An intracellular phase transition drives nucleolar assembly and size control

Transcription

1 An intracellular phase transition drives nucleolar assembly and size control Steph Weber Department of Biology McGill University

2 Membranes spatially organize eukaryotic cells

3 Cells also contain membraneless compartments Germ granules Stress granules C. elegans embryo U2OS cells Brangwynne et al 2009, Molliex et al 2015

4 Cell size changes through growth and development Reductive division Hypertrophic growth Nucleus Cell membrane 10 μm 200 μm S. Uppaluri L1 larva Adult 10 μm

5 How do cells establish and dynamically regulate spatial organization? How do membraneless organelles assemble?? How do cells coordinate and control the size of their organelles??

6 The nucleolus is a membraneless organelle Assembles around ribosomal DNA Synthesizes ribosomal subunits Ribosome C. elegans embryo Nucleus rdna Nucleolus Cytoplasm Nucleoli (FIB-1::GFP) Cell membrane (mcherry::ph PLCδ1 )

7 Membraneless organelles behave like liquids Droplet fusion Wetting and dripping Brangwynne et al 2011, Brangwynne et al 2009

8 Purified components condense into droplets and gels in vitro Multivalent domains Low complexity sequences [G/S]Y[G/S] 20 μm 3 mm Li et al 2012, Kato et al 2012

9 Hypothesis: membraneless organelles assemble via intracellular phase transitions Gas Liquid Solid Patel et al 2015 Weber and Brangwynne 2012

10 Phase diagram predicts behavior of a liquid mixture Temperature Phase boundary Two phases, separated One phase, mixed Saturation concentration All Red Concentration All Blue

11 Hallmarks of phase separation 1. Saturation concentration sets a threshold for droplet condensation 2. Size of condensed phase droplets increases with concentration Temperature C sat Concentration

12 Concentration can be tuned by manipulating embryo size ima-3(rnai) ani-2(rnai) Control C27D9.1(RNAi) Nuclear concentration, C n (µm) ima-3(rnai) ani-2(rnai) Control C27D9.1(RNAi) x 10 4 Embryo volume, V (µm 3 ) Weber and Brangwynne 2015

13 Nucleolar size increases with concentration Control C27D9.1(RNAi) ani-2(rnai) ima-3(rnai) Weber and Brangwynne 2015

14 Quantifying nucleolar size I μm I 2 Maximum nucleolar intensity per cell I o = I 1 + I 2 Weber and Brangwynne 2015

15 Nucleolar size increases with concentration Max nucleolar intensity, I o (a.u.) 10 x VC27D9.1(RNAi) n = 200 µm 3 Control ani-2(rnai) ima-3(rnai) sat V n = 200 μm Nuclear Concentration, C n (µm) Phase transition model organelle size I o = α[c n - C sat ]V n concentration nuclear volume Weber and Brangwynne 2015

16 Nucleolar size increases with concentration Max nucleolar intensity, I o (a.u.) 10 x VC27D9.1(RNAi) n = 200 µm 3 Control ani-2(rnai) ima-3(rnai) sat V n = 200 μm Nuclear Concentration, C n (µm) Temperature Phase transition model I o = α[c n - C sat ]V n C sat x C n Concentration degree of supersaturation

17 8-cell stage Nucleolar assembly depends on cell lineage, embryonic stage 10 μm 4-cell stage Nucleoli do not assemble in AB lineage until 8-cell stage ABp ABa P2 10 μm Small, transient nucleoli in EMS EMS Weber and Brangwynne 2015

18 Saturation concentration at the 4-cell stage Max nucleolar intensity, I o (a.u.) 10 x VC27D9.1(RNAi) n = 200 µm 3 Control ani-2(rnai) ima-3(rnai) 8-cell 2 sat 4-cell sat Nuclear Concentration, C n (µm) 4-cell Weber and Brangwynne 2015

19 Phase diagram for nucleolar assembly in vivo 1/χ Temperature Concentration Soluble phase NO NUCLEOLI small NUCLEOLI large χ = developmentally-regulated interaction parameter Droplet phase Nuclear Concentration, C n (µm)

20 Phase diagram for nucleolar assembly in vivo Max nucleolar intensity, I o (a.u.) 10 x VC27D9.1(RNAi) n = 200 µm 3 Control ani-2(rnai) ima-3(rnai) 2 sat 4-cell sat Nuclear Concentration, C n (µm) 4-cell sat 1/χ NO NUCLEOLI small NUCLEOLI large sat Nuclear Concentration, C n (µm)

21 Nuclear concentration is ~constant during early embryogenesis 8-cell 16-cell 32-cell 64-cell Onset of zygotic expression

22 Phase separation gives rise to organelle size scaling Max nucleolar intensity, I o (a.u.) 6 x 105 Control C n = 0.13 μm Nuclear Volume, V n (µm 3 ) I o = α[c n - C sat ]V n Cell stage: Weber and Brangwynne 2015

23 Summary of results 1. Nucleolar size increases with concentration (8-cell stage) 2. Saturation threshold for nucleolar assembly (4-cell stage) 3. Nucleolar size scales with nuclear size (8 64-cell stages)

24 So what? Function Membraneless organelles increase the local concentration of enzymes and substrates - accelerate biochemical reactions can also sequester reactants to inhibit activity

25 Nucleolar size, activity correlate with cell growth and proliferation Defects in ribosome biogenesis limit cell and organism size SFP1 overexpression Δsfp1 Misregulation of nucleolar size drives tumor growth Benign Malignant Jorgensen et al 2002, Montagne et al 1999; Derenzini et al 2009

26 Measuring nucleolar activity Ribosome Nucleus rdna Nucleolus Cytoplasm Ribosome biogenesis Transcription Processing Subunit assembly Nuclear export Fluorescence in situ hybridization rdna locus:

27 Large nucleoli produce more nascent transcripts 6 x 106 ITS1 ITS2 rrna FIB-1::GFP 5 18S 5.8S 26S Integrated intensity, rrna ITS rrna intensity, FISH internal transcribed spacer Hoescht Merge Integrated intensity, FIB-1::GFP x 10 6 Nucleolar intensity, FIB-1::GFP 10 m

28 rrna transcription increases with component concentration, nucleolar size AB lineage, 8-cell stage rrna FISH FIB-1::GFP FISH integrated intensity (a.u.) 15 x ima 3 ani 2 L4440 C27D9.1 C27D9.1 WT ani-2 ima-3 RNAi condition Nuclear concentration

29 ncl-1 mutants have enlarged nucleoli WT ncl-1 Yochem and Herman 2003 increased levels of rrna increased growth rate, body size WT ncl-1 Total RNA 26S 18S Mean worm volume (µm 3 ) WT ncl m Time (hrs)

30 Nucleolar size predicts organismal growth rate Uppaluri, Weber and Brangwynne 2016

31 Conclusions Supersaturation drives nucleolar assembly in vivo Phase separation gives rise to organelle size scaling Nucleolar size correlates with rrna transcription and organismal growth Transcription Phase transition 5 µm Growth 100 µm 1 nm Molecular 1 m Cellular 1 mm Organismal 1 m 1 nm Molecular 1 μm Cellular 1 mm Organismal 1 m

32 Princeton University Cliff Brangwynne Sravanti Uppaluri Damon Runyon Cancer Research Foundation Thank you! Anne-Marie Ladouceur Megan Couture Albright Kim Graydon Tope James Goldberg David Cohn