WNK kinases sense molecular crowding and rescue cell volume via phase separation

Boyd‐Shiwarski, Cary R.; Shiwarski, Daniel J.; Griffiths, Shawn E.; Beacham, Rebecca T.; Norrell, Logan; Morrison, Daryl E.; Wang, Jun; Mann, Jacob R.; Tennant, William; Anderson, Eric N.; Franks, Jonathan; Calderon, Michael; Connolly, Kelly; Cheema, Muhammad Umar; Weaver, Claire J.; Nkashama, Lubika J.; Weckerly, Claire C.; Querry, Katherine E.; Pandey, Udai Bhan; Donnelly, Christopher J.; Sun, Dandan; Rodan, Aylin R.; Subramanya, Arohan R.

doi:10.1016/j.cell.2022.09.042

Cited by 99 publications

(106 citation statements)

References 91 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…While we do not have precise structural data, we can hypothesize that FRET increases by, for example, assembling linear oligomers to form fibers, or the disordered domain starting from amino acids 45, 65, or 83, 24,31,45 generates fuzzy complexes leading to larger clusters. Crowding-triggered protein self-association in yeast and mammalian cells has been previously described for condensate-forming proteins, 10–12 suggesting that cluster formation may be especially sensitive to crowding, which may happen here as well.…”

Section: Discussionsupporting

confidence: 55%

Self-association of a nucleoid-binding protein increases with macromolecular crowding inEscherichia coli

Pittas

Boersma

2023

Preprint

View full text Add to dashboard Cite

Many proteins self-associate to achieve function. Macromolecular crowding enhances protein self-assembly in buffer experiments with added crowders, and crowding could therefore regulate protein function and organization in cells. In eukaryotic cells, protein condensation has been shown to increase with crowding. However, it is unclear what the effect of crowding is on native protein self-assembly in the highly crowded Escherichia coli cell. To determine the role of crowding in the self-assembly of a native protein, we study here the nucleoid-binding H-NS in E. coli and alter macromolecular crowding using a set of perturbations. We followed H-NS self-assembly using a FRET-based method for determining intermolecular interactions with a single genetic intervention. In dilute cell lysate, we see that H-NS self-assembly increases with salts, macromolecular crowding, and its own concentration. In E. coli, the oligomerization increases with crowding. We see that the response of H-NS oligomerization to a sudden crowding change is not immediate but requires time to adapt. Our findings implicate that in-cell crowding affects intracellular organization by promoting self-assembly.

show abstract

Section: Discussionsupporting

confidence: 55%

Self-association of a nucleoid-binding protein increases with macromolecular crowding inEscherichia coli

Pittas

Boersma

2023

Preprint

View full text Add to dashboard Cite

show abstract

“…This seemingly contradictory result seems to explain the mechanism of positive and negative regulation of YY1 better than previous theories. Phase separation is a state of dynamic equilibrium [53] and "moderate" LLPS has been proved important for many biological function, for example, WNK1 undergoes hypertonic pressure-induced dynamic phase separation to restore cell volume [54]; HSPB1 regulates the reversible TDP-43 phase separation from liquid to gel/solid [55]. In our study, broadly defined YY1 underwent LLPS, promoting the METTL3 expression and the proliferation of AML cells.…”

Section: Discussionmentioning

confidence: 59%

HDAC1/3-dependent moderate liquid–liquid phase separation of YY1 promotes METTL3 expression and AML cell proliferation

Meng

Xia

et al. 2022

Cell Death Dis

View full text Add to dashboard Cite

Methyltransferase-like protein 3 (METTL3) plays critical roles in acute myeloid leukemia (AML) progression, however, the mechanism of abnormal overexpression of METTL3 in AML remain elusive. In the current study, we uncovered that Yin Yang 1 (YY1) binds to the promoter region of METTL3 as a transcription factor and promotes its expression, which in turn enhances the proliferation of AML cells. Mechanistically, YY1 binds to HDAC1/3 and regulates METTL3 expression in a moderate liquid-liquid phase separation (LLPS) manner. After mutation of the HDAC-binding site of YY1 or HDAC inhibitor (HDACi) treatment, YY1 was separated from HDAC1/3, which resulted in an excessive LLPS state, thereby inhibiting the expression of METTL3 and the proliferation of AML cells. In conclusion, our study clarified the regulatory mechanism of the abnormal expression of METTL3 in AML, revealed the precise “Yin-Yang” regulatory mechanism of YY1 from the perspective of LLPS degree, and provided new ideas for the precise diagnosis and treatment of AML.

show abstract

“…This may explain recent findings that the WNK1 kinase responds to osmotic stress in cells by condensing into a functional biomolecular condensate. [15] We note here that our simulations probe the situation in which crowding polymers only exert steric and entropic forces on the IDPs in a concentration range similar to the cellular cytoplasm. The densest system we simulated had a crowder concentration of 120 mM, which is comparable to the estimates of 100 – 450 mg/ml for cellular cytoplasm.…”

Section: Discussionmentioning

confidence: 99%

“…Recent work has shown that some kinases respond to molecular crowding in the cytoplasm by condensing into a functional BC that facilitates a signalling pathway to regulate cell volume. [15] Crowding is also known to influence protein folding. [16] Aberrant phase transitions of IDPs are implicated in neurodegenerative diseases, cancer, prion protein diseases, and, more speculatively in ageing.…”

Section: Introductionmentioning

confidence: 99%

Macromolecular crowding is surprisingly unable to deform the structure of a model biomolecular condensate

Shillcock

Thomas

Ipsen

et al. 2022

Preprint

View full text Add to dashboard Cite

The crowded interior of a living cell makes experiments on simpler systems attractive. Although these reveal interesting phenomena, their biological relevance can be questionable. A topical example is the phase separation of intrinsically disordered proteins into biomolecular condensates, which is proposed to underlie the membraneless compartmentalisation of many cellular functions. How a cell reliably controls biochemical reactions in compartments open to the compositionally varying cytoplasm is an important question for understanding cellular homeostasis. Computer simulations are often used to study the phase behaviour of model biomolecular condensates, but the number of relevant parameters explodes as the number of protein components increases. It is unfeasible to exhaustively simulate such models for all parameter combinations, although interesting phenomena are almost certainly hidden in the jungle of their high dimensional parameter space. Here we have studied the phase behaviour of a model biomolecular condensate in the presence of a polymeric crowding agent. We used a novel compute framework to execute dozens of simultaneous simulations spanning the protein crowder concentration space. We then combined the results into a graphical representation for human interpretation, which provided an efficient way to search the high-dimensional parameter space. We found that steric repulsion from the crowder drives a near-critical system across the phase boundary, but the molecular arrangement within the resulting biomolecular condensate is rather insensitive to the crowder concentration and molecular weight. We propose that a cell may use the local cytoplasmic concentration to assist formation of biomolecular condensates, while relying on the dense phase reliably providing a stable, structured, fluid milieu for cellular biochemistry despite being open to its changing environment.

show abstract

WNK kinases sense molecular crowding and rescue cell volume via phase separation

Cited by 99 publications

References 91 publications

Self-association of a nucleoid-binding protein increases with macromolecular crowding inEscherichia coli

Self-association of a nucleoid-binding protein increases with macromolecular crowding inEscherichia coli

HDAC1/3-dependent moderate liquid–liquid phase separation of YY1 promotes METTL3 expression and AML cell proliferation

Macromolecular crowding is surprisingly unable to deform the structure of a model biomolecular condensate

Contact Info

Product

Resources

About