Biology subjects

Bland, T.

Publications and source records attributed to Bland, T..

4 recordsLinked to original sources

bioRxiv2023

Optimized dimerization of the PAR-2 RING domain drives cooperative and selective membrane recruitment for robust feedback-driven cell polarization

The behavior of cell polarity networks is defined by the 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 that arise during development. Using the PAR polarity network as a model, we demonstrate that these features are enabled by dimerisation of the polarity protein PAR-2 via ubiquitin-independent function of its N-terminal RING domain. Specifically, we combine theory and experiment to show that dimer affinity is optimized to achieve dynamic, selective, and cooperative recruitment of PAR-2 to the plasma membrane during polarization. Reducing dimerization results in loss of positive feedback and compromises robustness of symmetry-breaking, while enhanced dimerization renders the network less responsive due to kinetic trapping of PAR-2 on internal membranes and reduced sensitivity of PAR-2 to membrane displacement by the polarity kinase, aPKC/PKC-3. Thus, our data reveal how a dynamically oligomeric RING domain results in a cell polarity network that is both robust and responsive and highlight how tuning of oligomerization kinetics can serve as a general strategy for optimizing dynamic and cooperative intracellular targeting.

cell biology↗

bioRxiv2022

Cleavage furrow-directed cortical flows bias mechanochemical pathways for PAR polarization in the C. elegans germ lineage

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.

cell biology↗

bioRxiv2022

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 C. elegans zygote, anterior segregation of the polarity protein PAR-3 requires oligomerization. However, through direct assessment of advection of PAR proteins, we not only find no links between PAR-3 advection and oligomer size, but also observe efficient advection of both anterior and posterior PAR proteins. Consequently, differential cortex engagement cannot account for selective size-dependent PAR protein transport. Instead, combining experiment and theory we demonstrate that segregation efficiency of PAR proteins by cortical flow is determined by the stability of membrane association, which is enhanced by clustering and specifies persistence of transport. Indeed, stabilizing membrane association was sufficient to invert polarity of a normally posterior PAR protein. Our data therefore indicate that advection of membrane-associated proteins is more pervasive than anticipated and thus cells must tune membrane association dynamics to achieve differential transport by cortical flows.

cell biology↗

bioRxiv2022

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

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. Summary StatementImplemented as an easy-to-use Fiji Plugin, SAIBR provides effective autofluorescence correction for cells and tissues using standard imaging conditions.

cell biology↗