Direct Imaging of Aligned Neurofilament Networks Assembled Using In Situ Dialysis in Microchannels In collaboration with Prof. Cyrus R. Safinya (Materials Department)
		The goal of this research is to produce aligned neurofilament networks for direct imaging and diffraction studies using in situ dialysis in a microfluidic device. The alignment is achieved by assembling neurofilaments from protein subunits confined within microchannels. Resulting network structure was probed by polarized optical microscopy and atomic force microscopy, which confirmed a high degree of protein alignment inside the microchannels. This technique can be expanded to facilitate structural studies of a wide range of filamentous proteins and their hierarchical assemblies under varying assembly conditions. The microchannel devices were used to investigate the network structure of neurofilaments (NF’s), which, together with Actin and microtubules, form the scaffold for nerve cells. Although structural details and functions of NFs and their networks are still poorly understood, it is well-known that NFs consist of three different subunits that self-assemble into a flexible 10-nm-thick filament with unstructured polyampholyte brushes protruding perpendicularly out of the filamentous core. NFs are an excellent example of a filamentous protein with unstructured domains that prevent crystallization for high-resolution structural studies yet form well-ordered networks in vivo. The monomer subunits are synthesized in the neuronal cell perikarya and transported, either as subunits or filament, into the confinement of neuronal processes (axon and dendrite) with a diameter on the order of micrometers, where they form highly aligned network structures. In several neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Lewy-body-type dementia, and Parkinson disease, a disruption of the NF network structure as followed by protein aggregation can be observed.
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			Figure 1. Microchannel in situ dialysis chamber device. (a) Scanning electron micrograph of a 10-μm-wide, 50-μm-deep microchannel compartment opening out into one loading pool. Schematic (b) and cross sectional (c) view of the setup used for alignment. Top to bottom: (i) supporting stainless steel plate, (ii) Si wafer with microchannels, (iii) dialysis sheet, (iv) front plate, (v) microfluidic buffer exchange chamber.		Figure 2. (a, b) Cross-polarized optical microscopy images of an NF hydrogel formed in 50 × 50 μm2 channels. Images were taken at 45° with respect to the analyzer showing bright spots where a nematic hydrogel has formed. (c) NF nematic hydrogel inside a quartz capillary sample.
This work shows that a combination of a microchannel device and microfluidic dialysis allows us to align a hydrogel of filamentous proteins such as NFs. We demonstrated the quality of the macroscopic alignment using polarized microscopy. The details of the microscopic structure and the unexpected perpendicular alignment were probed using AFM inside the microchannels. Polymerizing the filaments in the microhannels, as demonstrated here, allows further in-depth investigations of the structure and interactions of NFs, using, for example, X-ray diffraction and motility assays.							Figure 3. AFM phase images on an aligned nematic hydrogel inside a 50-μm-wide microchannel. The right image close-up is 300 nm2. A Fourier transform analysis of image a is shown in Supporting Information, Figure S2.