parallel method that has been used very extensively to
localize proteins within cells is to use this thing called
green fluorescent protein, GFP.
And probably many of you have heard of it, and this was
discovered about 15, 20 years ago initially
from the GFP molecule.
The gene was isolated from jellyfish, and then people
realized that this a protein in the jellyfish that if you
shine a beam of blue light, and it'll give you a
fluorescence--
a yellowish green fluorescence.
OK.
So the original GFP is yellowish green.
But Roger Tsien's group, if you made a lot of mutations--
so they mutagenized.
They randomly introduced random mutations in the GFP
gene and looked for mutant forms that actually emit a
different wavelength.
And they now we have a variety, a series, of proteins
that are all derived from this carrying mutations from the
original GFP, but they are either yellow,
cyan, a blue, a red.
So there are many variants of this.
So the way that you--
this with this work was actually awarded the Nobel
Prize in 2008.
And when you use the GFP, what you do is you
fuse the GFP gene.
You take the DNA that encodes the GFP and then stitch it
together with the gene that you're interested in studying
the localization of.
And then you introduce this fusion, this artificially
fused piece of DNA, into the cell.
And because this is going to go glow, so you are basically
tethering a light bulb to the protein that you are
interested in.
So no matter where the protein goes, the GFP will show you
where it is.
OK.
Is that clear?
OK.
Cool.
And so there are advantages and disadvantages between
these two methods that we're not going to really go into.
OK.
So now with have these two methods, we can now start to
consider what is the process that sorts different proteins
into different compartments.
And this is somewhat similar to a post office situation,
where any mail that goes into a post
office has to be sorted.
The sorting signal is essentially the address that's
on the envelope.
Is it going to the US, the foreign Japan or
France, and so on.
So the sorting signal here is the address that's on the
envelope, and the way that these addresses are read out
is by human or by a machine.
And of course we have very sophisticated programs to
recognize what these things are, so how do cells do it?
What are the signals, and how are the signals read out?
These are the most critical questions that we'll discuss.
So the process of course starts from where the
proteins are made.
How are proteins made?
How are proteins made?
You guys should all know this.
On the ribosomes.
On the ribosomes, yes.
By translation and where does it happen is on ribosomes.
This is too easy for you guys.
And where in the cells are the ribosomes?
So this is a little bit trickier.
I don't know if you guys really--
so because of time I will just skip to the answer.
So there are two populations of ribosomes.
And there's the cytosolic ribosomes; these are
free-floating.
These are ribosomes that are just hanging out and ribosomes
that are not attached to anything.
And there are also ribosomes that are tethered to the
endoplasmic reticulum surface.
And the ER membrane that is decorated by ribosomes are
called the rough ER.
This is opposite to the smooth ER which are the ER membranes
that don't have ribosomes.
So the reason that I bring this up is because it turned
out that these two populations of ribosomes make proteins
that are destined to a different part of the cell.
So the ribosomes makes proteins that are eventually
going to either end up in the cytosol, in the nucleus, in
the mitochondria, the chloroplast, and the
peroxisomes.
And the membrane-attached, or the rough ER ribosomes, are
going to make proteins that are going to go into the ER,
into the Golgi, outside of the cell.
OK.
This is the goal of the next two lectures, so don't worry
if you're not getting it right away.
So now we're going to go into each of these sub-cellular
organelles and then trying to understand what is the signal
that target proteins into these organelles.
So we first start with the easiest one is the cytosol.
I mean, a cytosol is essentially the stuff between
the organelles.
And, as I said, it's a very concentrated solution of
proteins, metabolites.
It contains many enzymes that are required for various
metabolic processes, and it's the site of
cytosolic protein synthesis.
So protein synthesis occurs by ribosomes, and the ribosomes
read out messenger RNA and then add nucleic acid one by
one onto the polypeptide chain.
And these polypeptide chains are recognized by proteins
that help them to fold, these are chaperones.
And for cytosolic proteins, there is no special signal
that is required for these proteins.
So since the synthesis is occurring in a cytosol, since
the product after it's coming off the ribosomes, it's just
going to distribute within a cytosol.
There's no special signals required.
It's a default.
So the default pathway for cytosolic ribosomes is to
synthesize cytosolic proteins.
OK.
So the next one is much harder than the cytosol, and this is
the nuclear structure.
So the nucleus is of course a very
important essential organelle.
This is the organelle that houses
chromosomes, or genetic material.
This is the organelle that synthesized RNA.
All the transcriptional process
occurs within the nucleus.
So this is not only making the messenger RNA, but also the
tRNA and the ribosomal RNA.
And within the nucleus, there's a specific place with
the nucleus that makes the ribosomes.
This is called the nucleolus.
The nucleolus is a very dense structure, and it makes the
ribosomes and puts the ribosome proteins together
with the rRNAs to make ribosomes, so a
very important process.
So what separates the nucleus from the cytosol is a set of
membranes called nuclear membranes.
The nuclear membranes, there's two layers of it.
There's the inner nuclear membrane, the
outer nuclear membrane.
They're actually continuous.
It's all very, very confusing.
And this nuclear membrane is also continuous with the
endoplasmic reticulum.
So you don't really have to understand, I'm going to force
you guys to understand, all of these intricate connections,
but one thing that you really should take away from this
lecture is how molecules go inside and
outside of the nucleus.
It turned out that this is mediated through an
interesting structure called the nuclear pore complex.
So on this lipid membrane there are holes.
They're not simple holes, but they are actually a
multi-molecular apparatus that selects proteins of its desire
to import and to export.
This is pretty much the only way to go inside and outside
of the nucleus, so it's very important.
OK.
Let's take a closer look at this.
So first let's learn something about what is going to go into
the nucleus and what is going to come out of the nucleus
through these nuclear pore complexes.
And so the experiment is done as following.
Just for starters, you can purify different proteins,
some proteins are really large, other proteins are
really small.
Inject them into the cytosol.
And you can ask the question whether they go into the
nucleus or not go into the nucleus.
So through doing various experiments with different
proteins, it will become very clear quickly that very small
proteins, let's say 10 calcitonin peptides, they all
go very easily inside and outside the nucleus.
They can diffuse across the nuclear pore complex inside
and outside of the nucleus.
So small proteins are not blocked by the nuclear pore
complex, but larger proteins are prevented from going into
the nucleus unless it carries a specific signal.
So proteins are synthesized in the cytosol.
OK, so this is the ground truth, the first ground truth
that we have to establish.
There's no protein synthesis inside the nucleus.
So any proteins that go into the nucleus are synthesized in
the cytosoll, so they have to be
transported inside the nucleus.
So then it becomes very critical to figure out what is
the signal that carries this protein, tells the protein
that it should go into the nucleus.
And these experiments were done on this particular
protein called nucleoplasmin, and of course this
nucleoplasmin needs to be a large protein.
For very small proteins, they can just go into the nucleus
freely, then they don't need a signal.
So now for this large protein, this protein is as large as a
165 kilodaltons and is way bigger than the cut-off for
free diffusion, so it must contain a
special sorting signal.
So this is how the particular experiment is now done.
I realized that I told you guys about this
immunofluorescence and the GFP.
And these techniques actually came after this particular
experiment was done, so this is an old-school experiment
that was done with a different way of following the proteins.
And this way of following the proteins is to make the
protein radioactive.
So when you make the proteins, you actually feed the cells
with radioactive building blocks, amino acids.
So when the cells make these proteins, then it incorporates
into radioactivity into the protein.
And then you purify the protein, and then again you
can inject the proteins in the cytosol and you ask the
question whether this radioactivity can be detected
within the nucleus by putting a film on top of the cell.
And, in this case, the answer is yes.
So when you inject the whole protein labeled with
radioactivity, it goes into the cell, lights up the
nucleus when you put a film on it.
But now the question becomes which part of this--
do you need the whole protein in order
to go into the nucleus?
Or is it only a part of the protein that is directing the
protein to the nucleus?
Where is the signal?
So the researchers what they did is they used a technique
called limited proteolysis.
They added a protease, but the activity of these protease is
controlled so that it didn't chop up the protein completely
into little bits.
It only made one cut.
It cut the protein in two halves, the head and the tail.
Now, you can purify these hot head proteins, hot tail
proteins; inject each of them into the cell; and ask the
question whether do they go to the nucleus or not.
And indeed it seems that only the tail goes into the
nucleus, and each half actually is bigger than that
the free diffusion cut-off.
So this tells you that OK, maybe the tail is what
contains the signal to go into the nucleus.
And then another important experiment is that how do
these go into the nucleus?
I told you that they must go through the nuclear pore
complex, but that was not known by the time this
experiment was done.
So what experiment that they did is to coat the tail
protein with a gold particle.
These gold particles are metals, so they're metal, they
have very high contrast under EM, under electromicrospy.
So when they injected these gold particles that are
covered by the tail protein, which contains the
nucleoplasmin sequence, what they saw was not only proteins
and gold particles inside the nucleus, but they saw gold
particles right through these channels.
So that gives the hint that it's the channels that allows
the gold particles to go.
So it's sort of like if this is the door and we all come in
and outside of the room through the door, then if you
take a snapshot and people are exiting, you're going to catch
someone who's right at the door.
So that's what these experiments are for.
So through doing many of this type of experiment and
localizing more and more to the details of what is the
minimum requirement to target something into the nucleus,
people came up with a sequence.
This particular sequence for nucleoplasmin is required for
entering the nucleus.
It's called the nuclear localization sequence.
So after this nuclear localization sequence has been
characterized for multiple nuclear proteins, it becomes
clear that it's a commonality for what this
sequence looks like.
These are sequences that contain four
to eight amino acids.
And these four to eight amino acids contain a high
percentage of lysines and arginines.
What's in common between these two amino acids?
It's positively charged.
It's what?
It's positively charge.
It's positively charged.
Excellent.
OK.
So you need a lot of positive charges to
target these two proteins.
So now I told you this is the conclusion, but how do you
demonstrate that the nuclear localization sequence is
necessary and sufficient to target a particular protein to
the nucleus, what other experiment
that you need to do?
So protein expansion in the nucleus, and you add alleles
four or eight amino acids.
And then it goes [INAUDIBLE]
Great.
Sufficiency experiment.
Tag an NLS onto a protein that would otherwise not go to the
nucleus and ask the question whether that's sufficient to
target into the nucleus.
That's a sufficiency experiment.
What about necessity?
Yes.
Take a protein that usually is localized as [INAUDIBLE] and
then move that one sequence to see if--
Great.
So mutate away those NLS and ask whether that prevents the
otherwise endogenous portion that should go to the nucleus
from going there.
So those are the two experiments that were done to
establish that this positively charged stretch of amino acid
is indeed the nuclear localization sequence.
So there is a nuclear localization sequence.
So this is what's needed, what's to be read out, in
order to go into the nucleus.
The next question is, how do cells read this out.
So if there is a signal and a cell has to respond to this
signal, what is a way to read out to this particular NLS.
So because of time I will just skip.
And there has to be receptors.
There has to be receptors.
Any signal that the cells need to read out,
there has to be a receptor.
There has to be something that interacts with
this particular sequence.
And that receptor is a family of proteins
that are called importins.
This is a family a proteins that binds to a highly
positively charged stretch of amino acids.
And the importins which binds to the NLS and the targeting
protein go through the nuclear pore complex.
And of course this nuclear pore complex is a very
important concept that we have to learn.
It's a multi-protein complexes made up of
more than 30 proteins.
It has an eight-folded rotational symmetry, so when
you view this is purified nuclear pore complex from
using the EM, you see this eight-fold
symmetry from the top.
And this is not only important for proteins entering the
nucleus, but it's also important for new proteins
that are exiting the nucleus.
So proteins that need to exit the nucleus carries a
different sequence.
It's called nuclear export sequence, an ES, and they both
go through this door.
And the nuclear pore complex structure is not a symmetrical
structure in the sense that if you look on the outside of the
cytosolic side of the pore, it has these filaments.
And inside the nucleus, it has a ring structure.
It's just they're asymmetric.
So now I'm going to show you how this process occurs.
And not only did the system require the importins which
binds to the NLS, but it also has to have something, a
switch, that tells these two proteins to
separate from each other.
This is a sort of like a transportation system.
You need to hop on the train in order to go from here to
San Francisco, but when you get to San Francisco there has
to be a signal that tells you to hop off the train, but
otherwise the transport doesn't work.
So I told you that there's the binding between importin and
the NLS sequence that occurs in a cytosol.
That's great.
That's all good, but then when these proteins that are bonded
to each other go into the nucleus, the importin has to
let go of the cargo protein.
So that means there has to be a switch, and once the import
complex goes into the nucleus, the
switch has to be activated.
And the function of the switch is to let go, to dissociate
these two proteins.
And that's where small G protein, Ran, comes into play.
So essentially the Ran, it's a GTPase and
that has two states.
These GTPase are small G proteins, it binds to GTP, and
they have an intrinsic ability to hydrolyze GTP, the
triphosphate, taking away one of the last organic phosphate
and to generate GDP.
So the small G protein has the intrinsic ability to converge
the GTP-bound Ran and to make GDP, an inorganic phosphate.
So therefore there's two states of the small G protein.
There is a state that binds the GTP, and there's a state
that binds the GDP, and there's transitions between
these two states.
I told you that the Ran itself it's a small G protein, so it
has the intrinsic ability to hydrolyze GTP to make GDP.
But this process is very slow, and it needs the help of
another protein called Ran-GAP, the
GTP's activating protein.
So in the presence of this Ran-GAP, then this biochemical
reaction occurs much faster than otherwise.
So when you have a lot of Ran-GAP, the GDP-RAN will
accumulate.
In the reverse process, taking the GDP-Ran and converting it
to the GTP-Ran requires the GDP to dissociate from the
Ran, and then its nucleotide binding pocket be
replaced by a GTP.
And this process also occurs very slowly by itself, but
then in the presence of a nucleotide exchange factor it
occurs fast.
Now, let's go back to see how this works.
So in the cytosol, the importin binds to the
NLS-containing proteins, and this complex goes through the
nuclear pore complex.
And once it gets into the nucleus, the Ran-GTP comes
into play, and when it binds to the importin, it
let go of the cargo.
So the Ran-GTP is the signal that dissociates the importin,
the cargo from the vehicle.
What about the cytosol?
It turned out that in the cytosol, the Ran-GDP dominates
and the Ran-GTP bind to the importin that binds to the
cargo, and therefore it doesn't dissociate.
So why is the binding favored in the cytosol, but the
dissociation favored within the nucleus?
That becomes the key question.
Why there's a biochemical reaction that favors the
binding in the cytosol so then they can start to be
transported, but once it gets into the nucleus, why is it
the dissociation that is being favored?
That is because the localization
of the GEF and GAPs.
Inside, the nucleus, the GEF is present at a high
concentration, therefore there's a lot more Ran-GTP in
the nucleus.
And the Ran-GAP is targeted into the cytosol, and as a
result the GDP form is dominant.
And these go back and forth.
The Ran-GDP and GTP form goes back and forth.
And this type of switches is a theme in cell biology.
We'll come back to this again and again.
They're different classes of G proteins that are engineered
to regulate a different part of cell biology.
But they all have a commonality, which means that
they are switches.
When you need to turn things on and off, you use these
switching mechanisms.
And this is mediated by basically generating two
states of the GTP and GDP and being able to cycle them, and
then coming up with proteins that can
catalyze these reactions.
I think I'm going to stop here, and then we'll pick up
in the next lecture.