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.

A simplified counter-selection recombineering protocol for creating fluorescent protein reporter constructs directly from C. elegans fosmid genomic clones.

BACKGROUND: Recombineering is a genetic engineering tool that enables facile modification of large episomal clones, e.g. BACs, fosmids. We have previously adapted this technology to generate, directly from fosmid-based genomic clones, fusion gene reporter constructs designed to investigate gene expression patterns in C. elegans. In our adaptation a rpsL-tet(A) positive/negative-selection cassette (RT-cassette) is first inserted and then, under negative selection, seamlessly replaced with the desired sequence. We report here on the generation and application of a resource comprising two sets of constructs designed to facilitate this particular recombineering approach. RESULTS: Two complementary sets of constructs were generated. The first contains different fluorescent protein reporter coding sequences and derivatives while the second set of constructs, based in the copy-number inducible vector pCC1Fos, provide a resource designed to simplify RT-cassette-based recombineering. These latter constructs are used in pairs the first member of which provides a template for PCR-amplification of an RT-cassette while the second provides, as an excised restriction fragment, the desired fluorescent protein reporter sequence. As the RT-cassette is flanked by approximately 200 bp from the ends of the reporter sequence the subsequent negative selection replacement step is highly efficient. Furthermore, use of a restriction fragment minimizes artefacts negating the need for final clone sequencing. Utilizing this resource we generated single-, double- and triple-tagged fosmid-based reporters to investigate expression patterns of three C. elegans genes located on a single genomic clone. CONCLUSIONS: We describe the generation and application of a resource designed to facilitate counter-selection recombineering of fosmid-based C. elegans genomic clones. By choosing the appropriate pair of 'insertion' and 'replacement' constructs recombineered products, devoid of artefacts, are generated at high efficiency. Gene expression patterns for three genes located on the same genomic clone were investigated via a set of fosmid-based reporter constructs generated with the modified protocol.

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.

Seamless replacement of Autographa californica multiple nucleopolyhedrovirus gp64 with each of five novel type II alphabaculovirus fusion sequences generates pseudotyped virus that fails to transduce mammalian cells.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV), a member of the type I alphabaculoviruses, is able to transduce and deliver a functional gene to a range of non-host cells, including many mammalian lines and primary cells, a property mediated by the envelope fusion protein GP64. AcMNPV is non-cytopathic and inherently replication deficient in non-host cells. As such, AcMNPV represents a possible new class of gene therapy vector with potential future clinical utility. Whilst not a problem for in vitro gene delivery, the broad tropism displayed for non-host cells is less desirable in a gene therapy vector. The fusion protein F of type II alphabaculoviruses can substitute functionally for GP64, and such pseudotyped viruses display a severely impaired capacity for non-host-cell transduction. Thus, surface decoration of such an F-pseudotyped AcMNPV with cell-binding ligands may restore transduction competence and generate vectors with desirable cell-targeting characteristics. By seamlessly swapping the native gp64 coding sequence with each of five sequences encoding different F proteins, a set of F-pseudotyped AcMNPV was generated. This report details their relative abilities both to functionally replace GP64 in viral growth and to transduce human Saos-2 and HeLa cells. All five supported viable infections in insect cell cultures and one, the Mamestra configurata NPV (MacoNPV) F pseudotype, could be amplified to titres close to those of native AcMNPV. In contrast, none was able to transduce the Saos-2 and HeLa cell lines. The robust support provided by MacoNPV F in virus production makes the corresponding pseudotype a viable scaffold to display surface ligands to direct selective mammalian cell targeting.

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.

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.

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.

C. elegans flavin-containing monooxygenase-4 is essential for osmoregulation in hypotonic stress.

Studies in Caenorhabditis elegans have revealed osmoregulatory systems engaged when worms experience hypertonic conditions, but less is known about measures employed when faced with hypotonic stress. Inactivation of fmo-4, which encodes flavin-containing monooxygenase-4, results in dramatic hypoosmotic hypersensitivity; worms are unable to prevent overwhelming water influx and swell rapidly, finally rupturing due to high internal hydrostatic pressure. fmo-4 is expressed prominently in hypodermis, duct and pore cells but is excluded from the excretory cell. Thus, FMO-4 plays a crucial osmoregulatory role by promoting clearance of excess water that enters during hypotonicity, perhaps by synthesizing an osmolyte that acts to establish an osmotic gradient from excretory cell to duct and pore cells. C. elegans FMO-4 contains a C-terminal extension conserved in all nematode FMO-4s. The coincidently numbered human FMO4 also contains an extended C-terminus with features similar to those of FMO-4. Although these shared sequence characteristics suggest potential orthology, human FMO4 was unable to rescue the fmo-4 osmoregulatory defect. Intriguingly, however, mammalian FMO4 is expressed predominantly in the kidney - an appropriate site if it too is, or once was, involved in osmoregulation.