Limits...
Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles.

Nott TJ, Petsalaki E, Farber P, Jervis D, Fussner E, Plochowietz A, Craggs TD, Bazett-Jones DP, Pawson T, Forman-Kay JD, Baldwin AJ - Mol. Cell (2015)

Bottom Line: These bodies are stabilized by patterned electrostatic interactions that are highly sensitive to temperature, ionic strength, arginine methylation, and splicing.Moreover, the bodies provide an alternative solvent environment that can concentrate single-stranded DNA but largely exclude double-stranded DNA.We propose that phase separation of disordered proteins containing weakly interacting blocks is a general mechanism for forming regulated, membraneless organelles.

View Article: PubMed Central - PubMed

Affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON M5G 1X5, Canada; Physical and Theoretical Chemistry Laboratory, University of Oxford, Oxford OX1 3QZ, UK.

Show MeSH

Related in: MedlinePlus

Ddx4YFP Organelles Are Internally Mobile and Respond Rapidly to Changes in Environmental Temperature and Tonicity(A) Fluorescence recovery after photobleaching (FRAP) of a Ddx4YFP organelle in a live HeLa cell at 37°C. Sample bleaching is indicated with a gray bar. 50% of the fluorescence signal is recovered within approximately 2.5 s post-bleach, corresponding to a diffusion coefficient of 3 ± 1 × 10−13 m2 s−1.(B) Cold shock induces condensation of sub-nuclear Ddx4YFP droplets at low expression levels. Extended focus fluorescence intensity images showing the nucleus from a time series analysis of a HeLa cell expressing Ddx4YFP undergoing cold shock. Images are shown at 2-min intervals. Prior to cold shock treatment, Ddx4YFP had not reached the critical concentration for phase separation at 37°C and was diffuse in the nucleoplasm (first two frames). Rapid exchange of growth media at 37°C for media cooled on ice (time = 0) induced small Ddx4YFP droplets to condense rapidly within the nucleus (purple line, number of droplets; blue line, total volume of droplets). Following cold shock, the number of Ddx4YFP droplets decreased through a combination of coalescence and dissolution as the temperature rose. Scale bar, 5 μm (see Movie S2).(C) Extended focus fluorescence intensity image slices showing a section of the nucleus from a time series analysis of a HeLa cell containing Ddx4YFP droplets undergoing osmotic shock. Images are shown at 2-min intervals. Axis labels, data colors, and scale as in (B). See Movies S3 and S4.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4352761&req=5

fig2: Ddx4YFP Organelles Are Internally Mobile and Respond Rapidly to Changes in Environmental Temperature and Tonicity(A) Fluorescence recovery after photobleaching (FRAP) of a Ddx4YFP organelle in a live HeLa cell at 37°C. Sample bleaching is indicated with a gray bar. 50% of the fluorescence signal is recovered within approximately 2.5 s post-bleach, corresponding to a diffusion coefficient of 3 ± 1 × 10−13 m2 s−1.(B) Cold shock induces condensation of sub-nuclear Ddx4YFP droplets at low expression levels. Extended focus fluorescence intensity images showing the nucleus from a time series analysis of a HeLa cell expressing Ddx4YFP undergoing cold shock. Images are shown at 2-min intervals. Prior to cold shock treatment, Ddx4YFP had not reached the critical concentration for phase separation at 37°C and was diffuse in the nucleoplasm (first two frames). Rapid exchange of growth media at 37°C for media cooled on ice (time = 0) induced small Ddx4YFP droplets to condense rapidly within the nucleus (purple line, number of droplets; blue line, total volume of droplets). Following cold shock, the number of Ddx4YFP droplets decreased through a combination of coalescence and dissolution as the temperature rose. Scale bar, 5 μm (see Movie S2).(C) Extended focus fluorescence intensity image slices showing a section of the nucleus from a time series analysis of a HeLa cell containing Ddx4YFP droplets undergoing osmotic shock. Images are shown at 2-min intervals. Axis labels, data colors, and scale as in (B). See Movies S3 and S4.

Mentions: The internal order within the organelles was assessed using fluorescence recovery after photobleaching (FRAP) measurements. The half time to recovery of the fluorescence signal of a photo-bleached body of diameter 1.5 μm took approximately 2.5 s at 37°C (Figure 2A), corresponding to an approximate diffusion coefficient of 3 ± 1 × 10−13 m2 s−1, a value two orders of magnitude lower than that measured for free globular proteins of a similar size as determined by both FRAP and NMR (Figure S5B). These self-diffusion rates are consistent with those of other non-membrane organelles, such as nuclear speckles and nucleoli (Phair and Misteli 2000). While the observed diffusion within the droplets is substantially slower than the motion of free protein, the interior is nevertheless highly mobile, consistent with weak interactions between Ddx4 proteins within the droplet.


Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles.

Nott TJ, Petsalaki E, Farber P, Jervis D, Fussner E, Plochowietz A, Craggs TD, Bazett-Jones DP, Pawson T, Forman-Kay JD, Baldwin AJ - Mol. Cell (2015)

Ddx4YFP Organelles Are Internally Mobile and Respond Rapidly to Changes in Environmental Temperature and Tonicity(A) Fluorescence recovery after photobleaching (FRAP) of a Ddx4YFP organelle in a live HeLa cell at 37°C. Sample bleaching is indicated with a gray bar. 50% of the fluorescence signal is recovered within approximately 2.5 s post-bleach, corresponding to a diffusion coefficient of 3 ± 1 × 10−13 m2 s−1.(B) Cold shock induces condensation of sub-nuclear Ddx4YFP droplets at low expression levels. Extended focus fluorescence intensity images showing the nucleus from a time series analysis of a HeLa cell expressing Ddx4YFP undergoing cold shock. Images are shown at 2-min intervals. Prior to cold shock treatment, Ddx4YFP had not reached the critical concentration for phase separation at 37°C and was diffuse in the nucleoplasm (first two frames). Rapid exchange of growth media at 37°C for media cooled on ice (time = 0) induced small Ddx4YFP droplets to condense rapidly within the nucleus (purple line, number of droplets; blue line, total volume of droplets). Following cold shock, the number of Ddx4YFP droplets decreased through a combination of coalescence and dissolution as the temperature rose. Scale bar, 5 μm (see Movie S2).(C) Extended focus fluorescence intensity image slices showing a section of the nucleus from a time series analysis of a HeLa cell containing Ddx4YFP droplets undergoing osmotic shock. Images are shown at 2-min intervals. Axis labels, data colors, and scale as in (B). See Movies S3 and S4.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4352761&req=5

fig2: Ddx4YFP Organelles Are Internally Mobile and Respond Rapidly to Changes in Environmental Temperature and Tonicity(A) Fluorescence recovery after photobleaching (FRAP) of a Ddx4YFP organelle in a live HeLa cell at 37°C. Sample bleaching is indicated with a gray bar. 50% of the fluorescence signal is recovered within approximately 2.5 s post-bleach, corresponding to a diffusion coefficient of 3 ± 1 × 10−13 m2 s−1.(B) Cold shock induces condensation of sub-nuclear Ddx4YFP droplets at low expression levels. Extended focus fluorescence intensity images showing the nucleus from a time series analysis of a HeLa cell expressing Ddx4YFP undergoing cold shock. Images are shown at 2-min intervals. Prior to cold shock treatment, Ddx4YFP had not reached the critical concentration for phase separation at 37°C and was diffuse in the nucleoplasm (first two frames). Rapid exchange of growth media at 37°C for media cooled on ice (time = 0) induced small Ddx4YFP droplets to condense rapidly within the nucleus (purple line, number of droplets; blue line, total volume of droplets). Following cold shock, the number of Ddx4YFP droplets decreased through a combination of coalescence and dissolution as the temperature rose. Scale bar, 5 μm (see Movie S2).(C) Extended focus fluorescence intensity image slices showing a section of the nucleus from a time series analysis of a HeLa cell containing Ddx4YFP droplets undergoing osmotic shock. Images are shown at 2-min intervals. Axis labels, data colors, and scale as in (B). See Movies S3 and S4.
Mentions: The internal order within the organelles was assessed using fluorescence recovery after photobleaching (FRAP) measurements. The half time to recovery of the fluorescence signal of a photo-bleached body of diameter 1.5 μm took approximately 2.5 s at 37°C (Figure 2A), corresponding to an approximate diffusion coefficient of 3 ± 1 × 10−13 m2 s−1, a value two orders of magnitude lower than that measured for free globular proteins of a similar size as determined by both FRAP and NMR (Figure S5B). These self-diffusion rates are consistent with those of other non-membrane organelles, such as nuclear speckles and nucleoli (Phair and Misteli 2000). While the observed diffusion within the droplets is substantially slower than the motion of free protein, the interior is nevertheless highly mobile, consistent with weak interactions between Ddx4 proteins within the droplet.

Bottom Line: These bodies are stabilized by patterned electrostatic interactions that are highly sensitive to temperature, ionic strength, arginine methylation, and splicing.Moreover, the bodies provide an alternative solvent environment that can concentrate single-stranded DNA but largely exclude double-stranded DNA.We propose that phase separation of disordered proteins containing weakly interacting blocks is a general mechanism for forming regulated, membraneless organelles.

View Article: PubMed Central - PubMed

Affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON M5G 1X5, Canada; Physical and Theoretical Chemistry Laboratory, University of Oxford, Oxford OX1 3QZ, UK.

Show MeSH
Related in: MedlinePlus