Biology subjects

Scholz, J.

Publications and source records attributed to Scholz, J..

9 recordsLinked to original sources

bioRxiv2026

Conserved tyrosine Y16 acts as a phosphorylation switch controlling Ena/VASP recruitment to sites of actin assembly

Ena/VASP proteins are powerful actin polymerases that accumulate at sites of active actin assembly, including lamellipodial and microspike tips, focal adhesions and pathogen surfaces, where they drive processive filament elongation. However, the mechanisms controlling their recruitment remain elusive. Here, we used cells lacking all Ena/VASP-family members to systematically test phosphomimetic and nonphosphorylatable substitutions at conserved tyrosine and serine residues implicated in subcellular targeting. Combining cell-based reconstitution with biochemical assays, we identify phosphorylation of Y16 in vertebrate Ena/VASP proteins as the decisive inhibitory modification controlling subcellular Ena/VASP recruitment, in contrast to previously proposed roles for Y39 and S157. Corresponding phosphomimetic substitutions in the C. elegans orthologue UNC-34 phenocopied uncoordinated locomotion of UNC-34 null worms, supporting the evolutionary conservation of this regulatory mechanism. Thus, our findings identify a conserved phosphorylation-sensitive switch at the EVH1-FP4 ligand interface that links EVH1-dependent targeting to dynamic control of actin assembly in adhesion, migration and pathogenesis.

cell biology↗

bioRxiv2026

IqgD is a Rac1-interacting IQGAP required for efficient growth of Dictyostelium discoideum on bacterial lawns

IQGAPs are large multidomain scaffold proteins that interact with the Rho family GTPases Cdc42 and Rac1, functioning both as their effectors and as regulators by stabilizing their active GTP-bound state. In this study, we analyzed the function of IqgD, an IQGAP-related protein from the professional phagocyte Dictyostelium discoideum. IqgD contains a calponin homology domain (CHD), a GAP-related domain (GRD), and a RasGAP C-terminal (RGCt) domain. We show that the CHD is essential for F-actin binding and cortical localization, whereas the GRD and RGCt domains mediate interactions with Rac1 GTPases and the actin-bundling proteins cortexillins. Moreover, similar to mammalian IQGAPs, IqgD maintains Rac1 in its active conformation. IqgD is enriched in macropinocytic and phagocytic cups and co-localizes with F-actin and active Rac1 in the ring-like structure that forms around surface-bound particles at the cell bottom. Loss of IqgD results in markedly reduced growth on bacterial lawns and significantly smaller cell size. While mutant cells internalize bacteria from suspension as efficiently as wild-type cells, they display a strong defect in phagocytosis of surface-bound particles, accompanied by decreased adhesion to the cell substrate. Together, our data show that although IqgD localizes to macroendocytic cups, it is dispensable for macropinocytosis and phagocytosis of suspended particles. Instead, IqgD is specifically required for efficient phagocytosis of surface-bound particles, likely by facilitating robust F-actin polymerization at the cell bottom to generate the force necessary for detachment of surface-bound bacteria. Significance StatementPhagocytosis of surface-bound microbes is essential for host defense and environmental feeding strategies, yet its underlying mechanisms remain poorly understood. We identify the IQGAP-related protein IqgD in D. discoideum as a key factor required for efficient uptake of bacteria attached to solid surfaces. IqgD localizes to an F-actin- and Rac1-rich circular structure analogous to the phagocytic adhesion ring (PAR) recently described in mammalian macrophages, suggesting that this mode of force-driven particle detachment is evolutionarily conserved. Our findings provide mechanistic insight into substrate-dependent phagocytosis and establish IqgD as a central regulator of this process.

cell biology↗

bioRxiv2025

Profilin promotes lamellipodium protrusion by tuning the antagonistic activities of CP and VASP

Cell migration on rigid surfaces employs flat protrusions termed lamellipodia, which constitute the prime model system for branched actin filament networks that generate pushing forces for membrane movement. Branched actin filaments are nucleated by the Arp2/3 complex and play vital roles in various cell biological processes, such as organelle trafficking, phagocytosis and autophagy. Here we utilize genome editing to explore the functional connections between the actin monomer-binding protein profilin (Pfn), the filament nucleating Arp2/3 complex, its co-factor heterodimeric capping protein (CP) and the Ena/VASP family of actin filament polymerases in lamellipodial actin assembly. Individual and combinations of knockouts show that Pfn counters Ena/VASP but promotes Arp2/3 complex activity, while Ena/VASP and CP mutually antagonize each other. Notably, while Pfn is important for Arp2/3 complex activity irrespective of Ena/VASP, sensitivity of CP to Pfn removal is lost in the absence of Ena/VASP. Our findings establish Pfn as master regulator of Arp2/3 complex-dependent actin network formation, which differentially regulates VASP and its antagonizer CP. Finally, mathematical modeling of our data suggest that Ena/VASP and CP compete for binding at the lamellipodial edge, likely contributing to their functional antagonism at this subcellular site. This work provides critical insights into the molecular logic of branched actin network assembly in membrane protrusion and cellular force generation.

cell biology↗

bioRxiv2024

Recruitment of the cellular lipid transport protein CERT to C. psittaci inclusions regulates the timing of bacterial egress

Egress of intracellular pathogens is highly regulated and carefully timed. For the zoonotic bacterium C. psittaci, the predominant egress pathway is Chlamydia-containing sphere (CCS) formation, a calcium-dependent sequential mechanism including protease activity, inclusion membrane destabilization, intracellular calcium increase, and plasma membrane blebbing. How egress is regulated to ensure that it takes place only after C. psittaci intracellular development is thus far unknown. Here, we show that C. psittaci recruits the cellular ceramide transporter CERT to its inclusion during intracellular development, but this recruitment is reduced at late time points prior to egress. In addition, an early loss of CERT at the inclusion membrane induced by CERT-KO induces premature egress by CCS formation. Complementation of the CERT-KO with different CERT-GFP variants prevents premature egress, except of complementation with a variant lacking the inclusion targeting PH domain, showing that the localization of CERT is critical for CCS formation. The CERT-KO induced premature CCS are formed by the sequential process described for mature CCS, but they contain mostly RBs and are predominantly non-infectious. Thus, our findings suggest that the timing of C. psittaci egress by CCS formation is regulated by the recruitment of CERT to the inclusion. We propose that CERT stabilizes the chlamydial inclusion by the formation of ER-inclusion membrane contact sites during intracellular development, and the loss of CERT recruitment facilitates inclusion membrane destabilization and CCS formation.

microbiology↗

bioRxiv2023

The formation of Chlamydia-containing spheres, a novel non-lytic egress pathway of the zoonotic pathogen Chlamydia psittaci

Egress of intracellular bacteria from host cells and cellular tissues is a critical process during the infection cycle. This egress process is essential for bacteria to spread inside the host and can influence the outcome of an infection. For the obligate intracellular Gram-negative zoonotic bacterium Chlamydia psittaci little is known about the mechanisms resulting in chlamydial egress from the infected epithelium. Here, we describe and characterize a novel non-lytic egress pathway of C. psittaci by formation of Chlamydia-containing spheres (CCS). CCS are spherical, low phase contrast structures surrounded by a phosphatidylserine exposing membrane with specific barrier functions. They contain infectious progeny and morphologically impaired cellular organelles. The formation of CCS shares characteristics of apoptotic cell death including a proteolytic cleavage of the peptide DEVD albeit independent of active caspase-3, an increase in the intracellular calcium concentration of infected cells, followed by blebbing of the plasma membrane and rupture of the inclusion membrane. Finally, infected blebbing cells detach and leave the monolayer thereby forming CCS. These results support that Chlamydia psittaci egresses the epithelial cell by a novel non-lytic egress pathway, a process beneficial for the bacterium, which might influence the outcome of the infection in organisms. ImportanceHost cell egress is essential for intracellular pathogens to spread within an organism and for host-to-host-transmission. Here, we describe CCS formation as a novel egress pathway for the intracellular, zoonotic bacterial pathogen C. psittaci. This non-lytic egress pathway is fundamentally different from previously described Chlamydia egress pathways. Interestingly, CCS formation shares several characteristics of apoptotic cell death. However, the sequence of proteolytic activity, followed by plasma membrane blebbing and the final detachment of a whole phosphatidylserine exposing former host cell is unique for C. psittaci. Thus, CCS formation represents a new egress pathway for intracellular pathogens that could possibly be linked to C. psittaci biology including host tropism, protection from host cell defense mechanisms or bacterial pathogenicity.

microbiology↗

bioRxiv2022

Highly-Automated, High-Throughput Replication of Yeast-based Logic Circuit Design Assessments

We describe an experimental campaign that replicated the performance assessment of logic gates engineered into cells of S. cerevisiae by Gander, et al. Our experimental campaign used a novel high throughput experimentation framework developed under DARPAs Synergistic Discovery and Design (SD2) program: a remote robotic lab at Strateos executed a parameterized experimental protocol. Using this protocol and robotic execution, we generated two orders of magnitude more flow cytometry data than the original experiments. We discuss our results, which largely, but not completely, agree with the original report, and make some remarks about lessons learned.

synthetic biology↗

bioRxiv2021

Data Representation in the DARPA SD2 Program

1Modern scientific enterprises are often highly complex and multidisciplinary, particularly in areas like synthetic biology where the subject at hand is itself inherently complex and multidisciplinary. Collaboration across many organizations is necessary to efficiently tackle such problems [6, 15], but remains difficult. The challenge is further amplified by automation that increases the pace at which new information can be produced, and particularly so for matters of fundamental research, where concepts and definitions are inherently fluid and may rapidly change as an investigation evolves [7]. The DARPA program Synergistic Discovery and Design (SD2) aimed to address these challenges by organizing the development of data-driven methods to accelerate discovery and improve design robustness, with one of the key domains under study being synthetic biology. The program was specifically organized such that teams provided complementary types of expertise and resources, and without any team being in a dominant organizational position, such that subject-matter investigations would necessarily require peer-level collaboration across multiple team boundaries. With more than 100 researchers across more than 20 organizations, several of which ran experimental facilities with high-throughput automation, participants were forced to confront challenges around effective data sharing. The default architecture for scientific collaboration is essentially one of anarchy, with ad-hoc bilateral relations between pairs of collaborators or experimental phases (Figure 1(a)). This was by necessity the case during early phases of the SD2 program as well, in which incorporating new tools into pipelines was ad-hoc and time-consuming, and data was generally disconnected from genetic designs and experimental plans. The other typical approach for collaboration is one of "command and control", in which a dominant organization determines the data sharing content and format for all participants (Figure 1(b)). This can be efficient, but tends to be limited in flexibility and extensibility, rendering it unsuitable for research collaboration, as indeed was found when we attempted this approach during the first year of the SD2 program. We addressed these problems with the application of distributed standards to create a "flexible rendezvous" model of collaboration (Figure 1(c)), enabling information flow to track evolving collaborative relationships, improving the sharing and utility of information across the community and supporting accelerated rates of experimentation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/460644v1_fig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@fc7371org.highwire.dtl.DTLVardef@1ff2733org.highwire.dtl.DTLVardef@66af53org.highwire.dtl.DTLVardef@1809640_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1:C_FLOATNO Architectures for data sharing: bilateral relations (a), command and control (b), and flexible rendezvous (c). C_FIG

synthetic biology↗

bioRxiv2021

A Novel in vitro Model for Mature Toxoplasma gondii Bradyzoites Reveals their Metabolome and a Diminished Role of the Mitochondrial Tricarboxylic Acid Cycle

The apicomplexan parasite Toxoplasma gondii forms bradyzoite-containing tissue cysts that cause chronic and drug-tolerant infections. However, current in vitro models do not allow long-term culture of these cysts to maturity. Here, we developed a human myotube-based in vitro culture model of functionally mature tissue cysts that are orally infectious to mice and tolerate exposure to a range of antibiotics and temperature stresses. Metabolomic characterization of purified cysts reveals global changes that comprise increased levels of amino acids and decreased abundance of nucleobase- and tricarboxylic acid cycle-associated metabolites. In contrast to fast replicating tachyzoite forms of T. gondii these tissue cysts tolerate exposure to the aconitase inhibitor sodium fluoroacetate. Direct access to persistent stages of T. gondii under defined cell culture conditions will be essential for the dissection of functionally important host-parasite interactions and drug evasion mechanisms. It will also facilitate the identification of new strategies for therapeutic intervention.

microbiology↗

bioRxiv2017

Structural covariance of brain region volumes is associated with both structural connectivity and transcriptomic similarity

An organizational pattern seen in the brain, termed structural covariance, is the statistical association of pairs of brain regions in their anatomical properties. These associations, measured across a population as covariances or correlations usually in cortical thickness or volume, are thought to reflect genetic and environmental underpinnings.\n\nHere, we examine the biological basis of structural volume covariance in the mouse brain. We first examined large scale associations between brain region volumes using an atlas-based approach that parcellated the entire mouse brain into 318 regions over which correlations in volume were assessed, for volumes obtained from 153 mouse brain images via high-resolution MRI. We then used a seed-based approach and determined, for 108 different seed regions across the brain and using mouse gene expression and connectivity data from the Allen Institute for Brain Science, the variation in structural covariance data that could be explained by distance to seed, transcriptomic similarity to seed, and connectivity to seed.\n\nWe found that overall, correlations in structure volumes hierarchically clustered into distinct anatomical systems, similar to findings from other studies and similar to other types of networks in the brain, including structural connectivity and transcriptomic similarity networks. Across seeds, this structural covariance was significantly explained by distance (17% of the variation, up to a maximum of 49% for structural covariance to the visceral area of the cortex), transcriptomic similarity (13% of the variation, up to maximum of 28% for structural covariance to the primary visual area) and connectivity (15% of the variation, up to a maximum of 36% for structural covariance to the intermediate reticular nucleus in the medulla) of covarying structures. Together, distance, connectivity, and transcriptomic similarity explained 37% of structural covariance, up to a maximum of 63% for structural covariance to the visceral area. Additionally, this pattern of explained variation differed spatially across the brain, with transcriptomic similarity playing a larger role in the cortex than subcortex, while connectivity explains structural covariance best in parts of the cortex, midbrain, and hindbrain. These results suggest that both gene expression and connectivity underlie structural volume covariance, albeit to different extents depending on brain region, and this relationship is modulated by distance.

neuroscience↗