34 THE SCIENTIST | the-scientist.com
I
have a clear memory of presenting my
initial results about a “failed” protein
at a lab meeting with my postdoctoral
advisor Angus Lamond at the University
of Dundee in Scotland and the rest of his
group. It was the summer of 2000, and
for the first few months of my postdoc
I had been fusing green fluorescent pro-
tein with novel proteins that had recently
been identified by mass spectrometry as
residing in the nucleolus. I engineered
HeLa cells to produce copious amounts
of these fusion proteins, and watched
where they went. Most migrated to the
nucleolus, as expected, but one protein
steadfastly refused. Instead, it formed
nuclear dots that were much smaller than
the large and obvious nucleoli.
I was really worried about the messi-
ness of this result, but also intrigued. To
my relief, instead of being disappointed
that the protein was not doing what we
had expected, Angus and my lab mates
encouraged me to explore it further. The
group had access to antibodies against
many cellular structures, so I quickly
established that these nuclear dots were
different from any known nuclear bodies.
But having generated much of my data
with overexpressed protein, it was critical
to make sure that the endogenous form
of the protein also localized to the same
nuclear dots, and that what I had seen
were not simply artifacts of my approach.
We created an antibody against the
protein and incubated it with HeLa cells.
It was an incredibly nerve-wracking
moment looking down the microscope
to see what the antibody had stained.
Thankfully, it worked. I was ecstatic
when I saw that it had picked out the
same small nuclear dots that I had
identified with GFP. In 2002, I pub-
lished a manuscript introducing the
scientific community to paraspeckles—
orbs of protein and nucleic acid 360
nanometers in diameter, squeezed next
to the more famous and larger struc-
tures called nuclear speckles.^1
Since then, paraspeckles have become
an established part of cell biology; there
are more than 250 articles on them, and
they have already found their way intosome textbooks. We now know they are
membraneless organelles seeded by a
long noncoding RNA (lncRNA), formed
through a well-characterized physical
phenomenon known as liquid-liquid
phase separation, and composed of
numerous proteins and RNA molecules.
We also know that they can alter gene
regulation when cells get stressed, an
important mechanism for maintaining
cell homeostasis and one that appears to
be disrupted in many diseases.
In 2006, I left the UK to establish my
own paraspeckle lab in my home coun-
try of Australia. Reflecting on my post-
doc reminds me of just how far we have
come in such a short period of time. In
addition to all we have learned about
paraspeckle biology from in vitro work,
many studies have now established that
these structures appear in cells biopsied
from human patients and from healthy
mouse tissue samples. To have discov-
ered a new cellular structure and have
watched the birth of a research field
focused on understanding that struc-
ture is an honor and a privilege. Look-
ing ahead, I can see some big opportuni-
ties for paraspeckle biology, from using
them as a model to understand lncRNAs
and phase separation in the cell to devel-
oping therapeutics that modulate them
in different diseases.Discovering paraspeckles
When I joined Lamond’s lab in late
1999, I initially chose a project within
my molecular biology expertise, clon-
ing complementary DNAs for a bunch
of proteins identified in the human
nucleolus. But as I dove further into
the problematic protein that wouldn’t
localize to this known nuclear struc-
ture, I was quickly pulled out of my
comfort zone. I had to learn new tech-
niques to figure out why the protein was
appearing in mass spectrometry analy-
ses of nucleoli, but not in the nucleoli of
my GFP-fusion–expressing cells.
I ended up adopting a technique
that involved laser-bleaching the flu-
orescence of the wayward GFP-fusion
protein. I then took many microscopic
images over time to track where the
bleached protein, which I named para-
speckle protein 1 (PSP1, subsequently
renamed PSPC1), went in the cell. This
method showed that it was travelling in
and out of nucleoli under steady-state
conditions, even though it was not sub-
stantially enriched within them. Mass
spec was sensitive enough to pick up
this trace of PSPC1 in the nucleoli that
we could not see under the microscope.
I later found that two other proteins—
originally termed P54nrb and PSF, now
called NONO and SFPQ, which are in theGLOWING GREEN:
Twenty years ago,
GFP-labeled proteins in
HeLa cells revealed the
presence of the nuclear
bodies now known as
paraspeckles. (Human
breast cancer cell from
the MDA-MB-231 cell
line shown here; scale
bar is 5 μm.)ARCHA FOXCytoplasmParaspecklesNucleus