Biology subjects

Klapholz, B.

Publications and source records attributed to Klapholz, B..

4 recordsLinked to original sources

bioRxiv2026

Highly conserved talin isoforms encode distinct mechanical responses and ligand-binding capabilities

Talin is a central mechanosensitive adaptor that links integrins to the actin cytoskeleton and converts mechanical force into biochemical signals. Vertebrates express two highly homologous talin isoforms, talin-1 and talin-2, yet the molecular basis for their nonredundant functions remains poorly understood. Here, we investigate how isoform-specific sequence differences alter the mechanical response and ligand-binding capabilities of the talin rod. Using single-molecule force spectroscopy and biochemical binding assays, we show that talin-2 preserves the overall mechanical architecture of talin-1 but exhibits distinct mechanical responses in key rod regions. Talin-2 R7-R9 exhibits a previously unobserved three-domain cooperative unfolding pathway, while an alternative sequential pathway reveals early unfolding of R9 at substantially lower force than in talin-1. Strikingly, talin-2 R4-R6 has acquired two additional mechanically regulated interactions: a previously unrecognized vinculin-binding site in R5 and an A-kinase anchoring protein (AKAP) region within R5-R6 that recruits protein kinase A (PKA). Together with the conserved R9 AKAP site, this establishes two spatially separated, mechanically regulated PKA-anchoring regions within the talin-2 scaffold. These findings establish that isoform-specific sequence divergence not only reshapes the mechanical response of the talin scaffold but also expands its repertoire of force-regulated interactions, providing a mechanism for the assembly of different structural and signaling complexes in an isoform-dependent manner.

biophysics↗

bioRxiv2023

Actin-Driven Nanotopography Enhances Integrin Molecular Clutch in Developing Tissue

Morphogenesis requires building stable macromolecular structures from highly dynamic proteins. Muscles are anchored by long-lasting integrin adhesions to resist contractile force. However, the mechanisms governing integrin diffusion, immobilization, and activation within developing tissues remain elusive. Here, we show that actin polymerisation-driven membrane protrusions form nanotopographies that enable strong adhesion at Drosophila muscle attachment sites (MAS). Super-resolution microscopy revealed that integrins assemble adhesive belts around Arp2/3-dependent actin protrusions, forming invadosome-like structures with membrane nanotopographies. Single protein tracking showed that, during MAS development, integrins became immobile and confined within diffusion traps formed by the membrane nanotopographies. Actin filaments also displayed restricted motion and confinement, indicating strong mechanical connection with integrins. Using isolated muscles cells, we show that substrate nanotopography, rather than rigidity, drives adhesion maturation by regulating actin protrusion, integrin diffusion and immobilization. These results thus demonstrate that actin-polymerisation driven membrane protrusions are essential for the formation of strong integrin adhesions sites in the developing embryo, and highlight the important contribution of geometry to morphogenesis.

cell biology↗

bioRxiv2023

The mechanical response of vinculin

Vinculin is a mechanosensitive adapter protein that links the actin network to cell-extracellular matrix adhesions and cell-cell adhesions. It is perhaps the best characterized mechanoeffector, as it is recruited to sites of adhesion in response to force on the mechanotransducers talin and alpha-catenin. Here we examined the mechanical properties of vinculin to assess its potential role as a mechanotransducer. We find that at physiological loading rates, the structural domains of vinculin unfold at forces in the 5-15 pN range and rapidly refold when forces are reduced back to 1 pN. Thus, vinculin domains also have the potential to act as force dependent molecular switches, akin to those in talin and alpha-catenin. As with the force dependent switches in talin, the unfolding of these domains in vinculin introduces large extension changes in the vinculin cytoskeletal linkage up to 150 nm with 20-30 nm steps of unfolding. Modelling of the tension-dependent interactions of the unstructured vinculin linker region with a model protein containing two SH3 domains indicated that even unstructured protein regions can mediate force-dependent interactions with ligands, where the binding of a dual-SH3 model protein is predicted to be significantly suppressed by forces greater than 10 pN. Together, these findings suggest that vinculin has a complex mechanical response with force-dependent interaction sites, suggesting it also acts as a mechanotransducer, recruiting partners in response to force.

biophysics↗

bioRxiv2021

A Bioinformatic and Empiric Exploration of Prokaryotic Argonautes as Novel Programmable Endonuclease Systems

Argonautes are nucleases that can be programmed by short oligonucleotides to cleave complementary sequences. Here, we performed an unbiased bioinformatic search to mine bacterial genomes for prokaryotic Argonautes (pAgos) harboring a PIWI domain. Our search identified 3,033 pAgos in total, of which 1,464 portend to the subgroup of long pAgos with more than 600 amino acids. We purified a subset of 49 pAgos which were found in proximity to helicases and tested their nuclease activity in vitro. Ten of these were active towards single-stranded DNA substrates and this activity could be programmed by exogenous guide DNAs or RNAs. Cleavage of double-stranded plasmid DNA was much less readily observed and was fostered by elevated temperatures or exogenous addition of a DNA single-strand binding protein (ET-SSB). The efficiency of pAgo-mediated plasmid cleavage was dependent on the DNA target sequence as well as the surrounding sequence, suggesting that unwinding of the DNA double helix was a limiting factor. Intriguingly, we identified a cluster of pAgos from the Clostridial clade which was active at 37{degrees}C and activity was enhanced by exogenous ET-SSB. This suggests that Clostridial pAgos may be particularly suited to catalyze DNA double-strand cleavage and implies that such pAgos may be repurposed as gene editing tools in future.

molecular biology↗