Hi, I'm Abby Dernburg.
In the previous two segments,
I've introduced you to the process of meiosis,
the process by which homologous chromosomes
separate to different daughter cells
to produce haploid gametes,
or sex cells.
And I've told you a little bit about
what homologous chromosomes have to do
to accomplish this segregation:
they have to pair, synapse,
and undergo crossover recombination.
And I introduced you to
these special regions that we call pairing centers,
which are on each of the 6 chromosomes in C. elegans,
that play critical roles in the process of pairing and synapsis.
These chromosome regions recruit special proteins
that have zinc fingers
that bind to short sequence motifs,
and they interact with the nuclear envelope,
and that's essential for chromosomes
to undergo pairing and synapsis.
And we wanna understand
what it is about these interactions
with the nuclear envelope
that promote the pairing and synapsis of homologous chromosomes.
So one of the first key observations
that pushed this work forward was kind of fortuitous,
and I'll show you what we saw.
When we localize the pairing centers,
here used antibodies against all of the zinc finger proteins,
in red,
what we see is that they're distributed
sort of apparently randomly
around the nuclear periphery.
And we wanted to know what they were interacting with. 37 00:01:34,228 --> 00:01:34,227 And our first clue was really just an accident.
We had a protein that we...
that had been tagged with GFP, Green Fluorescent Protein,
by other researchers who were working on the nuclear envelope
and its role in other processes,
and they were studying this protein called ZYG-12.
It stands for zygotic lethal,
because this is an essential protein
that's a component of the nuclear envelope.
When we looked at ZYG-12
in early meiotic nuclei,
it precisely colocalized with these pairing centers,
suggesting that there might be an interaction
between the pairing centers and ZYG-12.
So, what is ZYG-12?
So, it turns out that ZYG-12 is something
called a KASH domain protein,
and KASH domain proteins
all share this property that they are recruited and retained
in the nuclear envelope,
the outer nuclear envelope,
by interaction with something called the SUN domain protein.
So SUN domain proteins are an anchor,
they're in the inner nuclear envelope.
They bind to KASH domain proteins
and then KASH domain proteins can interact
with different elements in the cytoplasm,
such as the actin cytoskeleton
or the microtubule cytoskeleton,
and they connect...
they structurally connect things inside the nucleus
to outside the nucleus.
So we were very intrigued by this
and we wanted to know
what the nature of these connections was.
We could tell just by looking at
C. elegans germline stained with antibodies or GFP
that these interactions between the chromosomes
and the nuclear envelope
were very transient.
So, we see these...
we call them "patches" of ZYG-12
that colocalize with the pairing centers,
but they only appear for a brief window of time
at the beginning of meiotic prophase,
and then by the time the chromosomes complete pairing and synapsis,
ZYG-12 dissociates from the pairing centers
and redistributes throughout the nuclear envelope.
So, we wanted to know more about the architecture
of these interactions
and what they do.
And a lot of experiments from my lab
and other labs have revealed that,
in meiosis, what happens is these zinc finger proteins,
HIM-8 or the ZIM proteins,
are recruited to the pairing centers,
and by recruiting regulatory activities like kinases,
they induce the aggregation of SUN-1 and ZYG-12
within the nuclear envelope,
to form these patches that we can actually see.
And we also knew from previous work that ZYG-12
interacts with the microtubule cytoskeleton,
it interacts with the microtubule motor dynein
and with microtubules.
And this was very intriguing,
and it's very reminiscent of something
that we knew happens during meiosis
in many organisms.
So, these pairing centers are a little bit odd,
this is not a general feature of meiosis,
but this interaction between chromosomes
and the nuclear envelope
does seem to be a ubiquitous aspect
of meiotic prophase.
And the more typical way it looks like something like this.
These are actually some very old drawings
based on observations of meiosis,
in this case, in a flatworm,
but they exemplify the stage of meiosis
called the "bouquet" stage.
What early cytologists observed
is that the ends of chromosomes,
which we now call telomeres,
associate with the nuclear envelope,
and often they cluster closely together
near one portion of the nuclear envelope
during meiotic prophase.
And in work in the fission yeast
Schizosaccharomyces pombe
had revealed that
in fission yeast telomeres associate with the nuclear envelope
through a similar SUN/KASH domain pair,
Sad1 and Kms1,
basically orthologous to SUN-1 and ZYG-12.
So, we think it's very likely
that this reorganization that we see,
and the interaction between pairing centers
and the nuclear envelope,
is basically an evolutionary variant of the more common situation
where telomeres associate with the nuclear envelope.
So what does all of this do?
Well, one way that we have explored
the role of these interactions
in promoting pairing and synapsis
is to take advantage of the fact that
we can actual observe meiosis in living organisms,
and we can start to look at the chromosome dynamics
in real-time, using fluorescent protein tags.
So this shows you an early recording
that we made in my lab.
In case we're looking at the ZYG-12:GFP,
so this nuclear envelope protein
that's associated with all of the pairing centers.
And then in red is a histone protein
labeled with a red fluorescent protein,
just so that you can identify the individual nuclei.
And what you see is that in these early meiotic nuclei,
that are undergoing pairing and synapsis,
these patches of ZYG-12
and the associated pairing centers
are dramatically moving all around the nuclear envelope,
and that motion becomes much less obvious
and the patches dissipate
upon chromosome pairing and synapsis.
So, to make more sense of this motion
and to understand how it might drive the interaction
between homologous chromosomes,
we wanted to label just the X chromosome pairing centers.
And to do that,
Dave Wynne, a graduate student in my lab,
made a HIM-8:GFP fusion,
so now we can just observe
the pairing centers on the X chromosome.
And in this movie,
we've also taken advantage of the fact
that the X chromosome replicates later
than all the other chromosomes in the germline,
because it's transcriptionally silent and heterochromatic.
And so if we inject into worms a DNA precursor
-- a fluorescent DNA precursor,
a deoxynucleotide --
it gets incorporated into replicating DNA,
and in many nuclei you preferentially label the X chromosomes.
And I really love watching this movie
because it's just a view of individual chromosomes in motion,
which is just something that
we haven't had the opportunity to look at really before.
And I wanted to just point out a couple of interesting features.
So, in most of these meiotic nuclei,
what you see is that the HIM-8:GFP focus,
it's at the nuclear envelope,
and it's dramatically moving around the nuclear surface.
The rest of the chromosome is not really following it
like a rigid rod,
the chromosomes are very flexible and elastic,
and so really most of the motion
is at that left end of the X chromosome
where the pairing center is.
And in a couple of nuclei,
there's one example and there's one over here,
here the X chromosomes have paired at their pairing center,
but they haven't zipped up yet
through the formation of the synaptonemal complex,
so we're catching an intermediate
in this process of pairing and synapsis.
So, we really wanted to understand
the nature of these motions,
and that turned out to be a lot harder than we'd expected
to sort of analyze quantitatively,
and I'll show you why.
So we can observe these motions,
and we're using
fairly high-speed fluorescence microscopy here,
where we're taking 3D data,
because we really need to capture the full 3D volume,
but the fastest we could really image these samples
was at about a 5 second interval
between successive frames.
And at that frame rate,
what we would see is that these spots
were moving around dramatically,
and we could easily track these spots using software,
and we could tell very easily that in early meiosis,
in these transition zone nuclei which are undergoing pairing,
these spots are extremely active.
So the pairing centers are moving around very actively,
and much more so than in premeiotic nuclei.
And we could track these spots
and kind of demonstrate that in a quantitative way.
But what we also realized in the course of doing this work
was that we weren't imaging fast enough,
and by that I mean that
if you watch one of these spots...
and basically this trace shows its position
in each frame of the movie,
what you see is its position...
it doesn't have any kind of constant trajectory.
It's direction seems to change
with each successive frame.
And that means we're not sampling fast enough
to actually see motion, and to measure the rate of motion.
And that might be a little non-intuitive,
but I'm just gonna use this example of time-lapse photography
of people moving around Grand Central Station.
Now, if it takes someone, on average,
like a minute to go from one side of this hall to the other,
and you only take an image every minute
or every 30 seconds,
you're lucky to see that same person even twice,
in one frame and the next frame.
In order to really measure
the rate of someone's motion through space,
you have to actually be able to see them
through multiple frames
to be able to actually measure the rate of their motion.
And so we realized that we were undersampling the motion
that we were trying to capture.
And to fill in the gaps,
we went to a higher framerate,
where we were simply taking individual frames,
not trying to capture the full 3D volume of the nucleus.
And in these faster movies,
now we could see these persistent,
or processive chromosome motions
that we could track over multiple frames,
and so we could actually measure their velocity
and their duration and so forth.
And what we found is that they show
behavior typical of motor-driven motion,
in that the duration of these motions
shows an exponential decay.
We can't measure the shortest motions
because the duration is still too short
compared to our framerate,
but the longer motions, where we can track
a single pairing center moving over multiple frames,
we could measure their duration.
And from this curve,
we can calculate that these motions
last an average of a little under 2 seconds.
So really, to get the full picture of what's going on,
we'd really like to have even faster microscopy,
and that's something that is constantly being improved,
so we're hopeful that someday
we'll be able to understand this process in more detail.
We could tell that these long-range motions,
which sometimes move chromosomes
like halfway across the nucleus,
around the periphery,
depended on the microtubule motor dynein.
So, here what we've done is taken a worm
that has a temperature-sensitive mutation in one dynein component,
and we've done RNAi against another dynein component,
to try to knock down the activity as strongly as possible.
And what we see is that the long-range motions disappear,
and that's kind of shown here in this graph,
that the apparent motions
of more than about half a micron
from one frame to the next disappear.
Nevertheless, if you're looking at this,
you might we wondering, "Well, these spots don't look unpaired..."
So, even though these motions are
clearly upregulated during meiosis,
and the most obvious thing that they would be doing
is helping chromosomes to find their partners,
they are actually dispensable for homologue pairing.
We see a slight delay in homologue pairing
in the absence of dynein activity,
but it's not gone.
And that's sort of surprising,
but it's consistent with what we had seen through other assays.
So, what is it that makes chromosomes pair in this region,
even in the absence of dynein?
And we're still working on the answer to that question,
but one clue came from analysis of the kinds of movies
that I've shown you.
These are basically individual tracks
of a pairing center
from a collection of nuclei
from multiple movies.
And we've overlaid, in this case,
15 individual tracks
on the same sphere so that you can
get a feel for how much chromosomes
are moving around in the space of about 5 minutes.
And what I just wanna highlight is that
in premeiotic nuclei, which are on the left,
the chromosome motion is very constrained.
It's not even diffusive, so chromosomes are sort of constrained
to be in a small region of the nucleus;
they don't move much.
In early meiosis, suddenly things really take off,
and the chromosomes are just moving
all around the nuclear surface.
This probably promotes their interactions with each other.
We don't think it happens in a directed way,
because when we actually measure these motions,
they show now directionality,
and they separate homologous chromosomes
as often as they bring them closer together.
But they clearly facilitate the exploration of space
by the chromosomes.
The interesting thing that I wanna highlight here is that,
even in the absence of dynein,
the motion that we see
in meiotic nuclei is a lot less constrained
than in the premeiotic nuclei.
And what we think this is telling us is that
there's a change in the whole behavior of the nuclear surface,
either the lamina that underlies
the nuclear membranes becomes more fluid,
or other changes must be happening
to enable chromosomes to diffuse more readily
through the nucleus.
And we think that this, together with other factors,
is a major determinant of homologue pairing.
We're still very interested in exploring
the other roles for this motion
and how it promotes pairing and synapsis.
And that's very active work still going on in my lab,
so stay tuned.