Optimized PAR-2 RING dimerization mediates cooperative and selective membrane binding for robust cell polarity.

Cell polarity networks are defined by quantitative features of their constituent feedback circuits, which must be tuned to enable robust and stable polarization, while also ensuring that networks remain responsive to dynamically changing cellular states and/or spatial cues during development. Using the PAR polarity network as a model, we demonstrate that these features are enabled by the dimerization of the polarity protein PAR-2 via its N-terminal RING domain. Combining theory and experiment, we show that dimer affinity is optimized to achieve dynamic, selective, and cooperative binding of PAR-2 to the plasma membrane during polarization. Reducing dimerization compromises positive feedback and robustness of polarization. Conversely, enhanced dimerization renders the network less responsive due to kinetic trapping of PAR-2 on internal membranes and reduced sensitivity of PAR-2 to the anterior polarity kinase, aPKC/PKC-3. Thus, our data reveal a key role for a dynamically oligomeric RING domain in optimizing interaction affinities to support a robust and responsive cell polarity network, and highlight how optimization of oligomerization kinetics can serve as a strategy for dynamic and cooperative intracellular targeting.

Cleavage furrow-directed cortical flows bias PAR polarization pathways to link cell polarity to cell division.

During development, the conserved PAR polarity network is continuously redeployed, requiring that it adapt to changing cellular contexts and environmental cues. In the early C. elegans embryo, polarity shifts from being a cell-autonomous process in the zygote to one that must be coordinated between neighbors as the embryo becomes multicellular. Here, we sought to explore how the PAR network adapts to this shift in the highly tractable C. elegans germline P lineage. We find that although P lineage blastomeres exhibit a distinct pattern of polarity emergence compared with the zygote, the underlying mechanochemical processes that drive polarity are largely conserved. However, changes in the symmetry-breaking cues of P lineage blastomeres ensure coordination of their polarity axis with neighboring cells. Specifically, we show that furrow-directed cortical flows associated with cytokinesis of the zygote induce symmetry breaking in the germline blastomere P1 by transporting PAR-3 into the nascent cell contact. This pool of PAR-3 then biases downstream PAR polarization pathways to establish the polarity axis of P1 with respect to the position of its anterior sister, AB. Thus, our data suggest that cytokinesis itself induces symmetry breaking through the advection of polarity proteins by furrow-directed flows. By directly linking cell polarity to cell division, furrow-directed cortical flows could be a general mechanism to ensure proper organization of cell polarity within actively dividing systems.

Design principles for selective polarization of PAR proteins by cortical flows.

Clustering of membrane-associated molecules is thought to promote interactions with the actomyosin cortex, enabling size-dependent transport by actin flows. Consistent with this model, in the Caenorhabditis elegans zygote, efficient anterior segregation of the polarity protein PAR-3 requires oligomerization. However, through direct assessment of local coupling between motion of PAR proteins and the underlying cortex, we find no links between PAR-3 oligomer size and the degree of coupling. Indeed, both anterior and posterior PAR proteins experience similar advection velocities, at least over short distances. Consequently, differential cortex engagement cannot account for selectivity of PAR protein segregation by cortical flows. Combining experiment and theory, we demonstrate that a key determinant of differential segregation of PAR proteins by cortical flow is the stability of membrane association, which is enhanced by clustering and enables transport across cellular length scales. Thus, modulation of membrane binding dynamics allows cells to achieve selective transport by cortical flows despite widespread coupling between membrane-associated molecules and the cell cortex.

An analog sensitive allele permits rapid and reversible chemical inhibition of PKC-3 activity in C. elegans.

Engineered analog sensitive kinases provide a highly effective method for acute, controllable, and highly selective inhibition of kinase activity. Here we describe the design and characterization of an analog sensitive allele of the polarity kinase, PKC-3. This allele supports normal function as measured by its ability to exclude PAR-2 from the anterior membrane of zygotes, and is rapidly and reversibly inhibited in a dose-dependent manner by the ATP analog 1NA-PP1. This allele provides a new tool to explore the role of PKC-3 in diverse contexts within C. elegans , particularly those in which acute and reversible control of PKC-3 kinase activity may be desired.

SAIBR: a simple, platform-independent method for spectral autofluorescence correction.

Biological systems are increasingly viewed through a quantitative lens that demands accurate measures of gene expression and local protein concentrations. CRISPR/Cas9 gene tagging has enabled increased use of fluorescence to monitor proteins at or near endogenous levels under native regulatory control. However, owing to typically lower expression levels, experiments using endogenously tagged genes run into limits imposed by autofluorescence (AF). AF is often a particular challenge in wavelengths occupied by commonly used fluorescent proteins (GFP, mNeonGreen). Stimulated by our work in C. elegans, we describe and validate Spectral Autofluorescence Image Correction By Regression (SAIBR), a simple platform-independent protocol and FIJI plug-in to correct for autofluorescence using standard filter sets and illumination conditions. Validated for use in C. elegans embryos, starfish oocytes and fission yeast, SAIBR is ideal for samples with a single dominant AF source; it achieves accurate quantitation of fluorophore signal, and enables reliable detection and quantification of even weakly expressed proteins. Thus, SAIBR provides a highly accessible low-barrier way to incorporate AF correction as standard for researchers working on a broad variety of cell and developmental systems.

The first steps in the life of a worm: Themes and variations in asymmetric division in C. elegans and other nematodes.

Starting with Boveri in the 1870s, microscopic investigation of early embryogenesis in a broad swath of nematode species revealed the central role of asymmetric cell division in embryonic axis specification, blastomere positioning, and cell fate specification. Notably, across the class Chromadorea, a conserved theme emerges-asymmetry is first established in the zygote and specifies its asymmetric division, giving rise to an anterior somatic daughter cell and a posterior germline daughter cell. Beginning in the 1980s, the emergence of Caenorhabditis elegans as a model organism saw the advent of genetic tools that enabled rapid progress in our understanding of the molecular mechanisms underlying asymmetric division, in many cases defining key paradigms that turn out to regulate asymmetric division in a wide range of systems. Yet, the consequence of this focus on C. elegans came at the expense of exploring the extant diversity of developmental variation exhibited across nematode species. Given the resurgent interest in evolutionary studies facilitated in part by new tools, here we revisit the diversity in this asymmetric first division, juxtaposing molecular insight into mechanisms of symmetry-breaking, spindle positioning and fate specification, with a consideration of plasticity and variability within and between species. In the process, we hope to highlight questions of evolutionary forces and molecular variation that may have shaped the extant diversity of developmental mechanisms observed across Nematoda.

Intracellular morphogens: Specifying patterns at the subcellular scale.

The notion that graded distributions of signals underlie the spatial organization of biological systems has long been a central pillar in the fields of cell and developmental biology. During morphogenesis, morphogens spread across tissues to guide development of the embryo. Similarly, a variety of dynamic gradients and pattern-forming networks have been discovered that shape subcellular organization. Here we discuss the principles of intracellular pattern formation by these intracellular morphogens and relate them to conceptually similar processes operating at the tissue scale. We will specifically review mechanisms for generating cellular asymmetry and consider how intracellular patterning networks are controlled and adapt to cellular geometry. Finally, we assess the general concept of intracellular gradients as a mechanism for positional control in light of current data, highlighting how the simple readout of fixed concentration thresholds fails to fully capture the complexity of spatial patterning processes occurring inside cells.

Patterning and polarization of cells by intracellular flows.

Beginning with Turing's seminal work [1], decades of research have demonstrated the fundamental ability of biochemical networks to generate and sustain the formation of patterns. However, it is increasingly appreciated that biochemical networks also both shape and are shaped by physical and mechanical processes [2, 3, 4]. One such process is fluid flow. In many respects, the cytoplasm, membrane and actin cortex all function as fluids, and as they flow, they drive bulk transport of molecules throughout the cell. By coupling biochemical activity to long-range molecular transport, flows can shape the distributions of molecules in space. Here, we review the various types of flows that exist in cells, with the aim of highlighting recent advances in our understanding of how flows are generated and how they contribute to intracellular patterning processes, such as the establishment of cell polarity.

A cell size threshold limits cell polarity and asymmetric division potential.

Reaction-diffusion networks underlie pattern formation in a range of biological contexts, from morphogenesis of organisms to the polarisation of individual cells. One requirement for such molecular networks is that output patterns be scaled to system size. At the same time, kinetic properties of constituent molecules constrain the ability of networks to adapt to size changes. Here we explore these constraints and the consequences thereof within the conserved PAR cell polarity network. Using the stem cell-like germ lineage of the C. elegans embryo as a model, we find that the behaviour of PAR proteins fails to scale with cell size. Theoretical analysis demonstrates that this lack of scaling results in a size threshold below which polarity is destabilized, yielding an unpolarized system. In empirically-constrained models, this threshold occurs near the size at which germ lineage cells normally switch between asymmetric and symmetric modes of division. Consistent with cell size limiting polarity and division asymmetry, genetic or physical reduction in germ lineage cell size is sufficient to trigger loss of polarity in normally polarizing cells at predicted size thresholds. Physical limits of polarity networks may be one mechanism by which cells read out geometrical features to inform cell fate decisions.

Switching states: dynamic remodelling of polarity complexes as a toolkit for cell polarization.

Polarity is defined by the segregation of cellular components along a defined axis. To polarize robustly, cells must be able to break symmetry and subsequently amplify these nascent asymmetries. Finally, asymmetric localization of signaling molecules must be translated into functional regulation of downstream effector pathways. Central to these behaviors are a diverse set of cell polarity networks. Within these networks, molecules exhibit varied behaviors, dynamically switching among different complexes and states, active versus inactive, bound versus unbound, immobile versus diffusive. This ability to switch dynamically between states is intimately connected to the ability of molecules to generate asymmetric patterns within cells. Focusing primarily on polarity pathways governed by the conserved PAR proteins, we discuss strategies enabled by these dynamic behaviors that are used by cells to polarize. We highlight not only how switching between states is linked to the ability of polarity proteins to localize asymmetrically, but also how cells take advantage of 'state switching' to regulate polarity in time and space.

Guiding self-organized pattern formation in cell polarity establishment.

Spontaneous pattern formation in Turing systems relies on feedback. Patterns in cells and tissues however often do not form spontaneously, but are under control of upstream pathways that provide molecular guiding cues. The relationship between guiding cues and feedback in controlled biological pattern formation remains unclear. We explored this relationship during cell polarity establishment in the one-cell-stage C. elegans embryo. We quantified the strength of two feedback systems that operate during polarity establishment, feedback between polarity proteins and the actomyosin cortex, and mutual antagonism amongst polarity proteins. We characterized how these feedback systems are modulated by guiding cues from the centrosome. By coupling a mass-conserved Turing-like reaction-diffusion system for polarity proteins to an active gel description of the actomyosin cortex, we reveal a transition point beyond which feedback ensures self-organized polarization even when cues are removed. Notably, the baton is passed from a guide-dominated to a feedback-dominated regime significantly beyond this transition point, which ensures robustness. Together, this reveals a general criterion for controlling biological pattern forming systems: feedback remains subcritical to avoid unstable behaviour, and molecular guiding cues drive the system beyond a transition point for pattern formation.

Regulated Activation of the PAR Polarity Network Ensures a Timely and Specific Response to Spatial Cues.

How do cells polarize at the correct time and in response to the correct cues? In the C. elegans zygote, the timing and geometry of polarization rely on a single dominant cue-the sperm centrosome-that matures at the end of meiosis and specifies the nascent posterior. Polarization requires that the conserved PAR proteins, which specify polarity in the zygote, be poised to respond to the centrosome. Yet, how and when PAR proteins achieve this unpolarized, but responsive, state is unknown. We show that oocyte maturation initiates a fertilization-independent PAR activation program. PAR proteins are initially not competent to polarize but gradually acquire this ability following oocyte maturation. Surprisingly, this program allows symmetry breaking even in unfertilized oocytes lacking centrosomes. Thus, if PAR proteins can respond to multiple polarizing cues, how is specificity for the centrosome achieved? Specificity is enforced by Polo-like and Aurora kinases (PLK-1 and AIR-1 in C. elegans), which impose a delay in the activation of the PAR network so that it coincides with maturation of the centrosome cue. This delay suppresses polarization by non-centrosomal cues, which can otherwise trigger premature polarization and multiple or reversed polarity domains. Taken together, these findings identify a regulatory program that enforces proper polarization by synchronizing PAR network activation with cell cycle progression, thereby ensuring that PAR proteins respond specifically to the correct cue. Temporal control of polarity network activity is likely to be a common strategy to ensure robust, dynamic, and specific polarization in response to developmentally deployed cues.

Anterior-enriched filopodia create the appearance of asymmetric membrane microdomains in polarizing C. elegans zygotes.

The association of molecules within membrane microdomains is critical for the intracellular organization of cells. During polarization of the C. elegans zygote, both polarity proteins and actomyosin regulators associate within dynamic membrane-associated foci. Recently, a novel class of asymmetric membrane-associated structures was described that appeared to be enriched in phosphatidylinositol 4,5-bisphosphate (PIP2), suggesting that PIP2 domains could constitute signaling hubs to promote cell polarization and actin nucleation. Here, we probe the nature of these domains using a variety of membrane- and actin cortex-associated probes. These data demonstrate that these domains are filopodia, which are stimulated transiently during polarity establishment and accumulate in the zygote anterior. The resulting membrane protrusions create local membrane topology that quantitatively accounts for observed local increases in the fluorescence signal of membrane-associated molecules, suggesting molecules are not selectively enriched in these domains relative to bulk membrane and that the PIP2 pool as revealed by PHPLCδ1 simply reflects plasma membrane localization. Given the ubiquity of 3D membrane structures in cells, including filopodia, microvilli and membrane folds, similar caveats are likely to apply to analysis of membrane-associated molecules in a broad range of systems.

aPKC Cycles between Functionally Distinct PAR Protein Assemblies to Drive Cell Polarity.

The conserved polarity effector proteins PAR-3, PAR-6, CDC-42, and atypical protein kinase C (aPKC) form a core unit of the PAR protein network, which plays a central role in polarizing a broad range of animal cell types. To functionally polarize cells, these proteins must activate aPKC within a spatially defined membrane domain on one side of the cell in response to symmetry-breaking cues. Using the Caenorhabditis elegans zygote as a model, we find that the localization and activation of aPKC involve distinct, specialized aPKC-containing assemblies: a PAR-3-dependent assembly that responds to polarity cues and promotes efficient segregation of aPKC toward the anterior but holds aPKC in an inactive state, and a CDC-42-dependent assembly in which aPKC is active but poorly segregated. Cycling of aPKC between these distinct functional assemblies, which appears to depend on aPKC activity, effectively links cue-sensing and effector roles within the PAR network to ensure robust establishment of polarity.

Intracellular Scaling Mechanisms.

Organelle function is often directly related to organelle size. However, it is not necessarily absolute size but the organelle-to-cell-size ratio that is critical. Larger cells generally have increased metabolic demands, must segregate DNA over larger distances, and require larger cytokinetic rings to divide. Thus, organelles often must scale to the size of the cell. The need for scaling is particularly acute during early development during which cell size can change rapidly. Here, we highlight scaling mechanisms for cellular structures as diverse as centrosomes, nuclei, and the mitotic spindle, and distinguish them from more general mechanisms of size control. In some cases, scaling is a consequence of the underlying mechanism of organelle size control. In others, size-control mechanisms are not obviously related to cell size, implying that scaling results indirectly from cell-size-dependent regulation of size-control mechanisms.

PAR polarity: from complexity to design principles.

The par-titioning-defective or PAR proteins comprise the core of an essential cell polarity network that underlies polarization in a wide variety of cell types and developmental contexts. The output of this network in nearly every case is the establishment of opposing and complementary membrane domains that define a cell׳s polarity axis. Yet, behind this simple pattern is a complex system of interactions, regulation and dynamic behaviors. How these various parts combine to generate polarized patterns of protein localization in cells is only beginning to become clear. This review, part of the Special Issue on Cell Polarity, aims to highlight several emerging themes and design principles that underlie the process of cell polarization by components of the PAR network.

Cell polarity: mechanochemical patterning.

Nearly every cell type exhibits some form of polarity, yet the molecular mechanisms vary widely. Here we examine what we term 'chemical systems' where cell polarization arises through biochemical interactions in signaling pathways, 'mechanical systems' where cells polarize due to forces, stresses and transport, and 'mechanochemical systems' where polarization results from interplay between mechanics and chemical signaling. To reveal potentially unifying principles, we discuss mathematical conceptualizations of several prototypical examples. We suggest that the concept of local activation and global inhibition - originally developed to explain spatial patterning in reaction-diffusion systems - provides a framework for understanding many cases of cell polarity. Importantly, we find that the core ingredients in this framework - symmetry breaking, self-amplifying feedback, and long-range inhibition - involve processes that can be chemical, mechanical, or even mechanochemical in nature.

Organelle growth control through limiting pools of cytoplasmic components.

The critical importance of controlling the size and number of intracellular organelles has led to a variety of mechanisms for regulating the formation and growth of cellular structures. In this review, we explore a class of mechanisms for organelle growth control that rely primarily on the cytoplasm as a 'limiting pool' of available material. These mechanisms are based on the idea that, as organelles grow, they incorporate subunits from the cytoplasm. If this subunit pool is limited, organelle growth will lead to depletion of subunits from the cytoplasm. Free subunit concentration therefore provides a measure of the number of incorporated subunits and thus the current size of the organelle. Because organelle growth rates are typically a function of subunit concentration, cytoplasmic depletion links organelle size, free subunit concentration, and growth rates, ensuring that as the organelle grows, its rate of growth slows. Thus, a limiting cytoplasmic pool provides a powerful mechanism for size-dependent regulation of growth without recourse to active mechanisms to measure size or modulate growth rates. Variations of this general idea allow not only for size control, but also cell-size-dependent scaling of cellular structures, coordination of growth between similar structures within a cell, and the enforcement of singularity in structure formation, when only a single copy of a structure is desired. Here, we review several examples of such mechanisms in cellular processes as diverse as centriole duplication, centrosome and nuclear size control, cell polarity, and growth of flagella.

Polarization of PAR proteins by advective triggering of a pattern-forming system.

In the Caenorhabditis elegans zygote, a conserved network of partitioning-defective (PAR) polarity proteins segregates into an anterior and a posterior domain, facilitated by flows of the cortical actomyosin meshwork. The physical mechanisms by which stable asymmetric PAR distributions arise from transient cortical flows remain unclear. We present evidence that PAR polarity arises from coupling of advective transport by the flowing cell cortex to a multistable PAR reaction-diffusion system. By inducing transient PAR segregation, advection serves as a mechanical trigger for the formation of a PAR pattern within an otherwise stably unpolarized system. We suggest that passive advective transport in an active and flowing material may be a general mechanism for mechanochemical pattern formation in developmental systems.

PAR proteins diffuse freely across the anterior-posterior boundary in polarized C. elegans embryos.

Polarization of cells by PAR proteins requires the segregation of antagonistic sets of proteins into two mutually exclusive membrane-associated domains. Understanding how nanometer scale interactions between individual PAR proteins allow spatial organization across cellular length scales requires determining the kinetic properties of PAR proteins and how they are modified in space. We find that PAR-2 and PAR-6, which localize to opposing PAR domains, undergo exchange between well mixed cytoplasmic populations and laterally diffusing membrane-associated states. Domain maintenance does not involve diffusion barriers, lateral sorting, or active transport. Rather, both PAR proteins are free to diffuse between domains, giving rise to a continuous boundary flux because of lateral diffusion of molecules down the concentration gradients that exist across the embryo. Our results suggest that the equalizing effects of lateral diffusion are countered by actin-independent differences in the effective membrane affinities of PAR proteins between the two domains, which likely depend on the ability of each PAR species to locally modulate the membrane affinity of opposing PAR species within its domain. We propose that the stably polarized embryo reflects a dynamic steady state in which molecules undergo continuous diffusion between regions of net association and dissociation.