Experimental Characterization and Theoretical Modeling of Circular Dorsal Ruffles
Final Report Abstract
Circular Dorsal Ruffles (CDRs) are dynamic actin structures that propagate on the dorsal cell surface, playing a crucial role in growth factor endocytosis and cytoskeletal reorganization. Our research aims to investigate the fundamental mechanisms of CDR propagation through comprehensive experimental characterization and theoretical modeling. We investigate the spatiotemporal dynamics of actin in its free, branched, and polymerized states, as well as CDR ultrastructure, with particular emphasis on characterizing and modeling actin polymerization inhibition. Our experimental approach employs a multi-level intervention strategy, targeting: i) upstream signaling pathways, ii) direct actin regulators, and iii) actin itself through both biochemical and optogenetic methods. We analyze phenotypic statics and dynamics as functions of growth factor concentration, biochemical inhibitor levels, and protein expression. Using disk-shaped micro-contact printed adhesion patches, we constrain CDRs to propagate as lateral waves along ring-like trajectories in the closed circular space between the nucleus and cellular periphery. This controlled system enables precise characterization of CDR propagation through measurements of mean velocity, lifetime, and repeat frequency. We aim to validate existing models that describe CDRs as bistable states while investigating the role of fluctuations through two distinct mechanisms. The first involves temporal fluctuations in protein activity and density at constant molecule numbers, primarily affecting short time scales of minutes. The second encompasses variations in gene expression that alter molecular copy numbers, becoming significant over longer time periods. We hypothesize that random gene expression and drift influence trajectories in state space, suggesting that total protein concentrations vary dynamically, resulting in an evolving rather than fixed phase space. Our methodology combines multiple experimental techniques, including various modes of optical microscopy (fluorescent, PH, DIC, RICM), microfluidics, microcontact printing, and optogenetics. The preparation of defined cell morphologies through adhesion to disk-like domains has proven critical for obtaining reproducible data. Optogenetic tools enable in vitro manipulation of protein expression while allowing observation of in vivo phenotypic variations. Our theoretical analysis integrates image correlation analysis, numerical solution fitting via parameter matching, and AI-based cluster analysis. Particular emphasis is placed on identifying bifurcation types and locations in relation to experimental observations, enabling the construction of comprehensive phase diagrams. This integrated approach provides novel insights into the complex dynamics of CDR formation and propagation, advancing our understanding of cellular membrane dynamics and signal transduction mechanisms.
Publications
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“Mechanobiological Control of Circular Dorsal Ruffle Dynamics.” PhD thesis. Universität Bremen, 2019
Julia Lange
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Biomechanical Aspects of Actin Bundle Dynamics. Frontiers in Cell and Developmental Biology, 8.
Lange, Julia; Bernitt, Erik & Döbereiner, Hans-Günther
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“Dynamics of morphological cell states.” PhD thesis. Universität Bremen
Malte Ohmstede
