Biology subjects

Sidor, C.

Publications and source records attributed to Sidor, C..

4 recordsLinked to original sources

bioRxiv2024

Genetically encoded reporters of actin filament organization in living cells and tissues

The cytoskeletal protein actin is crucial for cell shape and integrity throughout eukaryotes. Actin filaments perform essential biological functions, including muscle contraction, cell division and tissue morphogenesis. These diverse activities are achieved through the ability of actin filaments to be arranged into precise architectures. Much progress has been made in defining the proteome of the actin cytoskeleton, but a detailed appreciation of the dynamic organizational state of the actin filaments themselves has been hindered by available tools. Fluorescence polarization microscopy is uniquely placed for measuring actin filament organization by exploiting the sensitivity of polarized light excitation to the orientation of fluorophores attached to actin filaments. By engineering fusions of five widely used actin localization reporters to fluorescent proteins with constrained mobility, we have succeeded in developing genetically-encoded, green- and red-fluorescent-protein-based reporters for non-invasive, quantitative measurements of actin filament organization in living cells and tissues by fluorescence polarization microscopy.

cell biology↗

bioRxiv2023

Theory of sarcomere assembly inferred from sequential ordering of myofibril components

Myofibrils in striated muscle cells are chains of regular cytoskeletal units termed sarcomeres, whose contractions drive voluntary movements of animals. Despite the well characterized order of the sarcomere components in mature sarcomeres, which explains the sarcomere contraction mechanism, the mechanism of molecular ordering during sarcomere assembly remains debated. Here, we put forward a theoretical framework for the self-assembly of sarcomeres. This theory is based on measurements of the sequential ordering of sarcomere components in developing Drosophila flight muscles, identified by applying a novel tracking-free algorithm: myosin, -actinin and the titin homologue Sallimus form periodic patterns before actin. Based on these results, we propose that myosin, Sallimus, and sarcomere Z-disc proteins including -actinin dynamically bind and unbind to an unordered bundle of actin filaments to establish an initial periodic pattern. As a consequence, periodicity of actin filaments is only established later. Our model proposes that non-local interactions between spatially extended myosin and titin/Sallimus containing complexes, and possibly tension-dependent feedback mediated by an -actinin catch-bond, drive this ordering process. We probe this hypothesis using mathematical models and derive predictive conditions for sarcomere pattern formation, guiding future experimental analysis.

biophysics↗

bioRxiv2019

Rho-kinase planar polarisation at tissue boundaries depends on phospho-regulation of membrane residence time

Rho-kinase (Rok) is a major myosin II activator during morphogenesis. In the Drosophila embryonic salivary gland placode Rok is planar polarised at the tissue boundary, through a negative regulation by the apical polarity protein Crumbs that is anisotropically localised at the boundary. However, in inner cells of the placode both Crumbs and Rok are isotropically enriched at junctions. We propose a model that reconciles both behaviours through modulation of Rok membrane residence time by Crumbs and downstream effectors. Using FRAP in embryos expressing endogenously-tagged Rok combined with in silico simulations, we find that the lower membrane dissociation rate (koff) of Rok at the tissue boundary, where Crumbs membrane levels are lower, explains this boundary-specific effect. The S/T-kinase Pak1 negatively affects Rok membrane association in vivo within the epidermis, and in vitro can phosphorylate Rok near the PH domain that mediates membrane association. Pak1 is recruited to the membrane by Cdc42 which, like its binding partner Crumbs, shows anisotropic localisation at the boundary. These data reveal an important mechanism of modulation of Rok membrane residence time via affecting the koff that may be widely employed during tissue morphogenesis.

developmental biology↗

bioRxiv2019

Mask family proteins ANKHD1 and ANKRD17 regulate YAP nuclear import, stability and phase separation.

The Mask family of multiple ankyrin repeat and KH domain proteins were discovered in Drosophila to promote the activity of the transcriptional coactivator Yorkie (Yki), the sole fly homolog of mammalian YAP (YAP1) and TAZ (WWTR1). The molecular function of Mask, or its mammalian homologs Mask1 (ANKHD1) and Mask2 (ANKRD17), remains unclear. Mask family proteins contain two Ankyrin repeat domains that bind Yki/YAP as well as a conserved nuclear localisation sequence (NLS) and nuclear export sequence (NES), suggesting a role in nucleo-cytoplasmic transport. Here we show that Mask acts to promote nuclear import of Yki, and that addition of an ectopic NLS to Yki is sufficient to bypass the requirement for Mask in Yki-driven tissue growth. Mammalian Mask1/2 proteins also promote nuclear import of YAP, as well as stabilising YAP and driving colloidal phase separation into large liquid droplets. Mask1/2 and YAP normally colocalise in a granular fashion in both nucleus and cytoplasm, and are co-regulated during mechanotransduction. Our results suggest that Mask family proteins promote YAP nuclear import and phase separation to regulate YAP stability and transcriptional activity.

developmental biology↗