Tuomas P. J. Knowles, Duncan A. White, Adam R. Abate, Jeremy
J. Agresti, Samuel I. A. Cohen, Ralph A. Sperling, Erwin J. De Genst,
Christopher M. Dobson, and David A. Weitz
The crucial early stages of amyloid
growth, in which normally soluble proteins are converted into fibrillar
nanostructures, are challenging to study using conventional techniques yet are
critical to the protein aggregation phenomena implicated in many common
pathologies. As with all nucleation and growth phenomena, it is difficult to
track individual nuclei in traditional macroscopic experiments, which probe the
overall temporal evolution of the sample, but do not yield detailed information
on the primary nucleation step as they mix independent stochastic events into
an ensemble measurement. To overcome this limitation, we have developed
microdroplet assays enabling us to detect single primary nucleation events and
to monitor their subsequent spatial as well as temporal evolution, both of
which we find to be determined by secondary nucleation phenomena. By deforming
the droplets to high aspect ratio, we visualize in real-time propagating waves
of protein assembly emanating from discrete primary nucleation sites. We show
that, in contrast to classical gelation phenomena, the primary nucleation step
is characterized by a striking dependence on system size, and the filamentous
protein self-assembly process involves a highly nonuniform spatial distribution
of aggregates. These findings deviate markedly from the current picture of
amyloid growth and uncover a general driving force, originating from
confinement, which, together with biological quality control mechanisms, helps
proteins remain soluble and therefore functional in nature.
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