Finally, I want to go in the other direction. And solve two possible
problems. One of them was that, as you remember, GFP
absolutely needs the presence of oxygen to mature.
What about if you're forced to work on an obligate anaerobe?
Also, how do we go to yet higher spatial resolution? And
there are tricks to do so with optical microscopy, but the fundamental
way that most of our high resolution spatial information in cell biology
has come about has been through electron microscopy.
And up till now, electron microscopy has lacked the genetically
encodable tag that does what GFP does. In other words, where
you can just put in a gene, fuse it to the protein you care about,
and then follow it around by your favorite form of microscopy. It could
be great if we could do that in electron microscopy. And here
the trick is again, to go to another completely different protein
family. This is Arabidopsis phototropin, which binds flavins, in particular
flavin mononucleotide. And flavins are also ubiquitous in biochemistry,
and essentially all organisms that we care about have them, including
the obligate anaerobes. And so, this protein as it came out of the plant
doesn't do any fluorescence, it only uses light to trigger phototransduction,
but again, via somewhat similar trick -- a somewhat analogous trick
to the infrared fluorescence protein, we can frustrate this signal
transduction, turn it into a fluorophore, and this fluorophore
happens, by the way, to make something called singlet oxygen
as a byproduct. It does it actually quite well, and these curves
represent the data showing that the fluorescence switches rather like
GFP in wavelengths, but admittedly, not terribly bright.
And plus, it's singlet oxygen. Singlet oxygen is normally a bad
thing for us. And microscopists don't like it, because it's
responsible for a lot of the photobleaching. So we try to make
as little singlet oxygen as possible. But when you have a protein
that makes singlet oxygen well, you can use a special form of histochemistry
that's been known for a long time, and singlet oxygen will polymerize
a molecule that we supply in dead fixed cells. So remember, electron
microscopy has to be done, and has generally only been done on
dead fixed cells. So we don't mind after fixation, supplying diaminobenzidine,
and this molecule is instantly polymerized very locally wherever
the singlet oxygen is made. It's polymerized into a precipitate
that then is stainable by osmium, and osmium is the counterstain that we use so much
in electron microscopy anyway. So wherever this protein was fused,
when we excite it with light in the presence of oxygen gas, and
maybe I should explain here, oxygen gas that you and I are breathing
is a very unusual molecule, in the sense that it's a triplet.
It has unpaired electrons, and when it encounters the excited
state of this singlet oxygen generating protein, the regular oxygen
gets excited to this singlet oxygen state, which is an excited state of
oxygen still diffusible like regular oxygen. Can cross membranes,
but it is ravening beast in its chemical reactivity, and it loves to attack methionines,
tryptophans, histidines, and so on. But also will attack diaminobenzidine
to make this polymer. So here, we've transfected in miniSOG and targeted
the mitochondria by fusing it to a piece from cytochrome c, which is
a well known mitochondrial protein. And you can see the mitochondria by fluorescence
here. And they look like regular mitochondria. In a live cell they look sort of like
these little wispy threads. But then the crucial thing is we can
fix the cells and turn up the light and bubble pure oxygen to help
efficiency and include diaminobenzidine, and wherever there was fluorescence
before, we turn it into this black precipitate, and that's not too impressive yet. But
that black precipitate can be looked at under electron microscope at higher
and higher magnification. So this is a blowup of one of those mitochondria
down to the scale where you can see 200nm and we can see
all the cristae and the spaces between the inner and outer membrane.
This is the sort of classic appearance of a mitochondrion that you would've seen
from a textbook. But this mitochondrion has been picked out genetically.
And here is another test case, this is the gap junction that I mentioned.
And we fused connexin43, one of the major constituents of gap junctions,
to this so-called single oxygen generating protein, miniSOG. And by the way,
I forgot to say "mini" refers to the fact that this protein is only 106 amino acids,
it's less than half the size of GFP. And there are times when it is
a better fusion partner just because it's small. And as I said, it doesn't
need oxygen to fluoresce, because it uses the flavins that the cells provide.
Of course when we want to make the precipitate, we have to provide
oxygen, but that's done anyway, after the cell is dead and fixed.
So, these are the gap junctional stripes. These are in ordinary fluorescence
microscope level, these sort of boundaries between individual
cells that are lit up as gap junctions. This is after we have illuminated them
in the presence of diaminobenzidine and oxygen, and converted them
into black precipitates. And then when we blow it up, we can
blow it up to the scale where we can actually see what we believe individual
hexamers. As these white shadows in what looks like the machine gun
belt of precipitate. That's its sort of crude appearance. It looks like
bullets periodically spaced. And these white blobs may be
the leftover connexin that is blocking the formation of precipitate
everywhere else. In other words, the miniSOG up here are spitting out
precipitate and they got every they can, but the protein where the
connexin is sort of blocks it and keeps it away, because the space
is already occupied. And then when the knife that makes the section
cuts through this, if you happen to cut right through the center
of this region, you can get the periodic array and see things at very
much higher resolution. By comparison, old fashion techniques, still
useful in many cases but more difficult, which is immunogold
electron microscopy. Only light captures a very small fraction of the proteins because
here, we are trying to diffuse antibodies through a fixed tissue.
And the fixation tends to destroy a lot of antigenic reactivity, and
antibody has a hard time getting into the section. Also any excess
has to be washed out. So we're lucky when we can at least see a few
dots at the gap junction. But a picture like this, which is a good quality
immuno EM, doesn't give you any impression of how densely
packed this crystalline array of connexins is. And we know this
from many other experiments, that the connexins look something like this,
according to this model that I'm showing up here.