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, due 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 plugin 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, and 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.

We investigate planar cell polarity (PCP) in the Drosophila larval epidermis. The intricate pattern of denticles depends on only one system of PCP, the Dachsous/Fat system. Dachsous molecules in one cell bind to Fat molecules in a neighbour cell to make intercellular bridges. The disposition and orientation of these Dachsous–Fat bridges allows each cell to compare two neighbours and point its denticles towards the neighbour with the most Dachsous. Measurements of the amount of Dachsous reveal a peak at the back of the anterior compartment of each segment. Localization of Dachs and orientation of ectopic denticles help reveal the polarity of every cell. We discuss whether these findings support our gradient model of Dachsous activity. Several groups have proposed that Dachsous and Fat fix the direction of PCP via oriented microtubules that transport PCP proteins to one side of the cell. We test this proposition in the larval cells and find that most microtubules grow perpendicularly to the axis of PCP. We find no meaningful bias in the polarity of microtubules aligned close to that axis. We also reexamine published data from the pupal abdomen and find no evidence supporting the hypothesis that microtubular orientation draws the arrow of PCP.

10We investigate the mechanisms of planar cell polarity (PCP) in the larval epidermis of 11 Drosophila. Measurements of the amount of Dachsous across the segment find a peak 12 located near the rear of the anterior compartment. Localisation of Dachs and 13 orientation of ectopic denticles reveal the polarity of every cell in the segment. We 14 discuss how well these findings evidence a zigzag gradient model of Dachsous activity. 15 Several groups have proposed that Dachsous and Fat fix the direction of PCP via 16 oriented microtubules that transport PCP proteins to one side of the cell. We test this 17 proposition in the larval cells and find that most microtubules grow perpendicularly 18 to the axis of PCP. We find no significant bias in the polarity of those microtubules 19 aligned close to that axis. We also reexamine published data from the pupal abdomen 20 and fail to find evidence supporting the hypothesis that microtubular orientation 21 draws the arrow of PCP. 22As cells construct embryos and organs they need access to vectorial information that 24 informs them, for example, which way to migrate, divide, extend axons or how to 25 orient protrusions. In Drosophila there are (at least) two conserved genetic systems 26 Page 2 of 32 that generate vectorial information, and here we are concerned with only one of those, 27 the Dachsous/Fat system. Dachsous (Ds) and Fat (Ft) are large atypical cadherin 28 molecules that form heterodimeric bridges from cell to cell that help build planar cell 29 polarity (PCP) in one cell and also convey polarity between cells (Ma et al., 2003; 30 Matakatsu and Blair, 2004; Casal et al., 2006; Lawrence and Casal, 2018). The 31 activity of Ft is increased and Ds is reduced when they are phosphorylated by a third 32 molecule, Four-jointed (Fj), a Golgi-based kinase (Ishikawa et al., 2008; Brittle et al., 33 2010; Simon et al., 2010). The distribution of Ds, Ft and Fj and the interaction 34 between these molecules together determine what we describe as "Ds activity", by 35 which we mean the propensity of Ds on one cell to bind to Ft in the neighbouring cell. 36 Experiments suggest that, using the disposition and orientation of Ds-Ft bridges, each 37 cell compares the Ds activity of its two neighbours and points its denticles towards the 38 neighbour with the higher Ds activity. In this way, the local slopes in a landscape of Ds 39 activity determine polarities of all the cells (Casal et al., 2002; see Lawrence and 40 Casal, 2018 for more explanation). 41 A model: the ventral epidermis of the Drosophila larva 42Each segment of the larva is divided by cell lineage into an anterior (A) and a 43 posterior (P) compartment. In the ventral epidermis, a limited region of the segment 44 makes rows of thorny denticles that are polarised in an almost invariant pattern, while 45 the larger part of each segment makes no denticles and therefore its polarity is not 46 known (Figure 1-figure supplement 1). The Ds/Ft system is alone effective in 47 48 the "core" or Stan/Fz system (Casal et ...

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.

Clustering of membrane-associated molecules has long been proposed to promote size-dependent interactions with the actomyosin cortical network, thereby allowing them to be transported by actin flow. Consistent with such a model, clustering of the conserved polarity protein PAR-3 in the C. elegans zygote is essential for its polarization by cortical actomyosin flows, and clusters of PAR-3 visibly move with flows. However, here we show that advection by cortical flow is independent of clustering. Clustered and non-clustered PAR-3 variants are advected equally well over short timescales by cortical actin flow as are other PAR proteins. Moreover, we see no strong links between advection and either cluster size or diffusivity that would be consistent with size-dependent interactions with the actin cortex. Instead, using a combination of experiment and theory, we find that efficient long range transport of PAR proteins by cortical flow is primarily tuned by the stability of membrane association, which is enhanced by clustering and determines the persistence of both transport by flow and the resulting asymmetries once flows cease. Consistent with this model, stabilizing membrane association was sufficient to induce segregation of a non-segregating PAR protein, effectively inverting its polarity. We conclude that the impact of advection on membrane-associated proteins is much broader than previously anticipated and thus cells must appropriately tune membrane association dynamics to achieve selectivity in the long range transport of membrane-associated molecules by cortical flows.

During development, the conserved PAR polarity network is continuously redeployed, requiring that it adapts to changing cellular contexts and environmental cues. How it does so and the degree to which these adaptations reflect changes in its fundamental design principles remain unclear. Here, we investigate the process of PAR polarization within the highly tractable C. elegans germline P lineage, which undergoes a series of iterative asymmetric stem cell-like divisions. Compared to the zygote, we observe significant differences in the pattern of polarity emergence, including an inversion of the initial unpolarized state, changes in symmetry breaking cues, and the timings with which anterior and posterior PARs segregate. Beneath these differences, however, polarity establishment remains reliant on the same core pathways identified in the zygote, including conserved roles for cortical actin flows and PAR-dependent self-organization. Intriguingly, we find that cleavage furrow-directed cortical actin flows play a similar symmetry-breaking role for the germline cell P1 as centrosome-induced cortical flows in the zygote. Through their ability to induce asymmetric accumulation of PAR-3 clusters, these furrow-directed flows directly couple the geometry of polarization to cell division, which could be a general strategy for cells to ensure proper organization within dynamically growing systems, such as embryos. In summary, our data suggest that coupling of novel symmetry-breaking cues with highly adaptable core mechanochemical circuits enable robust PAR polarity in response to changing developmental contexts.

Biological systems are increasingly viewed through the lens of mathematics, physics, and systems approaches that demand accurate quantification of gene expression and local protein concentrations. Such approaches have benefited greatly from the revolution in genetic engineering sparked by CRISPR/Cas9. By facilitating the tagging of genes at their genomic loci, CRISPR/Cas9 allows us to use fluorescence to monitor proteins that are expressed at or near endogenous levels under native regulatory control. However, due to their typically lower expression levels, quantitative experiments using endogenously-tagged genes can run into limits imposed by autofluorescence (AF). AF is often a particular challenge in the illumination bands occupied by the most efficient 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 associated GUI-based FIJI plugin to correct for autofluorescence using standard filter sets and illumination conditions. Fully validated for use in C. elegans embryos and tested in diverse systems, including starfish oocytes and fission yeast, SAIBR 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.

Plants have evolved genome complexity through iterative rounds of single gene and whole genome duplication. This has led to substantial expansion in transcription factor numbers following preferential retention and subsequent functional divergence of these regulatory genes. Here we review how this simple evolutionary network rewiring process, regulatory gene duplication followed by functional divergence, can be used to inspire synthetic biology approaches that seek to develop novel phenotypic variation for future trait based breeding programs in plants.