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Investigation of the relationship between the material properties of insoluble, protein aggregates known as amyloids and common forms of age-related dementia such as Alzheimer’s and Parkinson’s

Final Report Summary - NANOMECHAMYLOID (Investigation of the relationship between the material properties of insoluble, protein aggregates known as amyloids and common forms of age-related dementia such as Alzheimer’s and Parkinson’s.)

Objective (1) in the proposal was to determine the stiffness of amyloids as this material property is thought to play an important role in amyloid-related diseases. This objective was achieved by measuring the stiffness of amyloid on three different samples and finding that it is indeed very stiff;
almost as rigid as spider silk! This result, and its possible implications for the pathology of amyloid-related diseases, was published in Proceedings of the National Academy of Sciences (“Exceptional rigidity and biomechanics of amyloid revealed by 4D electron microscopy” Fitzpatrick, A.W.P.; Park, S.T.; Zewail, A.H. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 10976.)

A tremendously exciting offshoot of this work was to use amyloid fibrils as a model system to demonstrate the feasibility of 4D cryo-electron microscopy. Traditionally, cryo-electron microscopy is a form of transmission electron microscopy that has been used to determine the (static) 3D
structure of biological specimens in the hydrated state and with high resolution. In the Journal of the American Chemical Society (“4D Cryo-Electron Microscopy of Proteins” Fitzpatrick, A.W.P.; Lorenz, U.J.; Vanacore, G.M.; Zewail, A.H. J. Am. Chem. Soc. 2013, 135, 19123.), we reported the
development of 4D cryo-electron microscopy by integrating the fourth dimension, time, into this powerful technique. From time-resolved diffraction of amyloid fibrils (as proposed in objective (1)) in a thin layer of vitrified water at cryogenic temperatures, we were able to detect picometer movements of protein molecules on a nanosecond time scale-unprecedented spatiotemporal resolution! Potential future applications of 4D cryo-electron microscopy are numerous, and were discussed in the ground-breaking paper.

During the period 01/03/2014 to 27/02/2015, we achieved the final, and most challenging, aim of objective (1), namely, to determine the nanoscale Young moduli and nanomechanics of amyloid. Precisely measuring the magnitude of the forces that hold amyloid together is crucial to
understanding the behavior of this persistent protein aggregate in living organisms. Through a series of meticulous experiments, we used four-dimensional electron microscopy to systematically dissect the nanoscale origins of amyloid elasticity by measuring the bond stiffnesses of the intermolecular forces stabilizing each of its three characteristic packing interfaces. We found that amyloid has a pronounced mechanical anisotropy with longitudinal, hydrogen bonding 20 times stiffer than transverse, amphiphilic, and electrostatic interactions. We proposed that such strongly
anisotropic elastic properties are likely to give rise to length-dependent mechanical behavior with short fibrils possessing significantly different material properties than longer fibrils. This is of great importance in understanding fibril-cell membrane interactions and fragmentation mechanisms, both of which are thought to play a crucial role in the spread of amyloid diseases. We published this significant result in the Proceedings of the National Academy of Sciences (“Nanomechanics and intermolecular forces of amyloid revealed by four-dimensional electron microscopy” Fitzpatrick,
A.W.P.; Vanacore, G.M.; Zewail, A.H. Proc. Natl. Acad. Sci. U.S.A. 2015, 112, 3380.)

Objective (2) was to develop an amyloid biosensor with a view to determining the strength of binding of chaperones or viruses to amyloid. The amyloid cantilever was developed and chaperone molecules were sprayed onto the cantilever using a microfluidic spraying device to investigate
whether the deposited mass was sufficient to be detected. The results of this experiment indicated that the mass of the deposited chaperone molecules was insufficient to significantly alter either the frequency or amplitude of oscillation of the amyloid cantilever, thus making them undetectable.

Objective (3) was to perform “optical tweezer” type experiments on Abeta fibrils within the 4D electron microscope. The objective here was twofold: (1) develop an exciting new technique which I call “4D optical force electron microscopy” with vastly superior spatiotemporal resolution than conventional optical tweezers and (2) measure the fracture mechanics of both Abeta(1-40) and Abeta(1-42) amyloid fibrils. Since these fibrils are central to the pathogenesis of Alzheimer’s disease, but display different cytotoxicities thought to be related to their distinct biophysical
properties, by pulling on these fibrils in a controlled manner using a laser beam, and imaging the displacements using an electron beam, we hoped to learn why these fibrils display such disparate physical behaviors. Experiments focusing on this objective were performed during the period 01/03/2014 to 27/02/2015.

One important component of this objective was to develop a way to perform the pulling experiments in the fibrils’ native, aqueous environment-even within the vacuum of the electron microscope! This was achieved by making micron-sized pockets of water encapsulated by two layers of graphene
(Figure 1, top) known as graphene liquid cells (GLC). Secondly, I had to perform a nano-construct in order to attach the polarizable gold bead exclusively to the ends of the amyloid fibrils (Figure 1, bottom), so that an offset “pump” laser beam could pull an individual fibril. This objective was also attained and fibrils with part of the fibril-gold complex contained within the aqueous environment of the graphene liquid cell were identified using conventional electron microscopy. Unfortunately, the presence of the water strongly attenuates the signal of the “probe” electron beam making it currently impossible to image movements of the optically pulled gold bead, and hence the extended amyloid fibril. My hope is that in the future, the brightness of the electron source can be improved, making this exciting experiment possible.

Objective (4) was to investigate whether cataract-associated amyloid plaques could be broken up using a pulsed laser within the electron microscope. To test this, I precisely determined the power and duration of laser pulse required to break up amyloid fibril plaques formed by insulin (owing to its commercial availability). Since I have returned to Cambridge for the “incoming phase” of my Fellowship, the details of the extremely low-dose laser bursts required to break up the insulin plaques are currently being used to investigate whether cataract-associated gamma-crystallin amyloid fibrils can be broken up in aqueous buffer within a test tube. Should the in vitro experiments prove successful, we could propose to test the approach in vivo.

During the period 01/03/2015 to 29/02/2016, I have successful reintegrated into European scientific research by returning to the University of Cambridge for the return phase of my Marie Curie Fellowship. I have used this period to fulfill the objectives of the return phase by bringing my knowledge of the unique time-resolved electron microscope (ultrafast electron microscope/4D electron microscope) technology back to Europe from Caltech. I have won a competition to become a Principal Investigator in the Department of Chemistry at the University of Cambridge with a set of ideas directly related to the development of an ultrafast electron microscope here in Cambridge (see attached .pdf for further details).

While applying for grants/fellowships related to starting my own research group here in Cambridge, I have also taken the opportunity to train on high-end cryo-electron microscopes at the nearby MRC Laboratory of Molecular Biology with a view to learning more about state-of-the-art cryo-EM imaging of protein samples. This training and knowledge will only enhance the application of the ultrafast electron microscope to biological problems when my research group have built a designated ultrafast electron microscope to directly image protein dynamics.

Finally, in order to demonstrate the broad range of applications of an ultrafast electron microscope, I also recently published a review and perspective on the technology in a prominent research journal: "Four-dimensional electron microscopy: Ultrafast imaging, diffraction and spectroscopy in materials science and biology GM Vanacore, AWP Fitzpatrick, AH Zewail - Nano Today, 2016."

Please see the project website for further details: