Sequence determinants of in cell condensate morphology, dynamics, and oligomerization as measured by number and brightness analysis

Abstract

Background: Biomolecular condensates are non-stoichiometric assemblies that are characterized by their capacity to spatially concentrate biomolecules and play a key role in cellular organization. Proteins that drive the formation of biomolecular condensates frequently contain oligomerization domains and intrinsically disordered regions (IDRs), both of which can contribute multivalent interactions that drive higher-order assembly. Our understanding of the relative and temporal contribution of oligomerization domains and IDRs to the material properties of in vivo biomolecular condensates is limited. Similarly, the spatial and temporal dependence of protein oligomeric state inside condensates has been largely unexplored in vivo.

Methods: In this study, we combined quantitative microscopy with number and brightness analysis to investigate the aging, material properties, and protein oligomeric state of biomolecular condensates in vivo. Our work is focused on condensates formed by AUXIN RESPONSE FACTOR 19 (ARF19), a transcription factor integral to the auxin signaling pathway in plants. ARF19 contains a large central glutamine-rich IDR and a C-terminal Phox Bem1 (PB1) oligomerization domain and forms cytoplasmic condensates.

Results: Our results reveal that the IDR amino acid composition can influence the morphology and material properties of ARF19 condensates. In contrast the distribution of oligomeric species within condensates appears insensitive to the IDR composition. In addition, we identified a relationship between the abundance of higher- and lower-order oligomers within individual condensates and their apparent fluidity.

Conclusions: IDR amino acid composition affects condensate morphology and material properties. In ARF condensates, altering the amino acid composition of the IDR did not greatly affect the oligomeric state of proteins within the condensate. Video Abstract.

Keywords: Biomolecular condensates; Fluorescence microscopy; Fluorescence recovery after photobleaching; Intrinsically disordered regions; Number and brightness analysis.

Fig. 1

Altering composition of the ARF19 IDR impacts the morphology of ARF19 condensates. A Schematic of the ARF19 protein showing the location of the DBD and PB1 domains. The graph below shows predicted prion-like domains within the ARF19 IDR, which were predicted using PLAAC [61]. B Schematic showing a subsection of the ARF19 IDR highlighting differences in IDR composition for the QtoS (middle) or QtoG (bottom) variants. While this schematic only shows a subsection of the IDR, for the QtoG or QtoS variants, all glutamines were changed to glycine or serine, respectively. C Images showing the range of condensate morphologies formed by wild-type ARF19 or ARF19 with the altered IDR compositions. Images were chosen to represent the breadth of condensate morphology and sizes that are frequently observed across protoplasts when expressing the various constructs. All images were taken in different protoplasts approximately 16 h after transfection. Scale bars represent 5 microns

Fig. 2

IDR composition impacts the fluidity of ARF19 condensates. A Average fluorescence recovery curves for the three ARF19 variants. Error bars report standard error of the mean. B Each point represents the percent recovery 2 min post-photobleaching of an individual condensate. For all FRAP experiments, only one half of the condensate was photobleached. N = 17 (WT), N = 14 (QtoS), N = 20 (QtoG). C A table summarizing data from panel A. P-values were calculated using a two-sided t-test comparing the values from wild-type to each of the two IDR variants

Fig. 3

IDR composition has little impact on the oligomeric state of ARF19 condensates. A Each point represents the percent of monomers and dimers out of the total number of measured oligomers for an individual condensate. B Each point represents the percent of 10+-mers out of the total number of measured oligomers for an individual condensate. C A table summarizing the data from figure panel A. D A table summarizing the data from figure panel B. Statistical testing is not shown because none of the comparisons were statistically significant

Fig. 4

ARF19 condensates are initially liquid-like and lack higher-order oligomers. A Time lapse images of wild-type ARF19 condensates in protoplasts. The top images are from the beginning of a time-lapse series started shortly after protoplast transfection (early ARF19 condensates) and show an example where the condensates have liquid-like behavior. The bottom images are from a later time point in a time lapse series (late ARF19 condensates) and show an example where the condensates appear to partially fuse but ultimately are unable to fully fuse resulting in a ‘grape-bunch’ like morphology. Note, time intervals between the bottom panels are not equal from panel to panel. B Each point shows the percent values for either lower order oligomers (left) or higher order oligomers (right) for individual ARF19 condensates approximately two or sixteen hours after protoplast transfection N = 16 (3 h) and N = 17 (16 h). C Table summarizing the data from figure panel B

Fig. 5

Distribution of oligomeric species in ARF19 condensates over space and time. A N&B analysis showing the spatial distribution of various oligomeric species in early (left) and late (right) ARF19 condensates. Each oligomeric species corresponds to a different color. The size of the early condensate relative to the late condensate can be seen in the box towards the top left of the late condensate. B N&B analysis showing the average percentage of higher-order and lower-order oligomers in ARF19 condensates in early condensates (left) and late condensates (right)

Fig. 6

IDR composition-dependent relationship between condensate oligomeric state and fluidity. A–F Each point shows N&B and FRAP data for an individual condensate. Panels on the left show the relationship between the percent of lower order oligomers and the fluorescence recovery two minutes post-photobleaching for individual condensates whereas panels on the right show the relationship between higher order oligomers and percent recovery for individual condensates. Dashed lines are linear fit lines. R² values are shown near the linear fit lines. N = 17 (WT), N = 14 (QtoS), and N = 20 (QtoG). G Equations for linear fit lines shown for each graph