Biology subjects

Ditlev, J. A.

Publications and source records attributed to Ditlev, J. A..

6 recordsLinked to original sources

bioRxiv2026

An optopharmacological tool for on-demand perturbation of branched actin polymerization

Regulated actin cytoskeletal organization and dynamics are essential for life-sustaining processes in eukaryotic cells. The spatiotemporal regulation of filamentous actin can generate a staggering variety of higher-order architectures to meet diverse cellular needs. However, connecting a specific actin substructure to the selective cellular function remains challenging because tools to specifically inhibit F-actin in a spatiotemporally controlled manner are currently limited. To address this crucial technical gap, we have synthesized and characterized a photocrosslinkable small molecule inhibitor of a major and evolutionarily conserved actin nucleation factor, Arp2/3 complex. The new inhibitor "AZ" combines the specificity of a preexisting inhibitor CK666 with photosensitive crosslinking by an Azido group, such that a light-induced, instantaneous, on-demand, irreversible inhibition of Arp2/3 complex-dependent actin nucleation is achieved in a spatiotemporally defined manner at a subcellular, supracellular, and organismal scale. Whole cell treatment with AZ, followed by subcellular photoirradiation, revealed a divergent actin nucleation homeostasis in the cortex of different cells. Thus, AZ could be a powerful tool to infer actin polymerization homeostasis and network properties in live cells-processes which otherwise remain obscure.

cell biology↗

bioRxiv2024

Disease-linked mutations dysregulate neuronal condensate physical properties, composition, and RNA translation

Local RNA translation is essential for development. In neurons, deficient local translation linked with mutations in scaffold proteins results in dysregulated dendrite and dendritic spine growth. However, mechanisms by which these proteins control translation and how disease-linked mutations induce aberrant translation were unclear. We use biochemical reconstitution and neuronal assays to show that mutations to the neuronal condensate scaffold shank2 cause physical hardening and altered composition of condensates; a key RNA translation-modulator FMRP is excluded from mutant condensates. Functionally, shank2 condensates repress translation while condensates composed of shank2 with intrinsically disordered region-localized missense mutations promote translation. These results demonstrate that disease-linked dysregulation of condensate physical properties and composition is an underlying mechanism of aberrant RNA translation often observed in disease. One Sentence SummaryDisease-linked missense mutations in the postsynaptic density scaffold protein shank2 dysregulate phase separated biomolecular condensate physical properties and composition resulting in aberrant RNA translation.

biophysics↗

bioRxiv2024

Protein interactions, calcium, phosphorylation, and cholesterol modulate CFTR cluster formation on membranes

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a chloride channel whose dysfunction leads to intracellular accumulation of chloride ions, dehydration of cell surfaces, and subsequent damage to airway and ductal organs. Beyond its function as a chloride channel, interactions between CFTR, ENaC, and SLC transporter family membrane proteins and cytoplasmic proteins, including calmodulin and NHERF-1, co-regulate ion homeostasis. CFTR has also been observed to form mesoscale membrane clusters. However, the biophysical mechanisms that regulate the formation of CFTR clusters are unknown. Using a combination of computational modeling and complex biochemical reconstitution assays, we demonstrate that multivalent protein-protein interactions with CFTR binding partners, calcium, and membrane cholesterol can induce CFTR cluster formation on model membranes. Phosphorylation of the intracellular domains of CFTR also promotes cluster formation in the absence of calcium, indicating that multiple mechanisms can regulate CFTR cluster formation. Our findings reveal that coupling of multivalent protein and lipid interactions promote CFTR cluster formation consistent with membrane-associated biological phase separation. SIGNIFICANCE STATEMENTMutations in the cystic fibrosis transmembrane conductance regulator (CFTR) membrane protein underlie cystic fibrosis. It is thought that molecular "hubs" of CFTR and its binding partners co-regulate ion homeostasis and that disruption of these clusters can result in disease. However, the physical basis for molecular hub formation is unclear. In this study, we present evidence that multivalent protein and lipid interactions drive the formation of mesoscale CFTR-containing clusters or "hubs" on model membranes in a manner consistent with biological phase separation. These data provide important insights into physical mechanisms that modulate CFTR membrane organization and offer a new lens for the development of corrective therapeutics.

biophysics↗

bioRxiv2024

Measurement of solubility product in a model condensate reveals the interplay of small oligomerization and self-association

Cellular condensates often consist of 10s to 100s of distinct interacting molecular species. Because of the complexity of these interactions, predicting the point at which they will undergo phase separation into discrete compartments is daunting. Using experiments and computation, we therefore studied a simple model system consisting of 2 proteins, polySH3 and polyPRM, designed for pentavalent heterotypic binding. We tested whether the peak solubility product, the product of dilute phase monomer concentrations, is a predictive parameter for the onset of phase separation. Titrating up equal total concentrations of each component showed that the maximum solubility product does approximately coincide with the threshold for phase separation in both the experiments and models. However, we found that measurements of dilute phase concentration include contributions from small oligomers, not just monomers; therefore, a quantitative comparison of the experiments and models required inclusion of small oligomers in the model analysis. We also examined full phase diagrams where the model results were almost symmetric along the diagonal, but the experimental results were highly asymmetric. This led us to perform dynamic light scattering experiments, where we discovered a weak homotypic interaction for polyPRM; when this was added to the computational model, it was able to recapitulate the experimentally observed asymmetry. Thus, comparing experiments to simulation reveals that the solubility product can be predictive of phase separation, even if small oligomers and low affinity homotypic interactions preclude experimental measurement of monomer concentration.

biophysics↗

bioRxiv2019

The mechanisms of dynamin-actin interaction

Cell-cell fusion is an indispensable process in the conception, development and physiology of multicellular organisms. Here we demonstrate a direct and noncanonical role for dynamin, best known as a fission GTPase in endocytosis, in cell-cell fusion. Our genetic and cell biological analyses show that dynamin colocalizes within the F-actin-enriched podosome-like structures at the fusogenic synapse, which is required for generating invasive membrane protrusions and myoblast fusion in vivo, in an endocytosis-independent manner. Biochemical, negative stain EM and cryo-electron tomography (cryo-ET) analyses revealed that dynamin forms helices that directly bundles actin filaments by capturing multiple actin filaments at their outer rim via interactions with dynamins proline-rich domain. GTP hydrolysis by dynamin triggers disassembly of the dynamin helix, exposes the sides of the actin filaments, promotes dynamic Arp2/3-mediated branched actin polymerization, and generates a mechanically stiff actin network. Thus, dynamin functions as a unique actin-bundling protein that enhances mechanical force generation by the F-actin network in a GTPase-dependent manner. Our findings have universal implications for understanding dynamin-actin interactions in various cellular processes beyond cell-cell fusion.

cell biology↗

bioRxiv2018

A Composition-Dependent Molecular Clutch Between T Cell Signaling Clusters and Actin

Biomolecular condensates play important roles in eukaryotic cells by concentrating molecules into foci without a surrounding membrane. During T cell activation, biomolecular condensates form at the immunological synapse (IS) through multivalency-driven phase separation of the adaptor protein LAT and its binding partners Grb2, Sos1, SLP-76, Nck and WASP. These condensates move radially at the IS, traversing a radially-oriented and then a concentric actin network. To understand the persistent radial movement, we biochemically reconstituted LAT condensates with mobile actomyosin filaments. We found that basic regions of Nck and N-WASP promote strong association and co-movement of LAT condensates with actin. Condensates lacking these components were instead propelled by steric interactions. In cells, LAT condensates lost Nck while traversing the boundary between the two actin networks, and condensates engineered to constitutively bind actin moved aberrantly. We propose that Nck and WASP form a clutch between LAT condensates and actin, and changes in composition enable condensate movement by distinct actin networks in different regions of the IS.

cell biology↗