In 1993, he showed that two regions
of synaptotagmin bind calcium, and this property allows it to efficiently grasp phospholipids.
Both research groups began by crystallizing the BoNT / B serotype toxin in complex with its protein receptor,
called synaptotagmin II.
In the second approach, Südhof created a large number of genetically modified mice which
lacked synaptotagmin.
This regulation is spectacularly apparent in the exquisite speed and precision of synaptic exocytosis,
where synaptotagmin (the calcium - ion sensor for fusion) cooperates with complexin (the clamp activator) to control the precisely timed release of neurotransmitters that initiates synaptic transmission and underlies brain function.
The work by Johnson and collaborators in the Department of Physics and Department of Biochemistry and Biophysics provides a molecular - level explanation for the observation that otoferlin and
synaptotagmin don't have the same functional role.
Südhof's original observations
on synaptotagmin tantalized him because they indicated that the protein performs calcium - dependent activities.
In contrast to vesicle -
dwelling synaptotagmin and VAMP / synaptobrevin, syntaxin concentrates in the cell membrane — at spots where neurotransmitters are released from nerve cells.
These and other studies provided compelling genetic evidence that Ca2 + binding to
synaptotagmin drives fast Ca2 + - triggered transmitter release.
Synaptotagmin detects the sudden and brief rise in calcium concentration that occurs when an action potential arrives and allows fusion to proceed — all in a fraction of a millisecond.
The size of the otoferlin molecule and its low solubility have made it difficult to study, including how otoferlin works differently than another neuronal calcium sensor in the brain,
synaptotagmin.
He generated a series of mice, each of which carried
a synaptotagmin with altered calcium - binding affinity.
Südhof next purified and studied another vesicle protein,
synaptotagmin.
Synaptotagmin's affinity for the ion correlated with the calcium sensitivity of neurotransmission.
Augmenting neurotransmitter release by enhancing the apparent Ca2 + affinity of
synaptotagmin 1.
Synaptotagmin: A calcium sensor on the synaptic vesicle surface.
He discovered that one of the vesicle membrane proteins,
synaptotagmin, had separate calcium and phospholipid binding domains, suggesting it had a key role in transmitter release.
Südhof then made use of the emerging power of mouse genetics to delineate the functional role of a number of these vesicle proteins including the role of
synaptotagmin, which he demonstrated to be the critical calcium sensor for rapid neurotransmitter release.
Simultaneously, working in Brunger's group, lead author Rongsheng Jin, an HHMI postdoctoral fellow, in collaboration with Thomas Binz and Andreas Rummel of the Medizinische Hochshule Hannover in Germany crystallized the receptor - binding domain of BoNT / B in complex with the recognition domain of
synaptotagmin II, achieving a significantly higher resolution of the complex.
Both teams showed that they could disrupt this binding by introducing mutations that would subtly alter the shape of
the synaptotagmin receptor.
Rothman and Scheller collaborated and proposed that NSF and SNAP form part of the vesicle fusion machinery in neurons, and
that synaptotagmin served as a clamp which prevented the fusion process from happening until a calcium signal arrived.
The toxin induces a helix in
the synaptotagmin protein that fits precisely into a groove in the toxin molecule.
Working with Chapman's group, co-authors Raymond Stevens, Qing Chai and Joseph Arndt of the Scripps Research Institute crystallized full length BoNT / B in complex with the «recognition domain» of
synaptotagmin II, to which the toxin attaches.
Südhof used genetic engineering to «knock - out»
the synaptotagmin gene in mice, with the result that synapses from the forebrains of mice that lacked synaptotagmin lost their capacity for fast calcium - triggered release of neurotransmitter, thus confirming the importance of synaptotagmin in vivo.
Südhof performed X-ray crystallography and other studies to reveal how a different group of proteins, the complexins, bind to the SNARE scaffold and help to stabilise the structure until
synaptotagmin displaces them, calcium - activated synaptotagmin then sparking the fusion of the vesicle with the pre-synaptic membrane.
Scheller also provided the first evidence that a calcium sensing protein called
synaptotagmin — discovered earlier by Südhof — was the switch for neurotransmitter release, and proposed in a paper in Science in 1992 that the v - SNARE and t - SNARE proteins combine with synaptotagmin into a three - dimensional scaffold on which other molecules assemble, to form the machinery that initiates vesicle fusion and neurotransmitter release.
No significant changes in DCX, NeuN,
synaptotagmin, and synaptophysin were detected.