Fig. 4.

CLEM reveals that the exon 1 HTT proteins are recruited to single membrane bound organelles. a–d 6-month-old zQ175 coronal sections were treated with the HTT-exon1-43Q-AF647 protein and subjected to high pressure freezing and freeze substitution. a–c Fluorescence images were taken at 10 × magnification using settings for a Alexa Fluor 647 showing the HTT-exon1-43Q-AF647 signal in magenta and b Alexa Fluor 488 to show autofluorescence levels. c Merged image from a and b. d DIC at 10 × magnification. e–h Ultrathin sections were subsequently prepared and placed on holey carbon QUANTIFOIL EM finder grids. e LM image including both fluorescence and DIC. f White boxed area in e showing the overlay of the fluorescent signal with EM. g 2D EM image from the white box in f. h The corresponding tomogram section of g with segmentation of the tomogram shown in Additional File 2: Video S1. The recruitment signal (magenta) labelled a single membrane bound, vesicle rich organelle, next to a double membrane, phagophore-like structure (cyan). i–k CLEM of HTT-exon1-43Q on 12-week-old R6/2 sections with TetraSpeck beads. i LM image of an ultrathin brain section on a QUANTIFOIL grid with 50 nm TetraSpeck beads (shown as green puncta and indicated by orange arrowheads). j White-boxed area in i showing the overlay of fluorescent signal with EM. Orange arrowheads indicate the TetraSpeck beads. k EM image of the structure in the white boxed area in j. l the tomogram of the structure in k was used to generate this 3D segmentation. The recruitment signal (magenta) uncovered a single membrane-bound vesicle-rich organelle containing a large vacuole. This was adjacent to a double membrane structure (cyan). Solid white boxed insert is a side view of the dashed white box area. Scale bars: a = 100 μm, e = 5 μm, f = 300 nm, g = 200 nm, i = 5 μm, j = 450 nm, k = 200 nm. DIC = differential interference contrast, LM = light microscopy, CLEM = correlative light and electron microscopy, EM = electron microscopy, 3Dseg = 3D segmentation