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.

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.

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.

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.

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.

Rapid beta-lactam-induced lysis requires successful assembly of the cell division machinery.

Beta-lactam antibiotics inhibit penicillin binding proteins (PBPs) involved in peptidoglycan synthesis. Although inhibition of peptidoglycan biosynthesis is generally thought to induce cell lysis, the pattern and mechanism of cell lysis can vary substantially. Beta-lactams that inhibit FtsI, the only division specific PBP, block cell division and result in growth as filaments. These filaments ultimately lyse through a poorly understood mechanism. Here we find that one such beta-lactam, cephalexin, can, under certain conditions, lead instead to rapid lysis at nascent division sites through a process that requires the complete and ordered assembly of the divisome, the essential machinery involved in cell division. We propose that this assembly process (in which the localization of cell wall hydrolases depends on properly targeted FtsN, which in turn depends on the presence of FtsI) ensures that the biosynthetic machinery to form new septa is in place before the machinery to degrade septated daughter cells is enabled. Beta-lactams that target FtsI subvert this mechanism by inhibiting FtsI without perturbing the normal assembly of the cell division machinery and the consequent activation of cell wall hydrolases. One seemingly paradoxical implication of our results is that beta-lactam therapy may be improved by promoting active cell division.

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.

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.

FRAP analysis of membrane-associated proteins: lateral diffusion and membrane-cytoplasmic exchange.

Obtaining quantitative kinetic parameters from fluorescence recovery after photobleaching (FRAP) experiments generally requires a theoretical analysis of protein mobility and appropriate solutions for FRAP recovery derived for a given geometry. Here we provide a treatment of FRAP recovery for a molecule undergoing a combined process of reversible membrane association and lateral diffusion on the plasma membrane for two commonly used bleach geometries: stripes, and boxes. Such analysis is complicated by the fact that diffusion of a molecule during photobleaching can lead to broadening of the bleach area, resulting in significant deviations of the actual bleach shape from the desired bleach geometry, which creates difficulty in accurately measuring kinetic parameters. Here we overcome the problem of deviations between actual and idealized bleach geometries by parameterizing, more accurately, the initial postbleach state. This allows for reconstruction of an accurate and analytically tractable approximation of the actual fluorescence distribution. Through simulated FRAP experiments, we demonstrate that this method can be used to accurately measure a broad range of combinations of diffusion constants and exchange rates. Use of this method to analyze the plextrin homology domain of PLC-δ1 in Caenorhabditis elegans results in quantitative agreement with prior analysis of this domain in other cells using other methods. Because of the flexibility, relative ease of implementation, and its use of standard, easily obtainable bleach geometries, this method should be broadly applicable to investigation of protein dynamics at the plasma membrane.

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.

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.

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.

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.

Artificial septal targeting of Bacillus subtilis cell division proteins in Escherichia coli: an interspecies approach to the study of protein-protein interactions in multiprotein complexes.

Bacterial cell division is mediated by a set of proteins that assemble to form a large multiprotein complex called the divisome. Recent studies in Bacillus subtilis and Escherichia coli indicate that cell division proteins are involved in multiple cooperative binding interactions, thus presenting a technical challenge to the analysis of these interactions. We report here the use of an E. coli artificial septal targeting system for examining the interactions between the B. subtilis cell division proteins DivIB, FtsL, DivIC, and PBP 2B. This technique involves the fusion of one of the proteins (the "bait") to ZapA, an E. coli protein targeted to mid-cell, and the fusion of a second potentially interacting partner (the "prey") to green fluorescent protein (GFP). A positive interaction between two test proteins in E. coli leads to septal localization of the GFP fusion construct, which can be detected by fluorescence microscopy. Using this system, we present evidence for two sets of strong protein-protein interactions between B. subtilis divisomal proteins in E. coli, namely, DivIC with FtsL and DivIB with PBP 2B, that are independent of other B. subtilis cell division proteins and that do not disturb the cytokinesis process in the host cell. Our studies based on the coexpression of three or four of these B. subtilis cell division proteins suggest that interactions among these four proteins are not strong enough to allow the formation of a stable four-protein complex in E. coli in contrast to previous suggestions. Finally, our results demonstrate that E. coli artificial septal targeting is an efficient and alternative approach for detecting and characterizing stable protein-protein interactions within multiprotein complexes from other microorganisms. A salient feature of our approach is that it probably only detects the strongest interactions, thus giving an indication of whether some interactions suggested by other techniques may either be considerably weaker or due to false positives.

Diverse paths to midcell: assembly of the bacterial cell division machinery.

At the heart of bacterial cell division is a dynamic ring-like structure of polymers of the tubulin homologue FtsZ. This ring forms a scaffold for assembly of at least ten additional proteins at midcell, the majority of which are likely to be involved in remodeling the peptidoglycan cell wall at the division site. Together with FtsZ, these proteins are thought to form a cell division complex, or divisome. In Escherichia coli, the components of the divisome are recruited to midcell according to a strikingly linear hierarchy that predicts a step-wise assembly pathway. However, recent studies have revealed unexpected complexity in the assembly steps, indicating that the apparent linearity does not necessarily reflect a temporal order. The signals used to recruit cell division proteins to midcell are diverse and include regulated self-assembly, protein-protein interactions, and the recognition of specific septal peptidoglycan substrates. There is also evidence for a complex web of interactions among these proteins and at least one distinct subcomplex of cell division proteins has been defined, which is conserved among E. coli, Bacillus subtilis and Streptococcus pneumoniae.

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.