Polar Positioning of Phase-Separated Liquid Compartments in Cells Regulated by an mRNA Competition Mechanism.

P granules are non-membrane-bound RNA-protein compartments that are involved in germline development in C. elegans. They are liquids that condense at one end of the embryo by localized phase separation, driven by gradients of polarity proteins such as the mRNA-binding protein MEX-5. To probe how polarity proteins regulate phase separation, we combined biochemistry and theoretical modeling. We reconstitute P granule-like droplets in vitro using a single protein PGL-3. By combining in vitro reconstitution with measurements of intracellular concentrations, we show that competition between PGL-3 and MEX-5 for mRNA can regulate the formation of PGL-3 droplets. Using theory, we show that, in a MEX-5 gradient, this mRNA competition mechanism can drive a gradient of P granule assembly with similar spatial and temporal characteristics to P granule assembly in vivo. We conclude that gradients of polarity proteins can position RNP granules during development by using RNA competition to regulate local phase separation.

Membrane prewetting by condensates promotes tight-junction belt formation.

Biomolecular condensates enable cell compartmentalization by acting as membraneless organelles1. How cells control the interactions of condensates with other cellular structures such as membranes to drive morphological transitions remains poorly understood. We discovered that formation of a tight-junction belt, which is essential for sealing epithelial tissues, is driven by a wetting phenomenon that promotes the growth of a condensed ZO-1 layer2 around the apical membrane interface. Using temporal proximity proteomics in combination with imaging and thermodynamic theory, we found that the polarity protein PATJ mediates a transition of ZO-1 into a condensed surface layer that elongates around the apical interface. In line with the experimental observations, our theory of condensate growth shows that the speed of elongation depends on the binding affinity of ZO-1 to the apical interface and is constant. Here, using PATJ mutations, we show that ZO-1 interface binding is necessary and sufficient for tight-junction belt formation. Our results demonstrate how cells exploit the collective biophysical properties of protein condensates at membrane interfaces to shape mesoscale structures.

A condensate dynamic instability orchestrates actomyosin cortex activation.

A key event at the onset of development is the activation of a contractile actomyosin cortex during the oocyte-to-embryo transition1-3. Here we report on the discovery that, in Caenorhabditis elegans oocytes, actomyosin cortex activation is supported by the emergence of thousands of short-lived protein condensates rich in F-actin, N-WASP and the ARP2/3 complex4-8 that form an active micro-emulsion. A phase portrait analysis of the dynamics of individual cortical condensates reveals that condensates initially grow and then transition to disassembly before dissolving completely. We find that, in contrast to condensate growth through diffusion9, the growth dynamics of cortical condensates are chemically driven. Notably, the associated chemical reactions obey mass action kinetics that govern both composition and size. We suggest that the resultant condensate dynamic instability10 suppresses coarsening of the active micro-emulsion11, ensures reaction kinetics that are independent of condensate size and prevents runaway F-actin nucleation during the formation of the first cortical actin meshwork.

Morphogen gradient scaling by recycling of intracellular Dpp.

Morphogen gradients are fundamental to establish morphological patterns in developing tissues1. During development, gradients scale to remain proportional to the size of growing organs2,3. Scaling is a universal gear that adjusts patterns to size in living organisms3-8, but its mechanisms remain unclear. Here, focusing on the Decapentaplegic (Dpp) gradient in the Drosophila wing disc, we uncover a cell biological basis behind scaling. From small to large discs, scaling of the Dpp gradient is achieved by increasing the contribution of the internalized Dpp molecules to Dpp transport: to expand the gradient, endocytosed molecules are re-exocytosed to spread extracellularly. To regulate the contribution of endocytosed Dpp to the spreading extracellular pool during tissue growth, it is the Dpp binding rates that are progressively modulated by the extracellular factor Pentagone, which drives scaling. Thus, for some morphogens, evolution may act on endocytic trafficking to regulate the range of the gradient and its scaling, which could allow the adaptation of shape and pattern to different sizes of organs in different species.

Liquid-liquid phase separation in biology.

Cells organize many of their biochemical reactions in non-membrane compartments. Recent evidence has shown that many of these compartments are liquids that form by phase separation from the cytoplasm. Here we discuss the basic physical concepts necessary to understand the consequences of liquid-like states for biological functions.

Cortical dynein controls microtubule dynamics to generate pulling forces that position microtubule asters.

Dynein at the cortex contributes to microtubule-based positioning processes such as spindle positioning during embryonic cell division and centrosome positioning during fibroblast migration. To investigate how cortical dynein interacts with microtubule ends to generate force and how this functional association impacts positioning, we have reconstituted the 'cortical' interaction between dynein and dynamic microtubule ends in an in vitro system using microfabricated barriers. We show that barrier-attached dynein captures microtubule ends, inhibits growth, and triggers microtubule catastrophes, thereby controlling microtubule length. The subsequent interaction with shrinking microtubule ends generates pulling forces up to several pN. By combining experiments in microchambers with a theoretical description of aster mechanics, we show that dynein-mediated pulling forces lead to the reliable centering of microtubule asters in simple confining geometries. Our results demonstrate the intrinsic ability of cortical microtubule-dynein interactions to regulate microtubule dynamics and drive positioning processes in living cells.

A cytokinetic ring-driven cell rotation achieves Hertwig's rule in early development.

Hertwig's rule states that cells divide along their longest axis, usually driven by forces acting on the mitotic spindle. Here, we show that in contrast to this rule, microtubule-based pulling forces in early Caenorhabditis elegans embryos align the spindle with the short axis of the cell. We combine theory with experiments to reveal that in order to correct this misalignment, inward forces generated by the constricting cytokinetic ring rotate the entire cell until the spindle is aligned with the cell's long axis. Experiments with slightly compressed mouse zygotes indicate that this cytokinetic ring-driven mechanism of ensuring Hertwig's rule is general for cells capable of rotating inside a confining shell, a scenario that applies to early cell divisions of many systems.

Cell flow reorients the axis of planar polarity in the wing epithelium of Drosophila.

Planar cell polarity (PCP) proteins form polarized cortical domains that govern polarity of external structures such as hairs and cilia in both vertebrate and invertebrate epithelia. The mechanisms that globally orient planar polarity are not understood, and are investigated here in the Drosophila wing using a combination of experiment and theory. Planar polarity arises during growth and PCP domains are initially oriented toward the well-characterized organizer regions that control growth and patterning. At pupal stages, the wing hinge contracts, subjecting wing-blade epithelial cells to anisotropic tension in the proximal-distal axis. This results in precise patterns of oriented cell elongation, cell rearrangement and cell division that elongate the blade proximo-distally and realign planar polarity with the proximal-distal axis. Mutation of the atypical Cadherin Dachsous perturbs the global polarity pattern by altering epithelial dynamics. This mechanism utilizes the cellular movements that sculpt tissues to align planar polarity with tissue shape.

Co-condensation of proteins with single- and double-stranded DNA.

SignificanceBiomolecular condensates are intracellular organelles that are not bounded by membranes and often show liquid-like, dynamic material properties. They typically contain various types of proteins and nucleic acids. How the interaction of proteins and nucleic acids finally results in dynamic condensates is not fully understood. Here we use optical tweezers and fluorescence microscopy to study how the prototypical prion-like protein Fused-in-Sarcoma (FUS) condenses with individual molecules of single- and double-stranded DNA. We find that FUS adsorbs on DNA in a monolayer and hence generates an effectively sticky FUS-DNA polymer that collapses and finally forms a dynamic, reversible FUS-DNA co-condensate. We speculate that protein monolayer-based protein-nucleic acid co-condensation is a general mechanism for forming intracellular membraneless organelles.

Droplet Differentiation by a Chemical Switch.

A fundamental question about biomolecular condensates is how distinct condensates can emerge from the interplay of different components. Here we present a minimal model of droplet differentiation where phase separated droplets demix into two types with different chemical modifications triggered by enzymatic reactions. We use numerical solutions to Cahn-Hilliard equations with chemical reactions and an effective droplet model to reveal the switchlike behavior. Our work shows how condensate identities in cells could result from competing enzymatic actions.