Volume 63, Issue 6, Pages 951-964 (September 2016) Soluble Oligomers of PolyQ-Expanded Huntingtin Target a Multiplicity of Key Cellular Factors  Yujin E. Kim, Fabian Hosp, Frédéric Frottin, Hui Ge, Matthias Mann, Manajit Hayer-Hartl, F. Ulrich Hartl  Molecular Cell  Volume 63, Issue 6, Pages 951-964 (September 2016) DOI: 10.1016/j.molcel.2016.07.022 Copyright © 2016 Elsevier Inc. Terms and Conditions

Molecular Cell 2016 63, 951-964DOI: (10.1016/j.molcel.2016.07.022) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Characterization of Cell Populations in Progressive Stages of Aggregation (A) Representative images of N2a cells expressing HttEx1-eGFP constructs Q18, Q64, and Q150+ for 24 or 48 hr. Scale bar, 10 μm. (B) N&B analysis using fluorescence fluctuation software (SimFCS) on cells expressing Q18, Q64, and Q150+ for 24 and/or 48 hr. The left images show the pixel selection map on the cell analyzed. Typically, ∼30%–50% of a single cell was imaged for the N&B analysis (selection map image is 256 × 256 pixels; length × width = 12.8 μm × 12.8 μm). The histograms of the number of pixels per molecular brightness, B, are shown in the right images, and the pixels corresponding to areas of the cell containing small oligomers comprised of 6–12 molecules per particle (B ∼1.45) are highlighted in red in the selection maps (left). The green curve indicates monomers (B ∼1.05, calibrated by Q18 and the monomeric super folder GFP), which were present throughout the cell; thus they were not highlighted in the selection maps (left). Higher B values indicate higher order aggregates. Large oligomers comprised of 22–28 molecules per pixel were measured in a small fraction of the Q64 and Q150+ expressing cells (B ∼2.01), as shown for a cell expressing Q150+ for 48 hr by the blue curve in the molecular brightness histogram (right) and highlighted by blue pixels in the selection maps (left). (C) The fraction of cells containing small oligomers (red circles) and large oligomers (blue circles) were quantified using N&B; cells containing aggregates (gray circles) and inclusions (black circles) were quantified using conventional light microscopy. Cell counting experiments for N&B analysis were performed by imaging 20 or more cells in random fields of view with 4–6 independent biological replicates for each cell population. The data are averages ± SEM. Molecular Cell 2016 63, 951-964DOI: (10.1016/j.molcel.2016.07.022) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 Proteomic Analysis of Soluble and Insoluble HttEx1 (A) A summary of the interactors of soluble, polyQ-expanded HttEx1 (HttEx1SOL) obtained for Q64 and Q150+ at 24 and 48 hr of expression is shown by Venn representation. The total number of interactors and the overlap between interactomes are indicated (Table S1). (B) Venn representation of the interactomes of soluble HttEx1 and of the proteins enriched in the insoluble HttEx1 fractions (monomeric Q18, HttEx1SOL, HttEx1INSOL-TX100, and HttEx1INSOL-TX100) (see Table S1). Molecular Cell 2016 63, 951-964DOI: (10.1016/j.molcel.2016.07.022) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 GO Analysis of HttEx1 Interactomes Enrichment of GO categories of proteins in the interactomes of soluble forms of Q64 and Q150+ after 24 and 48 hr, as well as monomeric Q18 and of proteins enriched in insoluble aggregate fractions of Q150+ (TX-100 insoluble and SDS insoluble) after 48 hr of expression (Table S1G). The interacting proteins were categorized according to their functional annotations in the GO databases using David v. 6.7. Significantly overrepresented categories (p values < 0.05) for the various interactomes are plotted according to their significance (-log10 of the enrichment p values). Note that the GO term enrichments in the interactome sets may change for statistical reasons as the number of interactors increases from the 24 hr to 48 hr time points, although the proteins belonging to a specific GO term are common to both interactor sets (see Figure 2A; Table S1). Molecular Cell 2016 63, 951-964DOI: (10.1016/j.molcel.2016.07.022) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Soluble Forms of PolyQ-Expanded HttEx1 Disrupt Ribosome Biogenesis (A) CFP-tagged Q25 or Q97 was expressed in HeLa cells with inducible expression of Rps2-YFP (Wild et al., 2010). The two top images show representative examples of an untransfected cell and a cell transfected with Q25 (control) with normal Rps2-YFP export to the cytosol. Inhibition of Rps2-YFP export from the nucleus was observed in a fraction of cells cotransfected with Q97 (bottom). The white dotted lines indicate the circumference of the nucleus. The scale bar represents 10 μm. (B) The fraction of Q25 or Q97 expressing HeLa cells with nuclear accumulation of Rps2-YFP was quantified. The cells were counted in ten random fields of view per experiment. The data represent averages ± SEM from three independent biological repeats. p values were determined using the Welch two sample t test. (C) Expression of mCherry-Fbl in N2a cells in the presence of Q64 and Q150+ results in mislocalization of mCherry-Fbl to the cytosol as small foci. HttEx1 constructs Q18, Q64, and Q150+ were expressed for 24 and 48 hr. The white dotted lines indicate the circumference of the nucleus. The scale bar represents 10 μm. (D) The fractions of cells with mCherry-Fbl mislocalization to the cytosol and colocalization with cytosolic Q150+ inclusions in (C) were quantified as in (B) (∗p < 0.05 and ∗∗p < 0.01 relative to Q18 expressing control cells from Welch two sample t test). Molecular Cell 2016 63, 951-964DOI: (10.1016/j.molcel.2016.07.022) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 Role of LCRs in Mediating Protein Interactions with PolyQ-Expanded HttEx1 (A) Proteins with LCRs ≥35 residues in length are highly represented in the interactomes of polyQ-expanded HttEx1. The percentage of proteins with LCRs ≥35 residues in the different interactomes was determined using the program SEG (Table S1) (Wootton and Federhen, 1996). Numbers of LCR proteins are: 886/5909 (MS), 1/14 (Q18), 19/73 (Q64 24 hr), 151/540 (Q64 48 hr), 21/145 (Q150+ 24 hr), 210/836 (Q150+ 48 hr), 8/63 (TX-100), and 10/36 (SDS). The proteins identified in the cumulative SILAC-MS/MS experiments (5,909 proteins) (MS) served as reference. (B) Schematic representation of the domain organization of the Q150+ interactor Fus is shown with four LCRs (LCR1, LCR2, LCR3, and LCR4), an RNA recognition motif (RRM) containing a nuclear export signal (NES), a zinc finger domain (ZNF), and a C-terminal NLS. (C) Representative images of N2a cells coexpressing mCherry-Fus and eGFP-HttEx1 constructs Q18, Q64, or Q150+ for 48 hr. The large white dotted lines indicate the circumference of the nucleus and the small dotted lines indicate the outline of the cell. Scale bar, 10 μm. (D) Quantification of small HttEx1 foci (>200 nm) and inclusions (>2 μm) and their colocalization with mCherry (control), mCherry-Fus, or mCherry-LCR when coexpressed with Q64 or Q150+ for 24 and 48 hr. The data represent averages ± SEM from three independent biological repeats. (E) Representative images of cells coexpressing mCherry-LCR and Q18, Q64, or Q150+ for 48 hr. The white dotted lines indicate the circumference of the nucleus. Scale bar, 10 μm. (F) Representative images of cells coexpressing mCherry-LCR and Q150+. mCherry-LCR was expressed for 48 hr prior to inducing Q150+ expression for another 48 hr (top, control) or Q150+ was induced for 48 hr, followed by the blocking of Q150+ expression for 48 hr and then inducing expression of mCherry-LCR for 48 hr (bottom). The outline of the cells is indicated. Molecular Cell 2016 63, 951-964DOI: (10.1016/j.molcel.2016.07.022) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 Chaperone Overexpression Rescues Cellular Defect Caused by HttEx1 Oligomers (A) Representative images of N2a cells cotransfected with mCherry-Fbl and various chaperones or empty vector (vector) for 3 hr followed by expression of HttEx1 Q64 for 48 hr. The various chaperones were tagged at the N terminus with a FLAG tag (Hip, mutant HipD211K/Y212A, and Hsc70) or a V5 tag (DnaJB6b) and visualized by immunofluorescence. The white dotted lines indicate circumference of the nucleus. (B) Fraction of cells in (A) in which mCherry-Fbl was mislocalized to the cytosol. The averages ± SEM from 3–6 independent experiments are shown. p values relative to the vector control were determined by Welch two sample t test. Molecular Cell 2016 63, 951-964DOI: (10.1016/j.molcel.2016.07.022) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 7 A Multiple Hit Model of Cellular PolyQ Pathology The interactome of soluble oligomers of polyQ-expanded HttEx1 is highly complex, while the insoluble inclusions are less interactive. This decrease in interaction capacity correlates roughly with the ∼100-fold reduced surface to volume of the inclusions relative to oligomers. Interactions with the oligomers are dynamic and can affect multiple key cellular pathways. Multiple chaperones and UPS components interact with both oligomers and inclusions, but are overrepresented in the insoluble aggregates. The enrichment of chaperones is apparently due to irreversible coaggregation. In contrast, the UPS components associate with the inclusion surface. The numbers in brackets indicate the protein numbers in the different GO categories (Table S1) and by manual annotation of chaperones and their regulators, which were missed by the GO annotation software. Molecular Cell 2016 63, 951-964DOI: (10.1016/j.molcel.2016.07.022) Copyright © 2016 Elsevier Inc. Terms and Conditions