Real-time imaging of filamentous protein self-assembly

by structured illumination and TIRF microscopy

Michael Shaw*, Angelo Bella, Santanu Ray & Maxim Ryadnov
1

Analytical Science Division, National Physical Laboratory, Teddington, Middlesex, TW11 0LW, UK

*Email: mike.shaw@npl.co.uk

Protein self-assembly

The starting point (individual protein blocks) and end point

(protein filaments) of this process have been explored
extensively [1], however relatively little is known about the
precise self-assembly mechanisms by which such
structures form.

Many proteins are known to form filamentous structures, which

have varied biological and pathological functions. Examples:
cytoskeletal proteins (actin filaments, intermediate filaments
and microtubules) that mediate cell adhesion, migration and
proliferation;
amyloid fibrils which underpin neurodegenerative disorders
such as Alzheimer's, Huntington's and Parkinson's diseases;
extracellular matrices that support all cellular processes;
filamentous viruses whose capsids are thought to assemble
through a similar mechanism.

In order to gain an insight into the nature of filamentous

self-assembly we synthesized a protein model (SKI)
labelled with the fluorescent dye Alexa Fluor 488 to enable
fluorescent imaging. SKI was engineering to fold into an helical coiled-coil homo-dimer with oppositely charged
overhangs to nucleate bi-polar longitudinal assembly of
coiled-coil proto filaments able to bundle up into more
mature filaments (Fig. 1). The folding properties of the SKI
peptide
were
verified
using
circular
dichroism
spectroscopy.

Fig. 1. Schematic diagram showing longitudinal self-assembly of SKI peptide

subunits into a protofilament. Oppositely charged ends of the subunits are
coloured red and blue.

Despite their functional diversity, all of these structures are

products of directed self-assembly in which filaments are built
up from individual polypeptide units.

Microscopy & image analysis

Fig.
2.
Schematic
diagram
showing
excitation path in SIM
system.
Insets:
(I)
example phase gratings
displayed on SLM; (III)
the spatial filter in the
Fourier plane of the
SLM;
(IIII)
intensity
pattern in the focal
plane of the objective
lens.

(b)

segmentation
& tracking

Widefield SIM and TIRF images were acquired using a 60x/1.3

silicone immersion and a 100x/1.49 oil objective lens
respectively. SIM images were reconstructed by combining
weighted, shifted image components through a generalised
Wiener filter [3].

Imaging of the SKI filaments in solution was made possible

using TIRF illumination which limited the effective depth of the
excitation field to a few 100 nm from coverglass/solution
interface. In practice the relatively low signal-to-noise ratio in
the raw TIRF-SIM image frames meant that effective
reconstruction of superresolution images was often difficult.

top hat filtering

Sinusoidal excitation patterns were generated using a

ferroelectric liquid crystal on silicon spatial light modulator
(SLM) configured as a binary phase grating [2];
Illumination at 488 nm with a fibre coupled OPSL;
Images acquired with a scientific CMOS camera with the
global exposure period synchronised to the SLM.
A closed loop focus stabilisation module was fitted to the
microscope to minimise axial drift during time lapse imaging

Fig. 3 shows a fibrous carpet of individual SKI filaments

formed by the self-assembly process imaged using 2D SIM
after the solution had dried. Out of focus signals (and
associated image artefacts) were reduced by applying a
Gaussian weighting function to supress the zero order
passband except close to the excitation frequency where it
replaced the complimentary information from the displaced
order passbands [4]. The inset shows an AFM phase image of
a similar sample.

raw image

Labelled SKI filaments were imaged in aqueous solution using

a custom-built structured illumination microscope (Fig. 2):

(a)

Fig. 3. (a) Widefield 2D SIM image of the final self-assembled system. Inset shows AFM
phase image of the system. (b) TIRF images of a single filament illustrating principal
image processing and analysis operations.

Analysis of filament growth kinetics was carried out using diffraction limited
TIRF images formed from a scaled linear summation of the nine raw image
frames. Contrast of the filaments was enhanced using a morphological top hat
filter. Individual filaments within the filtered images were then segmented and
tracked over time using open active contours [5], with the object and
background pixel values set individually for each filament. The other contour
parameters were optimised once and assigned the same values for all
filaments in the image sequence. Filament positions and lengths were then
extracted from the fitted contours.

Self-assembly kinetics

(a)

(c)

t= 180 mins

Time lapse images of assembling filaments in a 100:1

mixture (SKI:SKI-Alexa488) were acquired every 30
seconds with an image acquisition time of ~ 1.8 seconds.
Fig. 4(a) shows example images of a single filament 1, 2, 3
and 4 hours after the start of imaging. Fig 4(b) shows the
change in position of the tips of a single filament indicating
that the longitudinal filament growth is bidirectional. Fig. 4
(c) shows the measured length of 7 individual filaments
(see inset image) from the same image sequence. After an
initial period of relatively rapid growth, the rate of length
increase gradually slows, reaching a near equilibrium
approximately 5 hours after first formation of the filament.
This behaviour is consistent with equilibration between the
monomers in solution and those incorporated into the
filament.

widefield

SR-SIM

OS-SIM

SROS-SIM

(b)
Fig. 4. (a) Images of a single protein filament 60, 120, 180 and 240 mins after start of imaging. Scale bar is 1 m. (b) Change in coordinates (left) and
movement of filament tips (right) over image sequence. (c) Growth kinetics of 7 individual filaments (shown in inset image) over image sequence.

Conclusions
We used SI and TIRF microscopy to image fluorescently labelled model protein
filaments. By analysing the images we were able to measure the growth rates of
individual filaments and show, for the first time, that their longitudinal growth is bidirectional. These techniques and results can provide insights into similar proteins,
formed by mechanistic self-assembly, in various biological systems.

References
[1] A. Bella, et. al., Angew. Chem. Int. Ed., 51, 428-431
(2012).
[2] P. Kner et. al., Nat. Methods, 6, 339-342 (2009)
[3] M. Gustafsson et. al., Biophys. J., 94, 4957-4970 (2008)
[4] K. O'Holleran, M. Shaw, manuscript under review
[5] M. Smith et. al., Cytoskeleton, 67, 693-705 (2010)

Acknowledgements
This work was funded by the UK National
Measurement System through the exploratory
strategic research and ChemBio programmes.

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