Biology subjects

Jacobsen, T.

Publications and source records attributed to Jacobsen, T..

6 recordsLinked to original sources

bioRxiv2026

Structural determinants of endopilus assembly, stability and functional specificity in bacterial type II secretion

Gram-negative bacteria employ the type II secretion system (T2SS) to transport folded protein effectors across the outer membrane. This multiprotein nanomachine assembles the endopilus, a periplasmic helical polymer composed of one major and four minor pilin subunits. Endopili are structurally related to type IV pili but exhibit distinct features including a conserved calcium-binding site stabilizing their major pilin subunits. Endopilus polymerisation is coupled to substrate translocation through a dedicated outer membrane channel. To investigate the structural basis of endopilus assembly, stability and functional specificity, we performed a comparative analysis of the Out T2SS from the plant pathogen Dickeya dadantii and the Pul T2SS from the human pathogen Klebsiella oxytoca. Although their major pilins OutG and PulG share over 77% sequence identity, these bacteria differ markedly in ecological niche and range of secreted effectors. We report here the NMR structure of calcium-bound OutG monomer and cryo-EM structures of OutG and PulG endopili at 3.6 [A] resolution. The integration of structural, mutational, and biophysical analyses with in vivo assays, allowed us to identify the molecular determinants of secretion specificity and endopilus stability. These findings demonstrate how subtle sequence variations in conserved nanomachines have evolved to optimize their function and adapt to their local environments.

microbiology↗

bioRxiv2026

Regulation of Nucleus Pulposus Cell Phenotype Through RhoA Signaling and Microenvironment

Intervertebral disc degeneration is associated with loss of nucleus pulposus (NP) cell phenotype and extracellular matrix, both processes linked to changes in cytoskeletal contractility and cell shape. Here, we tested whether microenvironment-specific modulation of RhoA signaling can restore NP-like morphology and gene expression in NP cells cultured in 2D and in 3D alginate. In 2D monolayer culture, where cells are spread and mechanically activated, pharmacologic inhibition of RhoA with CT04 reduced RhoA activity, decreased actomyosin contractility gene expression, and shifted morphology toward a smaller, more circular phenotype. Bulk RNA sequencing showed that CT04 treatment increased expression of NP phenotypic and matrix-related genes including ACAN, GDF5, CHST3, and MUSTN1 while decreasing expression of catabolic and fibroblast-associated genes including ADAMTS1/9 and COL1, consistent with enrichment of extracellular matrix pathways. In contrast, RhoA activation with CN03 in 2D culture increased actin and phosphorylated myosin light chain intensity but produced limited phenotypic improvement. In 3D alginate, which minimizes integrin-mediated adhesion, baseline actomyosin markers were reduced relative to 2D culture. In alginate, RhoA activation with CN03 increased the amount of actin, phosphorylated myosin light chain, and actomyosin gene expression, yet also promoted a more compact, circular morphology and increased NP markers, including ACAN and KRT19 with repeated dosing. Across culture conditions, increased cell roundness was consistently associated with increased ACAN expression, indicating strong coupling between cytoskeletal state, morphology, and NP matrix programs. Together, these findings demonstrate that RhoA pathway perturbation can promote NP phenotypic gene expression in both 2D and 3D culture, but the direction of optimal modulation depends on the microenvironment, supporting RhoA signaling as a context-dependent therapeutic target for disc regeneration.

cell biology↗

bioRxiv2020

Beauty and the brain: Investigating the neural and musical attributes of beauty during a naturalistic music listening experience

Evaluative beauty judgments are very common, but in spite of this commonality, are rarely studied in cognitive neuroscience. Here we investigated the neural and musical attributes of musical beauty using a naturalistic free-listening paradigm applied to behavioral and neuroimaging recordings and validated by experts judgments. In Study 1, 30 Western healthy adult participants rated continuously the perceived beauty of three musical pieces using a motion sensor. This allowed us to identify the passages in the three musical pieces that were inter-subjectively judged as beautiful or ugly. This informed the analysis for Study 2, where additional 36 participants were recorded with functional magnetic resonance imaging (fMRI) while they listened attentively to the same musical pieces as in Study 1. In Study 3, in order to identify the musicological features characterizing the passages that were consistently rated as beautiful or ugly in Study 1, we collected post-hoc questionnaires from 12 music-composition experts. Results from Study 2 evidenced focal regional activity in the orbitofrontal brain structure when listening to beautiful passages of music, irrespectively of the subjective reactions and individual listening biographies. In turn, the moments in the music that were consistently rated as ugly were associated with bilateral supratemporal activity. Effective connectivity analysis also discovered inhibition of auditory activation and neural communication with orbitofrontal cortex, especially in the right hemisphere, during listening to beautiful musical passages as opposed to intrinsic activation of auditory cortices and decreased coupling to orbitofrontal cortex during listening to ugly musical passages. Experts questionnaires indicated that the beautiful passages were more melodic, calm, sad, slow, tonal, traditional and simple than the ones negatively valenced. In sum, we identified a neural mechanism for inter-subjective beauty judgments of music in the supratemporal-orbitofrontal circuit, irrespectively of individual taste and listening biography. Furthermore, some invariance in objective musical attributes of beautiful and ugly passages was evidenced. Future studies might address the generalizability of the findings to non-Western listeners.

neuroscience↗

bioRxiv2019

Tunable self-cleaving ribozymes for modulating gene expression in eukaryotic systems

Advancements in the field of synthetic biology have been possible due to the development of genetic tools that are able to regulate gene expression. However, the current toolbox of gene regulatory tools for eukaryotic systems have been outpaced by those developed for simple, single-celled systems. Here, we engineered a set of gene regulatory tools by combining self-cleaving ribozymes with various upstream competing sequences that were designed to disrupt ribozyme self-cleavage. As a proof-of-concept, we were able to modulate GFP expression in mammalian cells, and then showed the feasibility of these tools in Drosophila embryos. For each system, the fold-reduction of gene expression was influenced by the location of the self-cleaving ribozyme/upstream competing sequence (i.e. 5 untranslated region (UTR) vs. 3UTR) and the competing sequence used. Together, this work provides a set of genetic tools that can be used to tune gene expression across various eukaryotic systems.

bioengineering↗

bioRxiv2018

Dorsal/NF-κB exhibits a dorsal-to-ventral mobility gradient in the Drosophila embryo

Morphogen-mediated patterning is a highly dynamic developmental process. To obtain an accurate understanding of morphogen gradient formation and downstream gene expression, biophysical parameters such as protein mobilities must be quantified in vivo. The dorsal-ventral (DV) patterning of early Drosophila embryos by the NF-{kappa}B homolog Dorsal (Dl) is an excellent system for understanding morphogen gradient formation. Dl gradient formation is controlled by the inhibitor Cactus/I{kappa}B (Cact), which regulates the nuclear import and diffusion of Dl protein. However, quantitative measurements of Dl mobility and binding are currently lacking. Here, we use scanning fluorescence correlation spectroscopy to quantify the mobility of GFP-tagged Dl. We find that the DNA binding of Dl-GFP, which affects its mobility, varies along the DV axis, with highest DNA binding on the ventral side. Moreover, we also observe that the time scale for Dl-GFP to exit the nucleus is longer in the ventral and lateral regions of the embryo, which is consistent with stronger DNA binding. Using analysis of mutant alleles of dl tagged with GFP, we conclude that Dl-GFP/Cact interactions in the nuclei are responsible for the variation in Dl-GFP/DNA binding along the DV axis, which impacts our understanding of the spatial range of the Dl gradient and the robustness and precision of downstream gene expression. Thus, our results highlight the complexity of morphogen gradient dynamics and the ability of quantitative measurements of biophysical interactions to drive biological discovery.

developmental biology↗

bioRxiv2017

Rapid and scalable characterization of CRISPR technologies using an E. coli cell-free transcription-translation system

CRISPR-Cas systems have offered versatile technologies for genome engineering, yet their implementation has been outpaced by the ongoing discovery of new Cas nucleases and anti-CRISPR proteins. Here, we present the use of E. coli cell-free transcription-translation systems (TXTL) to vastly improve the speed and scalability of CRISPR characterization and validation. Unlike prior approaches that require protein purification or live cells, TXTL can express active CRISPR machinery from added plasmids and linear DNA, and TXTL can output quantitative dynamics of DNA cleavage and gene repression. To demonstrate the applicability of TXTL, we rapidly measure guide RNA-dependent DNA cleavage and gene repression for single- and multi-effector CRISPR-Cas systems, accurately predict the strength of gene repression in E. coli, quantify the inhibitory activity of anti-CRISPR proteins, and develop a fast and scalable high-throughput screen for protospacer-adjacent motifs. These examples underscore the potential of TXTL to facilitate the characterization and application of CRISPR technologies across their many uses.

synthetic biology↗