Independent active and thermodynamic processes govern the nucleolus assembly in vivo

Abstract

Membraneless organelles play a central role in the organization of protoplasm by concentrating macromolecules, which allows efficient cellular processes. Recent studies have shown that, in vitro, certain components in such organelles can assemble through phase separation. Inside the cell, however, such organelles are multicomponent, with numerous intermolecular interactions that can potentially affect the demixing properties of individual components. In addition, the organelles themselves are inherently active, and it is not clear how the active, energy-consuming processes that occur constantly within such organelles affect the phase separation behavior of the constituent macromolecules. Here, we examine the phase separation model for the formation of membraneless organelles in vivo by assessing the two features that collectively distinguish it from active assembly, namely temperature dependence and reversibility. We use a microfluidic device that allows accurate and rapid manipulation of temperature and examine the quantitative dynamics by which six different nucleolar proteins assemble into the nucleoli of Drosophila melanogaster embryos. Our results indicate that, although phase separation is the main mode of recruitment for four of the studied proteins, the assembly of the other two is irreversible and enhanced at higher temperatures, behaviors indicative of active recruitment to the nucleolus. These two subsets of components differ in their requirements for ribosomal DNA; the two actively assembling components fail to assemble in the absence of ribosomal DNA, whereas the thermodynamically driven components assemble but lose temporal and spatial precision.

Keywords: Drosophilanucleologenesis; RNA granule; intracellular phase transition; liquid–liquid phase separation; membrane-less organelle.

Fig. 1.

First-time assembly of six nucleolar proteins at 22 °C. Structural features of six nucleolar proteins studied are depicted in A. The structural elements of Ns1 were previously reported (21). For other proteins, the structural features were determined using National Center for Biotechnology Information conserved domain search (22) and InterPro (23) as well as IUPRED (24) for detection of disordered regions. Fib is a methyltransferase that associates with C/D box small nucleolar RNAs to form small nucleolar ribonucleoprotein complexes; the inhibitory domain of Ns1 prevents its nucleolar localization when GTP is not bound (25). The DEADc domain in Pitchoune, characterized by the conserved motif Asp-Glu-Ala-Asp (DEAD), contains an ATP binding region. B, basic region; C, coiled coil; D, domain; RGG, R/G-rich domain; RRM, RNA recognition motif. (B) The first-time emergence of assemblies for different nucleolar proteins at 22 °C at NC12–NC13 is depicted. Each line represents the fraction of nuclei in a single embryo that has detectable foci of each nucleolar protein, with the dashed lines marking the median $t_{1/2}$ for the first-time formation of each GFP-tagged protein (number of embryos in each panel = 8) (details are in Methods). Median $t_{1/2}$ for Fib is 6 min into interphase of NC13. Time 0 marks the beginning of interphase.

Fig. S1.

The nucleoplasmic concentrations of the nucleolar proteins increase by NC. The normalized nucleoplasmic intensities (arbitrary units) per unit volume, excluding the nucleoli, for EGFP-Mod, EGFP-Ns1, EGFP-Nopp140, and Pit-EGFP are shown as $\varsigma$. Times 0 marks the beginning of interphase (I) and end of mitosis (M).

Fig. 2.

Temperature uncouples the first-time assembly of different nucleolar proteins. The temperature dependence of two possible mechanisms for the formation of assemblies is illustrated qualitatively in A and B. (A) The rate of active processes is determined by the rate of the enzyme that couples them to an energy source. Therefore, active assembly is faster at higher temperatures and slower at lower temperatures. (B) This behavior is the opposite of a thermodynamic LLPS, in which more condensation occurs at lower temperatures (exceptions are in Discussion). (C and D) An embryo coexpressing (C) EGFP-Mod and (D) RFP-Fib is imaged at 7 °C and 28 °C to determine the first-time emergence of assemblies. Images are maximum-projected. (E) The percentages of nuclei with or without assemblies at each temperature for each cell cycle are depicted in a pie chart for different nucleolar proteins. Number of embryos is three or more for each condition.

Fig. S2.

Schematic of temperature-control microfluidic device. Slight modifications have been applied to the previously reported microfluidic device (27) to allow for live confocal imaging with $63\times$ objective and monitoring of the temperature live. The embryo was placed on a polydimethylsiloxane (PDMS)-coated cover glass to accommodate the short working distance of the confocal objective, and a built-in thermometer was added to the designed for live monitoring of the temperature.

Fig. S3.

Differential temperature dependence of nucleolar proteins. Five different transgenic lines coexpressing RFP-Fib with one of the EGFP-tagged proteins Nopp140, Mod, Ns1, Pit, or Rpl135 were allowed to develop at different temperatures as discussed in Methods, and the maximum-projected images for NC11–NC13 for each temperature are shown. The brightness and contrast of the images have been modified for presentation purposes. (Scale bar: $10\mathrm{\mu }$m.)

Fig. 3.

An in vivo assay to test the reversibility of assembly formation by nucleolar proteins. (A and C) A microfluidic device was used to switch between cold and warm temperatures in less than 1 min during NC14. (B and D) Schematics qualitatively illustrate predicted trends in assembly size for (B) a thermodynamic LLPS and (D) an active assembly. (B) A thermodynamic LLPS is expected to reversibly condense and dissolve by changing the temperature, whereas (D) an active assembly is expected to be slow at low temperatures, fast at high temperatures, and irreversible. (E–I and E′–I′) The integrated intensity at each temperature/time for high-concentration assemblies of different nucleolar proteins is determined, and mean $\pm$ SEM for n > 10 nuclei is normalized to its maximum level. Time 0 is when the experiment begins. The changes in the integrated intensity of the assemblies of (E′–I′) EGFP-tagged nucleolar proteins compared with those of (E–I) RFP-Fib are shown. (J) During early NC13, (i) Fib and Nopp140 assemblies are first to form at 8 °C to 10 °C. (ii) As soon as Fib assemblies appear, the embryos are shifted to 22 °C to 25 °C, in which the critical concentrations for Fib and Nopp140 are higher than the nucleoplasmic concentrations of these two proteins. This shift, therefore, causes the assemblies to dissolve. (iii) As the concentrations of Fib and Nopp140 increase by time, the assemblies reappear. The schematic in Left qualitatively illustrates the state of each step (i–iii) in the phase diagram. Images are maximum-projected. Time 0 marks the time when the temperature shift was applied.

Fig. S4.

Fluorescent recovery after photobleaching. The assembly of different nucleolar proteins have been photobleached, and the recovery of the fluorescent signal for the nucleolus has been quantified at 7 °C and 22 °C. Mean $\pm$ SEM is depicted for three or more nucleoli.

Fig. 4.

Transcription of rRNA coordinates the active and thermodynamic assemblies. (A) Each column depicts a mutant embryo lacking rDNA repeats at 22 °C and NC14 and coexpressing RFP-Fib with another EGFP-tagged nucleolar protein (Lower). Fib, Nopp140, and Rpl135 colocalize to variable numbers of assemblies in a nucleation-limited process, whereas Pit forms one diffuse assembly at the apical side of the nucleus. Mod and Ns1 do not form any assemblies in the absence of rDNA. Images are maximum-projected. (B) Distribution of the number of assemblies per nucleus in WT embryos for each of the nucleolar proteins at NC13 is depicted at high and low temperatures. At 22 °C, assemblies form at two rDNA repeats located on X and Y chromosomes. More than two foci observed for Fib and Nopp140 at 8 °C are indicative of unseeded assembly formation, whereas other proteins can only assemble at rDNA repeats in WT embryos.

Fig. S5.

Schematic representation of different types of phase diagrams. A solution showing (A) phase separation with UCST, (B) closed loop where LCST is lower than UCST, (C) UCST lower than LCST, and (D) hourglass with no critical temperature (details are in Discussion).

Fig. S6.

IDR and hydrophobicity of six studied proteins. The hydrophobicity score (38) for each amino acid was determined using ExPASy, with red indicating hydrophilic residues and blue indicating hydrophobic residues. The disorderness of the proteins studied here was determined using IUPRED (24), with scores above 0.5 indicating disorder (light green). The shaded areas indicate the disordered regions.