12.2019 | THE SCIENTIST 19So Manjunath Rajagopal, a graduate
student in Sinha’s lab, set out to devise a
ne w, more accurate type of cellular ther-
mometer. He and Sinha used atomic
force microscopy (AFM), which deliv-
ers very precise measurements of the
forces at work between a probe and a
sample. Adapting the technology for use
inside a living cell took years. “We rede-
signed to make sure the probe is suffi-
ciently long and thin... which makes
it less damaging to the cell when it’s
entering the cell,” Rajagopal says. Cali-
brating the instrument posed another
challenge; rather than using hot and
cold water—the usual approach for cal-
ibrating temperature-measuring instru-
ments—they aimed for higher accuracy
with techniques taken from the semi-
conductor industry, where AFM is used
to measure the smoothness of surfaces.
For the device’s trial run, Sinha and
Rajagopal worked with neuroscientist
Rhanor Gillette, also at the University of
Illinois at Urbana-Champaign (UIUC),to measure the temperature of some
unusually large neurons—those found in
the abdomen of a sea slug known as the
California sea hare (Aplysia californica).
The team placed cultured neurons under
a microscope and inserted the custom
thermometer into one of them, as well
as a voltage-measuring device to moni-
tor the cell’s health.
“We were expecting to see the temp-
erature kind of go steady, without any
increments. And that would be confirm-
ation that our probe really works well,”
Sinha says. The researchers found the
temperature did stay uniform much of
the time, “but in addition to that we saw
this very sudden spike that happens.”
Indeed, the probe picked up temperature
blips of a few Kelvin (or degrees Celsius),
each lasting about a second.
The results didn’t seem to make
sense. In a 2014 paper, researchers in
France had calculated that, to raise its
temperature by just one Kelvin, a cell
would need to respire—that is, burnglucose—five orders of magnitude faster
than normal (Nat Methods, 11:899–901).
Claims of temperature changes of a few
Kelvin, therefore, were not realistic, the
French team concluded. Sinha agreed.
Sinha’s group performed some exper-
iments to see if some factor other than
temperature might account for their read-
ings, but came up empty-handed. So the
researchers considered whether the neu-
rons might be drawing power from some-
thing other than normal, glucose-fueled
respiration. It’s well-known that during
respiration, mitochondria generate a pro-
ton gradient across their membranes that
they harness to make AT P. That gradient
can also act as a battery through a process
called proton uncoupling, in which pro-
tons are temporarily allowed to flow across
the membrane, generating heat—although
researchers don’t fully understand how the
process is triggered.
To find out whether proton uncoup-
ling in mitochondria could explain the
temperature spikes they recorded, Sinha
and his colleagues tried artificially
inducing the process in A. californica
neurons by treating them with a chemi-
cal that shuttles protons across the mito-
chondrial membrane. The researchers
measured jumps of 7.5 Kelvin in these
cells, compared with changes of 2.3 Kel-
vin or less in control cells. So it’s likely,
the study authors conclude, that proton
uncoupling is responsible for the flashes
of heat the probe picked up (Comm Biol,
2:279, 2019).
Ambre Bertholet, a molecular biol-
ogist at the University of California,
San Francisco, who works on proteins
involved in proton uncoupling and was
not involved in the study, says that the
invention of the thermal probe is an
important contribution to the scien-
tific debate over whether large temper-
ature changes occur in single cells. But
she notes that the probe only measures
temperature at a specific location in the
cell, and the study doesn’t really dem-
onstrate that spikes detected at those
locations correspond to increases in the
whole cell’s temperature. “They mea-
ANDRZEJ KRAUZE sure the mitochondrial thermogenesis,