Article Figures & Data
Figures
- FIG 1
HSP48 is upregulated during Dictyostelium discoideum development. (A) HSP48 expression is upregulated during Dictyostelium development. Wild-type Dictyostelium cells were starved to induce development. Cells were harvested at 0 and 24 h for RNA extraction, and RT-PCR was performed to assess expression levels of α-crystallin domain-containing proteins (n = 2). (B) HSP48 is not induced upon heat stress. Wild-type Dictyostelium cells were grown at either 22°C or 30°C for 1 h and then harvested for RNA extraction. RT-PCR was performed to assess expression levels of the α-crystallin domain-containing proteins HSPG1, HSPG2, and HSP48 (n = 2).
- FIG 2
HSP48 phase separates to form a biomolecular condensate. (A) 3D volume-rendered HSP48. Dictyostelium cells expressing GFPHSP48 were imaged using a confocal microscope. 3D volumes were then produced using Imaris software. (B) HSP48 puncta are highly spherical. 3D volume images of HSP48 were used to obtain sphericity values using Imaris software. Values equal to 1 indicate sphericity (n = 20). (C) HSP48 colocalizes to defined puncta. Dictyostelium cells were electroporated with a plasmid that expresses GFPHSP48 and selected for a minimum of 2 weeks. Cells were fixed with methanol, stained with DAPI, and imaged with a fluorescence microscope (n = 3). (D) HSP48 colocalizes to defined puncta. Dictyostelium cells were electroporated with a plasmid that expresses GFPHSP48 and selected for a minimum of 2 weeks. Cells were fixed with methanol, stained with wheat germ agglutinin (WGA), and imaged with a fluorescence microscope (n = 3). (E) HSP48 colocalizes to defined puncta. Dictyostelium cells were electroporated with a plasmid that expresses GFPHSP48 and selected for a minimum of 2 weeks. Cells were stained with LysoTracker and imaged live with a fluorescence microscope (n = 3). (F and G) HSP48 FRAP analysis reveals two populations with different inherent mobilities. Dictyostelium cells were electroporated with a plasmid that expresses GFPHSP48, selected for a minimum of 2 weeks, and used for FRAP. For FRAP, half of each individual droplet was bleached and then imaged for the indicated time points. ImageJ was then used for analysis to obtain fluorescence intensity values (n = 18). HSP48 puncta do not require a lipid bilayer. Dictyostelium cells were electroporated with a plasmid that expresses GFPHSP48 and selected for a minimum of 2 weeks. Cells were either imaged as intact cells (H) or lysed (I) and imaged by fluorescence microscopy (n = 5).
- FIG 3
A region next to HSP48’s C terminus drives phase separation. A region next to HSP48’s C terminus is intrinsically disordered. HSP48’s amino acid sequence was analyzed for its probability of disorder using RONN (A), IUPred (B), PONDR (C), and FoldIndex (D). (E and F) HSP48’s intrinsically disordered region is necessary for liquid droplet formation. Dictyostelium cells were electroporated with plasmids that express either GFPHSP48, GFPHSP48ΔN-term, or GFPHSP48ΔC-term and selected for a minimum of 2 weeks. Cells were then imaged using confocal microscopy (n = 3). (G) HSP48 puncta do not require a lipid bilayer. Dictyostelium cells were electroporated with plasmids that express either GFPHSP48, GFPHSP48ΔN-term, or GFPHSP48ΔC-term and selected for a minimum of 2 weeks. Cells were then lysed and imaged using a fluorescence microscope (n = 5).
- FIG 4
Polyphosphate drives HSP48 phase separation and stabilization. (A) HSP48 has a highly basic C terminus. In silico analysis of HSP48 was performed to determine the isoelectric point. (B) HSP48 is not predicted to have a DNA or RNA binding motif. HSP48’s amino acid sequence was analyzed by MOTIF search against the Pfam database. (C) HSP48 is not predicted to bind RNA. HSP48’s amino acid sequence was analyzed by PRIdictor. (D) Polyphosphate levels increase during Dictyostelium development. Wild-type AX4 cells were developed and harvested at the indicated time points. To image polyphosphate, RNA was isolated, run on an acrylamide gel, and stained with DAPI (n = 3). (E, F) Polyphosphate promotes HSP48 phase separation in vivo. GFPHSP48 constructs were transformed into wild-type (C) and △PPK1 (PPK1−) (D) cells. Cells were selected and then imaged using a fluorescence microscope (n = 4). (G) Confocal image of GFPHSP48 puncta in wild-type cells. Wild-type cells electroporated with a plasmid that expresses GFPHSP48 and were imaged by confocal microscopy (n = 4). (H) Confocal image of GFPHSP48 puncta in △PPK1 cells. △PPK1 cells were electroporated with a plasmid that expresses GFPHSP48 and were imaged by confocal microscopy (n = 4). (I) GFPHSP48 protein levels are decreased in △PPK1 cells. Wild-type and △PPK1 cells expressing either GFPHSP48 or GFP were analyzed by Western blotting (n = 3). *, nonspecific band. (J) GFPHSP48 protein levels are decreased in △PPK1 cells. Quantification of HSP48 in wild-type and △PPK1 cells (n = 3). ****, P < 0.0001. (K) GFP protein levels are unchanged in △PPK1 cells. Quantification of GFP in wild-type and △PPK1 cells (n = 3).
- FIG 5
Polyphosphate and HSP48 levels dramatically decrease upon germination. (A) Wild-type AX4 cells were developed for 24 h, after which, spores were isolated and cultured in medium for the indicated time points. Cells were then imaged using bright-field microscopy. (B) Quantification of the numbers of amoeba and spores at 0 h (number of cells counted = 28) and 5 h postgermination (number of cells counted = 22). (C) Polyphosphate decreases rapidly after germination. Wild-type AX4 cells were developed for 24 h, after which, spores were isolated and cultured in medium for the indicated time points. RNA was extracted to obtain polyphosphate, run on an acrylamide gel, and stained with DAPI (n = 3). (D) HSP48 transcript levels decrease rapidly after germination. Wild-type AX4 cells were developed for 24 h, after which, spores were isolated and cultured in medium for the indicated time points. RNA was extracted, and RT-PCR was performed to assess expression levels of HSP48 (n = 3).
