Integrity of the cellular protein quality control machinery throughout the experiment can be regarded as a paramount requirement if protein fate is to be studied under conditions meaningful for pathology. We therefore employed direct cytosolic protein expression. A large body of evidence indicates that formation of N-terminal fragments of Htt containing exon 1 (ex1) by proteolytic cleavage plays an important role in pathogenesis and that mutant Htt-ex1 alone recapitulates the pathological features of HD in several mammalian cell culture models3, including PC12. We transiently transfected ex1 of Htt in NGF-treated sympathetic-neuron-like PC12m (subclone of PC12) cells (Fig. 1a). Htt-ex1 was visualized by C-terminal fusion to the enhanced yellow fluorescent protein, eYFP. The construct was overexpressed compared to endogeneous cytosolic huntingtin in order to produce a measurable aggregation response, where that propensity existed, within days after transfection.

Figure 1 Overview of fluorescence microscopy of sub-cellular huntingtin distributions. (a) Low-magnification white-light (WL) image of NGF-differentiated PC12m cell culture. (b) Schematic of full-length Htt (not to scale) and the Htt-ex1-xQ constructs fused at the C terminus to eYFP for expression experiments with x = 25, 46, 97. (c) Diffraction-limited (DL) fluorescence of a PC12m cell transfected with Htt-ex1-97Q-eYFP at high magnification (100x) with an evident inclusion body (IB, bright puncta). Before photo-bleaching, the same cell's image was dominated entirely by the brightness of the IB, reflecting its extremely high recruitment of Htt-ex1-97Q-eYFP. (CCD image taken without EM gain and at ~1% pump intensity for region of IB). Only after targeted partial photo-bleaching of the contents of the IB do other more intricate dimmer structures become appreciable (full image, composite of five regions). In general, perinuclear rings of compact aggregates and other small aggregate species can be discerned in many cells. Nu = Nucleus. (d) DL image and (e) super-resolution (SR) reconstruction (plotted as the number of localizations, or events, per pixel) of a perinuclear aggregate, showing the bundling of short fibers. DL examples of cells with (f) diffuse cytosolic distribution, (g) inclusion body and (h) small aggregate species (round and elongated, fibrillar appearance, SAS) in a perinuclear (I) or dispersed cell-wide (II) arrangement, where the IB has been partially photo-bleached. Scale bars: 20 μm (a,c), 1 μm (d,e), 5 μm (f–h). Full size image

In this paper, we define the intracellular distribution of Htt-ex1 for three distinct glutamine repeat lengths, 25Q, 46Q and 97Q (Fig. 1b). From our observation of many cells by methods described below, three distinctive phenotype classes emerged. Most commonly, transfected cells featured a uniformly diffuse (Diff) Htt-ex1 distribution throughout the cell body and dendritic extensions, with evident exclusion from the nucleus (Fig. 1f and Supplementary Fig. S1). Among cells transfected with constructs containing Q-repeats beyond the disease threshold, a fraction exhibited strongly fluorescent perinuclear and cytoplasmic IBs, which were dense aggregates typically ~3–7 μm in diameter (Fig. 1g and Supplementary Fig. S1). A majority of cells with IBs contained additional small aggregates, herein referred to as small aggregate species (SAS, Fig. 1c,h and Supplementary Fig. S1), which became visible by a specialized protocol. The Htt-ex1 SAS were imaged far beyond the optical diffraction limit (Fig. 1d,e) in cells for the first time.

Making small aggregate species visible

When an IB is present, its fluorescence is extremely bright so that little else can be observed in the cell. To visualize the SAS, we therefore employed a specific procedure to reduce the extreme brightness of the IB. This involved targeted photo-bleaching of the IB, followed by the selective illumination of sub-cellular regions of interest (Fig. 2a). Transfected cells (Fig. 2b) were positioned such that the strongly fluorescent inclusion was centered in the excitation beam (Fig. 2c). The IB was then singled out for photo-bleaching by narrowing an iris diaphragm in the beam path. This solely exposed the targeted region to high-intensity light (~2.5 kW/cm2 at peak), strongly reducing the IB's fluorescence signal by many orders of magnitude (Fig. 2d), but preserving nearby fluorescent structures. Only then did the SAS become detectable (Fig. 2e). The disparate fluorescent signal level of the SAS compared to the IB represents an experimental obstacle that may have prevented earlier studies from detecting the diversity of aggregate populations in close spatial proximity.

Figure 2 Targeted photo-bleaching of inclusion bodies and selective illumination of sub-cellular regions. (a) Schematic of the procedure employed for bleaching a sample region containing a strongly fluorescent inclusion body (IB), to reduce its detrimental signal interference with nearby dimmer structures. The IB was first moved to the center of the excitation beam profile. This location was then singled out for photo-bleaching at high intensity (~2.5 kW/cm2 at peak) by narrowing an iris diaphragm in the excitation beam path. (b) White-light (WL) image of a transfected PC12m cell. (c) The diffraction-limited (DL) epi-fluorescence image of eYFP-labeled Htt-ex1 before bleachdown (boxed region in b) was entirely dominated by the IB signal (image recorded without EM gain). Nu = Nucleus. (d) After 12 minutes of bleach-down, the IB's signal had essentially disappeared. (e) Small aggregate species (SAS) were subsequently perceived (shown here with EM gain in a composite image of two regions). The residual fluorescence from the IB still had to be excluded by delimiting the regions of interest with the iris in each case, because the IB brightness slowly recovered over time as eYFP returned from very long-lived dark states. Scale bars: 10 µm (b–d), 5 µm (e). Full size image

Quantifying phenotype

With the capability to detect SAS as well as IB, we were able to quantify phenotype evolution of Htt-ex1 localization in cells for the three Q-repeat lengths. Counting experiments were performed using low-power laser excitation at 514 nm in a wide-field epi-fluorescence configuration. Cells were fixed at times t = 16, 24, 48, 96, 144, 192 hours (Fig. 3). The columns represent fractions of total numbers of transfected cells examined. Of these, the largest fraction at any given time-point exhibited diffuse cytosolic distribution (light gray column). A subset of cells exhibited one or more prominent inclusion bodies (white column). Of these cells with inclusion bodies, most contained small aggregate species as well, thus the dark gray column is always less or equally as tall as the white one. For the non-pathogenic Htt-ex1-25Q, the diffuse phenotype was observed in 100% of cells for all time points (Fig. 3a), in line with expectations for a Q-length below the disease threshold. In contrast, for 97Q, the fraction of cells containing IBs increased from ~5% at t = 16 hours to ~40% at the 192-hour time point (Fig. 3c). IBs developed significantly later for Htt-ex1 with 46 Q-repeats (above threshold, Fig. 3b).

Figure 3 Phenotype counting analysis of cellular huntingtin distributions for three repeat-lengths (25,46,97 Q-repeats) by high-magnification examination of NGF-differentiated PC12m cells. (a,b,c) Count of Htt-ex1-transfected cells (fixed) classified as the three phenotypes defined in Fig. 1f–h for the three Q-repeat lengths, at time-points t = 16–192 hours post-transfection. Small aggregates were identified in a subset of the cells with bright inclusion bodies (indicated by brackets). (n = number of counting experiments of ~70–150 transfected cells each, N = total number of cells analyzed. Mean ± s.e.m.). Full size image

At low magnification, the bright IBs obscured any other, dimmer fluorescent features nearby in the sample. However, using high magnification, combined with targeted photo-bleaching of the region containing the IB (see Methods and Fig. 2), we were able to visualize the SAS as well. Some aggregates formed a perinuclear shell-like arrangement, similar to earlier descriptions13 (Fig. 1h (I) and Supplementary Video S3). In addition, SAS were frequently found to be dispersed throughout the entire cell (Fig. 1h (II), Fig. 4 and Supplementary Video S4). Thus, strikingly, aggregation is not exclusively defined by the intensely-fluorescent IBs and perinuclear aggregates alone, but also by a rich diversity of much smaller structures that can form within the same cell, as illustrated by the example in Fig. 1c.

Figure 4 Sub-cellular distributions of huntingtin aggregates. (a,b,c) Small aggregates dispersed throughout the cell bodies form for both 46Q (example e, from a different image plane in c) and 97Q (a,b,d) at time points beyond 16 hrs post-transfection. Their observation became possible after significantly reducing the brightness of the inclusion body (IB) through our targeted photo-bleaching and imaging protocol described in Fig. 2. Only remnants of the bright IB remain. Nu = nucleus. (d,e) Super-resolution (SR) reconstructions of fibrillar aggregates. Scale bars: 5 μm (a–c), 1 μm (d,e). Full size image

Super-resolution imaging of small aggregate species

In conventional epifluorescence imaging, SAS appeared as poorly-defined diffraction-limited (DL) objects with round or oblong shapes. To extract further structural detail, we subjected the small aggregate species to examination by SR fluorescence microscopy. The light-induced blinking of fluorescent proteins exploited here is one example of a suite of fluorescence-based single-molecule techniques that can deliver sub-diffraction-limit SR images10. Crucially, genetically expressed probes can be utilized to study protein super-structures in cells at high resolution14. Illuminated with 514 nm light (~2 kW/cm2), eYFP molecules were forced into long-lived dark states. Sparse subsets of eYFP then returned to emit bursts of ~2000 photons per molecule (on average). These bright “blinks” sampled the underlying spatial arrangements (structure) of Htt-ex1 proteins. In a continuous fluorescence recording of these blinking events, single molecules were imaged in each frame and localized with high spatial precision (σ ≈ 15–20 nm) by Gaussian fitting (Supplementary Fig. S2) and SR images of the aggregates were reconstructed (example in Fig. 1e). The increase in information over the diffraction-limited image (Fig. 1d) is substantial, revealing bundled shorter fibers as observed before in vitro9, suggestive of a three-dimensional structure. Fig. 4d shows the SR reconstruction of a small aggregate (from the field of view in Fig. 4a) with fiber-like linear segments. The diffuse localizations outside of the fiber in the SR reconstruction result from monomeric and possibly small oligomeric Htt-ex1 that was not cleared from the cytosol by addition to the IB or other aggregates. This residual monomeric/oligomeric background, which varied among cells, did not compromise our ability to image the SAS co-existing with this pool of very small species. SAS also formed in the axonal and dendritic processes extending from the PC12m cell bodies (Fig. 5a,b and Fig. 1c, top right). Blockage of neuronal processes (by larger aggregates) may have toxic effects, if anterograde and retrograde transport is impaired. Aggregates both in the cell body (Figs. 1 and 4) and the processes (Fig. 5c–e) strongly resembled the fibers examined by SR fluorescence and AFM in vitro9.