Hi.  I'm Sue Biggins
and I'm an investigator at the Fred Hutchinson Cancer Research Center
in the Division of Basic Sciences.
I'm also an investigator with the Howard Hughes Medical Institute.
Today, I'm going to talk about the process of
chromosome segregation,
which ensures that your cells inherit
the right chromosomes when they divide.
Cells must inherit the right chromosomes.
This is a karyotype of a normal cell
-- these are chromosome spreads --
and you can see that you have two copies
of every chromosome,
one from mom and one from dad.
This looks really different in a cancer cell.
Here is a cancer cell chromosome spread
and you can see a really large variation in chromosome number
-- there's entire gains and losses of chromosomes.
In addition, there are chromosomal rearrangements
that often occur.
This condition where cells have the wrong number of chromosomes
is called aneuploidy
and it's one of the most common hallmarks of cancer cells,
and you can learn a lot more about the consequences of aneuploidy
in the lectures from Angelika Amon.
To ensure that daughter cells
inherit the right chromosomes,
they undergo two major events during the cell cycle.
After G1 of interphase,
they enter S phase,
and the chromosomes replicate, or duplicate,
to form sister chromatids.
At mitosis, the chromosomes segregate
to opposite poles
and then when the cells undergo cytokinesis
to form daughter cells,
this ensures that each daughter cell inherits
one copy of each chromosome.
Now, a chromosome missegregation event during mitosis,
shown here,
results in aneuploidy,
as I just showed you on the previous slide,
which is a common hallmark of cancer,
and I should mention it's a common hallmark of other diseases,
such as birth defects.
And I want to remind you that you have
greater than a trillion cells in your body,
so this process has to happen correctly
for every pair of chromosomes in every cell
every time they divide.
So, mitosis was first observed
in the late 1800s by Walther Flemming,
and these are just drawings he did.
What he did is he stained
grasshopper cells or salamander cells
with a DNA dye,
and then he was able to deduce that
during the cell cycle chromosomes duplicate and partition.
And you can see here that they partition...
he even drew in fibers that we now know are microtubules.
Due to major advances in cell biology and microscopy,
we can actually visualize this process in real time.
I'm going to show you two movies
from Geert Kops' lab,
where he visualizes mitosis.
What he did, or what their lab did,
was they expressed
a fluorescent fusion to a histone
so that they could visualize the chromosomes,
and then they marked the microtubules
with a fluorescently tagged component
of the microtubules.
So, I'm going to start the movie
and what you're watching here are the microtubules
forming a spindle,
and those microtubules are capturing the chromosomes,
aligning them, and, there,
they're pulling them to opposite poles.
Now, this is a really dynamic and complicated process,
and it turns out it's actually also
very error-prone.
I'm going to show you an example of that in the next movie.
Here, most of the chromosomes get properly attached
to microtubules,
but in the upper-left you can see an entire set of chromosomes
doesn't get captured,
and therefore it doesn't segregate to opposite poles,
and that generates the aneuploidy
that I just told you is a hallmark of many diseases.
So, let's break down the events of chromosome segregation.
As I said, during S phase
chromosomes replicate to make sister chromatids,
shown here,
and then a complex called cohesin
physically links those sister chromatids together,
and that's important so that the cell
can distinguish pairs of chromosomes.
At metaphase, a bipolar spindle forms,
and this is due to the microtubules
that emanate from the spindle poles,
and some of those capture
a locus on the chromosome called the kinetochore,
and we're going to talk about the kinetochore in depth today.
Once every pair of chromosomes
is properly captured by microtubules,
a complex called the APC,
or anaphase promoting complex, is activated,
and it cleaves cohesin so that the linkage is destroyed,
and that allows the microtubules to
physically pull those chromosomes
to opposite poles at anaphase.
Now, the kinetochore, as I said,
is the structure that directs segregation.
This is a really enormous protein complex
that assembles on centromeric DNA,
and so this is just a cartoon here
so that you can see the kinetochore.
The centromere I'm going to define as
the DNA on the chromosome
where the kinetochore forms,
and in this image you can see that
the inner centromere is the underlying DNA
and then there's an inner kinetochore,
which are the proteins that directly bind to the centromeric DNA,
and then there's a whole 'nother layer of proteins
that assemble onto that
and they form the outer kinetochore.
And that is the microtubule attachment site.
And we now know that the human kinetochore
has more than 100 unique components,
so it's a really complicated structure
and most of these proteins are present
in multiple copies,
so there's hundreds and hundreds of proteins
in just a single kinetochore,
one of the most complicated protein structures in the cell.
The centromere is defined as
the chromosomal site of kinetochore assembly, as I said,
and you can see it, it's the primary constriction in these images
that you can see here.
When we look at this by electron microscopy,
the underlying chromatin is this
sort of fibrous mat underneath,
and then you can see the structure on top of that,
where the white arrows are, is the kinetochore,
and then you can actually see the microtubules
are attaching to that kinetochore.
And I think you can appreciate that
it's very difficult to really understand
the structure of this complex protein structure
from looking at EM (electron microscopy) images in cells.
There's three types of centromeres
-- a point centromere, a regional, and a holocentric --
and they're defined by the number of microtubules
that the kinetochore can bind.
So, point centromeres bind to just a single microtubule,
regional kinetochores are the most common,
and they consist of multiple microtubule binding sites
in a single kinetochore on the centromere,
and then holocentric chromosomes are really interesting
-- they're microtubule attachment sites over the entire chromosome,
and that's common in insects and worms.
Now, the centromeres vary a lot in size
and sequence as well,
and this is just representative centromeres
from a number of organisms
to make this point.
So, in budding yeast, at the top,
there's a 125 base pair centromere,
just 125 base pairs is sufficient
to ensure that that's a centromere.
And this is really different in other organisms,
such as humans and Arabidopsis,
that have megabases of DNA
that form the centromere.
They also vary a lot in sequence.
Budding yeast is exceptional
and it has a sequence-specific centromere
that's sufficient to drive kinetochore formation.
However, in most organisms,
such as the rest of them shown here,
the hallmark of the centromere
is really just repetitive DNA,
and so in humans it's just
repeats of alpha-satellite arrays.
So, the underlying sequence is highly repetitive
and it's usually AT-rich
and, because of this,
the last region of the genome
is usually the centromere to be sequenced,
because it's such highly repetitive DNA.
Now, because there's really no sequence specificity
to centromeres other than budding yeast,
it turns out that centromeres are normally
epigenetically defined or specified.
So, the site on the chromosome
that's going to become the kinetochore
is defined epigenetically
and the most common hallmark is this nucleosome
called CENP-A.
So, as you know, the chromosome
consists of chromatin,
which is built up of nucleosomes.
Normally, those nucleosomes
contain H2-A, H2-B, H3, and H4.
However, at the centromere
there's an interesting nucleosome called CENP-A,
where the two copies of H3
are replaced with this histone variant
called CENP-A.
And so what this means is that at the centromere
there's arrays of CENP-A nucleosomes
interspersed with H3 nucleosomes.
And this histone variant
is only present at centromeres,
so these nucleosomes are exclusively at the centromere
and are clearly the epigenetic mark.
Another hallmark of centromeres is that
they're embedded in heterochromatin.
In addition, there are specific modifications
to the histones in these centromeres,
so they have a lot of unique features,
and the field is still trying to understand
how this histone variant is deposited
specifically at the centromere
and how that's controlled,
and then what are the mechanisms that make sure that
it doesn't accidentally go into the rest of the chromosome,
where it could form additional kinetochores.
Okay, so let's turn to the kinetochore now.
The first kinetochore proteins
were identified by exploiting the fact that
it turned out that patients
who have some autoimmune diseases
have antibodies that recognize the centromere
or kinetochore locus.
And you can see that in this image from Bill Earnshaw.
In the top image of a cell,
an immunofluorescence image,
you can see tubulin, shown in blue,
and at the tip of those kinetochore microtubules,
you can see the red dots
that are the antibodies from these patients,
and that's called ACA -- anti-centromere antibodies.
So, Earnshaw and, independently,
another group, Goldner,
realized that they could take advantage of the fact
that these antibodies recognized kinetochores
to clone the first kinetochore proteins.
And actually, in this image below,
you can see CENP-A staining
-- it turned out that CENP-A,
that I just talked about,
was the first kinetochore protein that Earnshaw identified.
Over the years, biochemistry and genetics
have really identified the bulk of kinetochore proteins,
and these are immunoprecipitations from Ian Cheeseman,
where... you don't need to worry about what all these complexes are
or what all these proteins are,
the point I'm trying to make is one of the main approaches
to identifying kinetochore proteins
was to take one kinetochore protein
and immunoprecipitate it to identify its co-purifying proteins.
And over the years we learned that
one of the hallmarks of the kinetochore
is that it can be broken down into subcomplexes,
stable subcomplexes that can be purified on their own
and reconstituted on their own.
In addition, I should comment that genetics, of course,
contributed heavily to identifying these proteins as well.
Model organisms, such as budding yeast,
fission yeast,
Drosophila, worms...
people did genetic screens to look for things like
chromosome loss mutants,
and those screens additionally identified
many kinetochore proteins.
So, what we know now is that there are these...
there are many subcomplexes
and I won't talk about all of them,
and the basic idea is that they form
a hierarchical kinetochore structure,
and that's just cartooned here.
There are many, many copies of these subcomplexes,
and so in this cartoon this is greatly simplified,
but the things to remember are that,
as I said, there's a centromeric chromatin structure
at the bottom that's the foundation for the kinetochore
and a hallmark is the CENP-A nucleosome,
the inner kinetochore forms on top of that
and this is often called the CCAN,
for constitutive centromere-associated network.
These are proteins that then bind to
that centromeric chromatin
and form the foundation of the kinetochore.
They're present throughout the cell cycle.
And then at mitosis a number of outer kinetochore proteins
assemble onto that base,
and as I said there's many subcomplexes,
some of which directly bind to the microtubule.
Okay, so now that I've told you about the structure of the kinetochore,
let's talk about its function.
And there's three major functions
that kinetochores carry out:
one is a mechanical role,
where they maintain an attachment
to the tip of the dynamic microtubule;
a second is that they have
interesting biophysical properties
-- tension can stabilize these attachments,
as we'll talk about that;
and finally, they serve as signaling hubs
-- kinetochores can completely halt the cell cycle
if there's a defect in kinetochore microtubule attachments.
And so I'm going to talk about each of these functions.
Let's start with the mechanical role.
So, to remind you,
microtubules are the polymers that segregate the chromosomes,
and these are hollow protofilaments
that are assembled from alpha- and beta-tubulin dimers,
and they exist in one of the four states shown here.
They're either assembling due to the addition of tubulin subunits at the tip,
or they're disassembly due to their removal,
and when they switch from assembling to disassembling
it's called a catastrophe event,
and when they switch from disassembling back to assembling
it's called a rescue event.
And this process where microtubules in a population
are constantly growing and shrinking
is termed dynamic instability,
and was originally discovered
by Mitchison and Kirschner.
Okay, so the kinetochore has a hard job:
it has to stay attached to these microtubule tips.
Kinetochores have to make load-bearing attachments.
Now, the microtubules have an inherent polarity
-- the - end is embedded in the poles
and the + end is defined as the distal end.
So, remember, microtubules
are growing and shrinking in the population,
and at some point
a microtubule will capture a kinetochore.
And most of the time this is a lateral attachment
where the kinetochore initially is on the side of the microtubule.
Now, if things stayed like this,
this would not result in proper chromosome segregation.
Instead, it has to get converted
to a tip attachment, shown here,
where the kinetochores attach to microtubules
from opposite poles.
And remember, I told you
that thousands of tubulin subunits
are being added and lost from this tip
while that kinetochore has to stay bound,
so it's almost like you're trying to climb a rope
and someone is constantly pulling the rope out from under you.
So the kinetochore has to stay bound
to this really dynamic tip,
and so a huge question is, how can that happen?
So, over the years, we've identified proteins
within the kinetochore
that bind to the microtubule,
and I'm only going to talk about one of those today,
which is Ndc80,
because it's really considered to be
the major microtubule binding activity
within the kinetochore.
This is the structure of a domain
of the Ndc80 protein itself,
which was originally solved by the Harrison lab,
and you can see it's a CH domain,
which stands for calponin homology,
and this is commonly found in other microtubule binding proteins,
such as EB1.
Now, Andrea Musacchio's lab solved an engineered structure...
Ndc80 is a four-protein complex,
they made this engineered complex called the "bonsai"
and discovered that there's actually two calponin homology domains,
one in Ndc80 and one in Nuf2,
and then over the years we've realized that
Ndc80 has the major microtubule binding activity.
This is higher structural information
from Eva Nogales' lab.
The microtubule is shown in green
and you can see Ndc80 is shown in blue,
and you can see the calponin homology domain
directly binding to that microtubule.
The Nuf2 is shown in yellow
and you can see that it doesn't directly bind,
and I want to point out
the red is another part of Ndc80,
which is an N-terminal extension
that also has microtubule binding activity.
So, this is the complex that's really considered to be
the major microtubule binding activity,
however, I want to emphasize,
there really are other microtubule binding proteins in the kinetochore
and we're still trying to understand
how they all act together and what their relative contributions are.
So now, let's talk about the mechanism
that the kinetochore can use to
actually maintain an attachment to these dynamic tips.
So, there are two major models right now.
The first is the conformational wave model,
and the idea here is that the microtubule
stores a lot of energy
and when it switches from assembling into disassembling,
the tip structure peels back,
the protofilament curves back,
and as long as the kinetochore can maintain an attachment
to this curved protofilament,
it can harness the disassembling energy
of the microtubule to move the chromosome.
And there's two ways that you can imagine that
the chromosome can stay attached:
one is a ring model
where part of the kinetochore
forms a ring around the microtubule,
and as long...
and there's evidence in budding yeast that the Dam1 complex
does form a ring;
there's also a fibril-based mechanism,
where individual components of the kinetochore
can attach to the microtubule tip
and then maintain an attachment
through this conformational wave.
Now, Hill proposed a different model
called biased diffusion.
In this model, the idea is that the kinetochore
contains multiple weak microtubule binding elements
that rapidly bind and unbind to the microtubule tip,
and as long as this diffusion
is biased towards the tip,
which can occur due to thermal fluctuations
of the chromosome binding near the tip,
that then the kinetochore would stay attached
to the growing and the shrinking tip.
And there's evidence that this model might be true
because the Asbury and Davis lab
showed that the Ndc80 complex itself
can undergo biased diffusion.
Finally, there's hybrid models
that would take both of these into account,
and so we're still actively trying to understand
what the underlying mechanism for attachment really is.
Okay, so now let's talk about
the interesting biophysical properties
of the kinetochore,
where tension can stabilize, rather than destabilize,
an attachment.
Kinetochores have to make bioriented attachments,
so each sister kinetochore
has to bind to microtubules from opposite poles.
However, as I said at the beginning,
the process of making attachments
is actually very error-prone
and there's a number of incorrect attachments that can be made,
such as mono-oriented attachments,
where one or both sister kinetochores
attach to microtubules from a single pole,
there's even merotelic, shown here on the bottom,
where one kinetochore can attach to microtubules from both poles.
And it's essential that the cell can detect
and correct these errors.
And so how can cells detect and correct these errors?
It turns out that tension is the key.
Now, Dietz originally noticed that
chromosomes would continually reorient on the spindle
until they became stably bioriented,
and then Bruce Nicklas really did
the pioneering experiment
to show that that's because tension is stabilizing the attachments.
So, what he did is take advantage of the fact that
grasshopper spindles are really large
and that you can use micromanipulation techniques.
So, he used meiosis I spindles,
where now the homologues
are attached to opposite poles,
rather than the sister chromatids,
and he took a micromanipulator
and was able to convert a bioriented attachment,
where the homologues are attached to opposite poles,
into one where the homologues
are attached to the same pole.
And he noticed that this is highly unstable
and what would happen, then,
is that the kinetochore would detach.
And there were one of two fates:
if it went back and created the wrong attachment again,
that was unstable;
however, if it created the correct attachment,
shown in the top,
it was now stabilized.
And then to demonstrate that this is because of tension,
he actually used a needle
to artificially apply an opposing force.
So, he took that incorrect attachment
where both homologues are attached to the same pole,
applied an opposing force,
and now the incorrect attachment was stable.
And this really demonstrated
that that's because tension is stabilizing that attachment.
So, tension stabilizes attachments
and they're in vivo very stable
once they come under tension,
and in contrast any attachment
that's lacking tension is highly unstable.
So, how can tension stabilize attachments?
And we know that there's at least two mechanisms
that contribute to this.
There's work from a number of labs
that have shown there's an error correction pathway
that uses phosphorylation
by the Aurora B protein kinase.
So, the idea here is that
when there's an incorrect attachment that lacks tension,
the Aurora B kinase recognizes that
and can phosphorylate kinetochore proteins,
and I want to mention that Ndc80
is one of the major targets of the Aurora B kinase.
When these proteins are phosphorylated,
it destabilizes that incorrect attachment,
and then that gives the cell a chance
to go back and make that correct attachment again.
Now, there's a second mechanism,
and that's one where tension
directly stabilizes attachments,
and the idea here is that tension itself
promotes more microtubule binding elements
to bind to the tip of the microtubule.
And this is like those finger traps,
if you remember,
where as you pull on them things get even tighter.
And this is really just an intrinsic property
of the kinetochore-microtubule interaction
-- there is no Aurora B involved in this --
and in Part 2 of my talk
I'll tell you how we discovered this mechanism.
Now, let's turn to the signaling functions of the kinetochore
and, as I said, the kinetochore
can transmit a signal to completely halt the cell cycle.
Cell cycle progression has to be halted
when kinetochore-microtubule interactions are incorrect,
so when there's a lack of tension
or an unattached kinetochore,
the checkpoint has to recognize that and halt the cell cycle.
And the spindle assembly checkpoint
-- I'm going to call it the spindle checkpoint, for short --
is the signal transduction system
that can do that.
Genetic screens from Andy Hoyt
and Andrew Murray's labs
originally identified the checkpoint genes,
and the idea here is they used budding yeast
as a model organism.
As you know, the yeast will divide
and make a colony when they're grown under normal conditions.
If you add a microtubule depolymerizing drug,
such as benomyl,
this creates a difficulty for the cell
in making kinetochore-microtubule attachments,
because now the microtubules are less stable.
And then there's a mitotic delay
while the cell tries to make the proper attachments,
and that's due to the spindle checkpoint.
So, the Hoyt and the Murray labs
reasoned that mutants in the checkpoint
would not be able to delay,
would therefore go through the cell cycle with incorrect attachments,
and that would lead to cell death.
So mad2 is one of the checkpoint mutants,
and when they treated that with benomyl
there was no delay, and then they just
get a microcolony of dead cells.
So by looking for mutants that cannot grow on benomyl,
they were able to isolate, really,
the majority of spindle checkpoint genes.
We now know those spindle checkpoint genes
that were originally identified in budding yeast
are conserved,
and there are additional ones that they didn't identify,
but the bulk really were identified in these two classic screens
in the early '90s.
Okay, so where is checkpoint signal generated?
The first homolog of the yeast genes identified
was the Xenopus MAD2 gene,
and the Murray lab showed that
it's required for the checkpoint using frog egg extracts,
and then the made the striking finding that
Mad2 binds specifically to unattached kinetochores.
So you can see that in this image, here,
from their paper.
This is a newt lung cell,
and this was a collaboration with Ted Salmon's lab,
and you can see on the left the chromosomes that...
most of them have aligned at the metaphase plate
and are properly attached to microtubules,
and then on the right is
immunofluorescence against Mad2,
and you can see there's almost no Mad2 staining.
So the kinetochores that are properly attached
do not have any Mad2.
But if you look on the right-hand side...
or, on the left-hand side,
you'll see a mono-oriented chromosome.
One kinetochore is attached to the pole
and the other one is not,
and then if you look at the staining of Mad 2,
you can see if specifically
stains the unattached kinetochore
in that pair of chromosomes.
So this really was the evidence that Mad2
specifically binds to unattached kinetochores,
and over the years a lot of work
has shown that kinetochores
clearly generate the checkpoint signal.
And we now know that there's a phosphorylation cascade
that recruits checkpoint proteins
to unattached kinetochores to trigger the checkpoint,
and I'm not going to talk about all those details.
Now, what does the checkpoint monitor?
I've already told you that defects in tension
or attachment
will lead to aneuploidy and chromosome missegregation,
so it's critical that both of these defects are monitored.
And it's been really controversial
whether tension or attachment
is the key to triggering the checkpoint.
Work from Conly Rieder suggested that attachment is monitored.
So, he did a classic experiment
where there was a cell that had a mono-oriented,
unattached chromosome,
so there's a single kinetochore attached to the microtubules
and the other is unattached.
He went...
and those cells are arrested because the checkpoint is on
because there's a defect.
Now, he went in and he laser ablated
that unattached kinetochore,
and that satisfied the checkpoint
and the cell cycle continued,
even though the remaining kinetochore is attached
and not under tension.
And this was the work that really suggested that
unattached kinetochores were what the checkpoint was monitoring.
However, remember that classic experiment
I told you about from Bruce Nicklas,
where there was a mono-oriented chromosome attached...
where both sister kinetochores are attached to the same pole,
and in that situation,
where there's no tension but there is attachment,
it turns out that the cell cycle was stopped
due to the checkpoint.
And when he went in and micromanipulated
and applied the opposing tension,
it turned out that the cell cycle went ahead,
even though the kinetochores
were attached to the wrong pole.
And so this showed that tension
seemed to be sufficient
to tell the cell that everything was ready to go,
even though those attachments were incorrect.
So it's still really confusing
whether tension or attachment
is the primary signal,
and I just want to remind you that defects in tension
are converted to unattached kinetochores.
So remember, when kinetochores lack tension,
these attachments are released
due to error correction mechanisms,
and that generates unattached kinetochores.
So, the reason it's been so hard to understand
might just be because these two issues
are so interrelated.
Now, let's talk about how the checkpoint halts the cell cycle.
A clue to this came from
work from Ted Salmon's lab
that looked at the Mad2 protein
and found that it dynamically cycles on and off
the kinetochore.
So, what they did here is they took
cells expressing a Mad2-GFP fusion
so that the kinetochores...
they could see the kinetochores...
Mad2 on the kinetochores,
and they treated the cells with nocodazole
to generate lots of unattached kinetochores,
which is why all of these kinetochores
are lighting up with Mad2.
They then did what we call a photobleaching experiment,
where they ablated,
in a single kinetochore, shown there,
the Mad2 fluorescence,
and then they watched,
how long does it take for new Mad2
to bind to the kinetochore?
And what they learned from this experiment is that
a population of Mad2 rebinds within 25 seconds,
which is extremely rapid,
and I want to point out,
there was also a more stably bound population as well.
So, this dynamic localization
of Mad2 suggested that, perhaps,
it forms an inhibitory complex
when it goes to the kinetochore that can quickly diffuse
and then generate a cell cycle arrest.
We now know that the APC
is the target of the checkpoint.
I told you earlier that
this anaphase promoting complex
promotes the metaphase-to-anaphase transition,
and it turns out there's an activator called Cdc20
that triggers APC activity.
The spindle checkpoint directly inhibits this protein.
Mad2 binds to Cdc20,
and additional proteins do as well,
and this complex is called the MCC,
or the mitotic checkpoint complex,
and when that complex binds to Cdc20,
it prevents it from activating the APC.
So, Hong Tau Hu in Andrea Musacchio's lab
solved the structure of Mad2
and they found out there's actually two forms.
There's a closed form,
and that directly binds to Cdc20 and inhibits it,
and then there's an open form.
And so the idea here,
in this template model that Andrea proposed,
is that there's a cytoplasmic pool of Mad2
that's in the open form
and then there's actually a closed form of Mad2
sitting on the kinetochore.
When the open form binds to the closed form
on the kinetochore,
it gets converted to the closed form,
that then diffuses away and can bind to Cdc20 to inhibit it.
I just want to summarize the general model
for the checkpoint right now
-- a lot of details are missing,
but you'll get the overall picture.
The idea here is there's a Mad2 complex,
and Mad1 is its receptor.
That complex binds to the kinetochore,
such as an unattached kinetochore,
and then open mad Mad2
binds to the Mad2 that's closed on the kinetochore,
that converts it to closed Mad2,
that closed Mad2 binds to Cdc20,
and then that assembles into a mitotic checkpoint complex
that then can go inhibit the APC.
And so that keeps the cell cycle off
while the proper attachments are made.
Once everything is okay,
that checkpoint complex is relieved
and Cdc20 is then activated
to then go bind to the APC
and promote cell cycle progression.
And so we're still working out a lot of details of this model,
but that gives you a general picture
of how the checkpoint is thought to work.
Okay, so what I've told you about today are
the complex functions of kinetochores.
They play mechanical roles in directly coupling the kinetochore
and chromosome to microtubules
to move the chromosomes during the cell cycle.
We've identified some of the major players
that bind to microtubules, such as Ndc80,
but we're still trying to identify
what other microtubule binding proteins
are in the kinetochore,
what are their relative contributions
to maintaining an attachment,
and, really, what is the underlying mechanism
-- conformational wave, biased diffusion --
by which the kinetochore can maintain an attachment?
Kinetochores have these really interesting biophysical properties
where tension actually stabilizes attachments,
and we know that there's at least two mechanisms
that contribute to that
-- an Aurora B mechanism,
we still don't know how Aurora B
selectively works on kinetochores lacking tension
but doesn't affect those that have tension,
and then as I've said, we've identified a second mechanism,
an intrinsic mechanism,
that we're still trying to understand
the underlying mechanism for.
And then, finally, they can act as signaling hubs,
where a single unattached kinetochore
can completely halt the cell cycle
through the spindle checkpoint.
And we're still trying to understand
what the checkpoint is monitoring,
what state of kinetochore-microtubule attachment,
and then there's a lot of work to be done
to understand how that signal is amplified
to halt the cell cycle
and then, how,
when everything is set up correctly,
is that checkpoint silenced so that then the cell cycle can continue?
So, I'm going to talk, specifically, in my next talk
about the first two functions,
and I thank you for your time.
