Nucleation by rRNA Dictates the Precision of Nucleolus Assembly

Abstract

Membrane-less organelles are intracellular compartments specialized to carry out specific cellular functions. There is growing evidence supporting the possibility that such organelles form as a new phase, separating from cytoplasm or nucleoplasm. However, a main challenge to such phase separation models is that the initial assembly, or nucleation, of the new phase is typically a highly stochastic process and does not allow for the spatiotemporal precision observed in biological systems. Here, we investigate the initial assembly of the nucleolus, a membrane-less organelle involved in different cellular functions including ribosomal biogenesis. We demonstrate that the nucleolus formation is precisely timed in D. melanogaster embryos and follows the transcription of rRNA. We provide evidence that transcription of rRNA is necessary for overcoming the highly stochastic nucleation step in the formation of the nucleolus, through a seeding mechanism. In the absence of rDNA, the nucleolar proteins studied are able to form high-concentration assemblies. However, unlike the nucleolus, these assemblies are highly variable in number, location, and time at which they form. In addition, quantitative study of the changes in the nucleoplasmic concentration and distribution of these nucleolar proteins in the wild-type embryos is consistent with the role of rRNA in seeding the nucleolus formation.

Figure 1. Nucleation and growth in phase separation processes

Birth of a new phase is called nucleation. Smaller size assemblies are unstable due to their large surface to volume ratio, which increases the negative effect of surface tension. Therefore, smaller assemblies shrink rather than grow. However, if the assemblies reach a critical size, they will readily grow. If the nucleation barrier is large, then the formation of new assembly becomes nucleation-limited. Otherwise, this process becomes growth limited.

Figure 2. De novo nucleolus formation at n.c. 13

A Representative images of embryos expressing RpI135-GFP and RFP-Fib at n.c. 10-14. The nuclear signal for these two proteins is diffuse throughout the nucleus during n.c. 10-12, but both proteins colocalize to the nucleolus (bright foci) from n.c. 13. Asterisks show auto-fluorescence. Scale bar 10 μm. Numbers show the time in minutes from the end of mitosis. B. Schematic representation of the distribution of the nucleolar proteins during early embryogenesis. The nucleolus forms for the first time during mid-blastula transition (MBT), which coincides with the large-scale zygotic genome activation. Prior to MBT, no nucleolus can be detected in the Drosophila embryos. See also Movie 1.

Figure 3. The transcription of rDNA starts prior to the nucleolus formation in the Drosophila embryos

A. A schematic view of pre-rRNA, and the fragments used for detecting pre-rRNA. B. The pools of RNA present in wild-type embryos from three different collections were tested for the presence of pre-rRNA (ITS1), and three controls. The unfertilized embryos (UF) have only maternal RNA, the 0-1 hr collection has largely maternal or early zygotic RNA, and the 3-4 hr collection has mostly zygotically transcribed RNA. RT+ represents the samples that were reverse transcribed, while RT- is the negative control. hsp83 and fruhstart are maternal and zygotic, respectively. Tubulin is the loading control. C. The real-time PCR quantification of cDNAs from single embryo RNA extracts at different time points tested for the presence of 5′ETS shows a significant increase compared to the unfertilized embryos starting from n.c. 11. Data are presented as mean ± SEM of triplicate samples. Embryos were collected at seven minutes after the telophases of n.c. 10-12, 10 minutes of n.c. 13, and 10 and 20 minutes of n.c. 14. The mutants lacking rDNA were collected at n.c. 14. D. Likewise, FISH of ITS1 at n.c. 10-14 shows the emergence of bright foci starting from n.c. 11. For the purpose of presentation, the images in D and E have adjusted contrasts and changed saturation values, with the insets having lower saturation values. E. Immunolabeling of fibrillarin followed by FISH of ITS1 in embryos at n.c. 13. Scale bars 10 μm.

Figure 4. Fibrillarin and RNA pol I form high concentration assemblies in the absence of rDNA

A. Representative images of the nuclei for wild type versus mutant embryos lacking rDNA at n.c. 14 show that both RFP-Fib and RpI135-GFP can form high concentration assemblies in the absence of rDNA. B. FRAP of Fibrillarin assemblies for the nucleoli and HANPs show that both are dynamic structures (rate constant for unbinding is 0.34 ± 0.03 s^-1 for nucleoli and 0.27 ± 0.04 s^-1 for HANPs). C. Histogram of the integrated intensity of nucleolus and HANPs normalized to the mean intensity of HANPs is shown for RFP-Fib (top) and RpI135-GFP (bottom). The probabilities for the case of RpI135-GFP in nucleolus is multiplied by 10 for better visualization. D. The fraction of nuclei showing different numbers of nucleoli in the wild-type or HANPs in the absence of rDNA is depicted for embryos after 15 minutes into n.c. 14. E. Top: Lateral view of the wild type and mutant embryos lacking rDNA at n.c. 14, expressing RFP-Fib. Bottom: quantification of the distance from the chorion of the nucleoli in a wild-type and HANPs in the mutant embryos lacking rDNA. F. Formation of fibrillarin high concentration assemblies for ten wild type (bottom) and ten mutant embryos lacking rDNA (top) over time at n.c. 13-14. Each color depicts an individual embryo. Time zero marks the end of mitosis. Scale bars 10 μm. See also Movie 2 and Figure S1.

Figure 5. Nucleolus formation is concentration dependent

A. Phase separation processes are concentration dependent. The prediction of a first order phase separation is that at lower concentrations, only one low concentration phase, the nucleoplasm, should exist. After reaching a critical concentration (C_cr) a new high concentration phase, the nucleolus, should appear. The formation of this new phase is either nucleation-limited (ii) or growth-limited (iii). B. The normalized nucleoplasmic intensities (arbitrary units) per unit volume, excluding the nucleoli, for RFP-Fib, RpI135-GFP and GFP-NLS are shown as ς. The shaded areas in B and C depict the times that the nucleolus forms and coincides with a decrease in the nucleoplasmic concentration of RNA pol I and fibrillarin. C. Comparison of the ς for fibrillarin at n.c. 13 shown in B with its total intensity per nucleus. Although total nuclear amounts of fibrillarin increases until 14 min into n.c. 13, the nucleoplasmic concentration (ς) decreases upon nucleolus formation to reach a steady state. Both nucleoplasmic concentrations and total nuclear intensities have been normalized to their maximum values for presentation purposes. Zeros mark the end of mitosis (M) and the beginning of the interphase (I). Data are represented as mean ± SEM (n=10 for RpI135 and fibrillarin and n=3 for GFP-NLS). See also Figure S2.

Figure 6. Local Inhomogeneities prior to the Nucleolus Formation at Cycle 14, but Not Cycle 13

A. The emergence of local inhomogeneities in the whole nucleus and the nucleoplasm excluding the nucleolus has been calculated for fibrillarin as described in Supplemental Experimental Procedures. A decrease in the homogeneity shows the appearance of local inhomogeneities. As a comparison, the time at which the nucleoli are detectable are shown in terms of percent of nuclei with nucleolus (purple). Shaded areas highlight the time prior to nucleolus formation. The detectable decrease in the homogeneity at n.c. 13 is only due to the nucleolus formation, while at n.c. 14, inhomogeieties arise prior to nucleolus formation. B-C. Representative images of nuclei at n.c. 14 during the initial steps of nucleolus formation. While the only detectable bright foci at n.c. 13 are at NORs (C-C′), at n.c. 14 high-concentration assemblies appear ubiquitously in the nucleoplasm (D-D′). The nuclei in B′-C′ show the higher magnification of the regions depicted by red square in B-C, respectively. For the purpose of presentation, images have adjusted contrasts and saturation values. Scale bar for B-C, 10 μm and for B′-C′, 2 μm. See also Figure S3.

Figure 7. Reduction in rRNA transcription delays nucleolus formation

A. Injection of wild-type embryos with RNAi against RpI12 reduces the amount of this transcript to approximately 45% of that of un-injected embryos (Control-1) or embryos injected with RNAi against RpI135 (Control-2). B. The amount of pre-rRNA is determined by RT-qPCR on 25 individual control-1 embryos, 25 embryos injected with RNAi αRpI12, and nine of Control-2 embryos at 10 min into n.c. 13. The amount of pre-rRNA is reduced by nearly 2 fold in the embryos injected with RNAi for RpI12 compared to the two controls (P < 0.005 by Student's t-test). Red diamonds in A-B show mean ± SEM for each group. Representative images of n.c. 13 for a Control-2 embryo with normal nucleolus formation (C) and RpI12 RNAi embryo with delayed nucleolus formation (D). Scale bars, 10 μm. E. Formation of assemblies was measured for 39 individual RpI12 RNAi embryos (orange) and 39 un-injected embryos (blue) at 10 min into n.c. 13. Images shown in C and D score 0.9 and 0.3 for Formation of Assembly. F. Time evolution of formation of fibrillarin assemblies in the wild-type embryos (n=10, reproduced from Figure 4F) and G. RpI12 RNAi embryos (n=28) during n.c. 13-14. Each line depicts an individual embryo. Time zero marks the end of mitosis.