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Schaks, M.

Publications and source records attributed to Schaks, M..

12 recordsLinked to original sources

LOAM: A family of genomic language models trained on long-read soil metagenomes

Soil ecosystems represent a vast, largely uncharacterised reservoir of microbial diversity. Metagenomic assembly has unlocked access to this resource, and recent advances in long-read sequencing have improved the recovery and quality of microbial genomes from complex samples. While genomic language models have proven highly effective at capturing biological concepts from large sequence datasets, they have predominantly been trained on reference genomes or short-read assemblies. Here, we present LOAM, a family of decoder-only genomic language models ranging from 25 to 624 million parameters and trained exclusively on Oxford Nanopore long-read environmental metagenomes. LOAM model performance scales predictably with model size and training-token budget. Despite a relatively small training sequence corpus, LOAM models outperformed comparably sized models across biological benchmarks, and achieved performance competitive with substantially larger state-of-the-art models trained on much larger datasets. Context-intervention experiments further showed that LOAM models use genomic information over several kilobases, highlighting the potential value of increased contiguity provided by long-read metagenome-assembled genomes. For probe-based benchmark tasks, we systematically evaluated representations across hidden layers and revealed that task-relevant biological information was frequently more linearly accessible from intermediate than final model layers. Finally, we observed that variation in zero-shot variant-effect prediction was strongly associated with the presence of homologous target sequences in the pre-training corpus. Together, these results establish long-read environmental metagenomes as a viable foundation for training competitive genomic language models and demonstrate the importance of both model scale and training-corpus composition for biological generalisation.

genomics↗

Arp2/3 complex-dependent actin remodeling is required for efficient RSV uncoating in A549 cells

Productive human respiratory syncytial virus (RSV) cell-entry requires coordinated interactions between viral proteins and host-cell factors at the plasma membrane-actin cortex interface. Branched actin networks remodel this interface, but their precise contribution to the early stages of RSV infection remains unclear. Here, we interfered with Arp2/3 complex-dependent actin filament branching by generating A549 cell lines disrupted for expression of the essential Arp2 subunit by CRISPR/Cas9. Permanent loss of Arp2 reduced the infection of the RSV long GFP reporter virus as quantified over the first 24 h post-infection. Compromised infection efficiency in Arp2 knockout cells persisted at later time points and also resulted in reduced syncytia formation. Notably, these infection phenotypes were not accompanied by obvious changes in viral host cell attachment. Moreover, photoactivated localization microscopy (PALM) studies revealed comparable receptor diffusion and clustering in cells stably expressing mEos3.2-tagged insulin-like growth factor I receptor (IGF1R). Although Arp2/3-deficient cells displayed fewer albeit larger macropinosomes as compared to WT cells, no changes were observed for internalized RSV genome levels. In contrast, Arp2-deficient cells appeared suppressed in viral uncoating efficiency. Consequently, viral mRNA expression and the cellular type III interferon response were reduced. Together, these data reveal that Arp2/3 complex-dependent, branched actin networks contribute to the efficiency of RSV uncoating. ImportanceHuman respiratory syncytial virus (RSV) is a major cause of severe respiratory disease. The infection initiates at the plasma membrane-actin cortex interface, yet the role of actin in productive RSV entry has remained unclear. Using CRISPR/Cas9 disruption of the essential Arp2/3 complex subunit Arp2 in A549 cells, we show that branched actin networks are required for efficient RSV infection. Despite actin network remodeling, photoactivated localization microscopy showed unchanged diffusion and clustering of the RSV receptor IGF1R. Although macropinocytosis was affected in Arp2-deficient cells, RSV attachment and internalization were not influenced. In contrast, a {beta}-lactamase virus-like-particle-based assay revealed a defect in uncoating, followed by reduced viral gene expression and a weaker type III interferon response. These findings define Arp2/3 complex-dependent branched actin networks as a host determinant of RSV uncoating and provide a practical approach to quantify uncoating without engineering the RSV genome.

microbiology↗

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↗

Scientific reasoning driven by influential data: resuscitate dfstat!

In biomedical literature, one of the most widely employed statistical procedures to analyze and visualize the association between two variables is linear regression. Data points that exert influence on the fit and its parameters are routinely, but not as often as required, identified by established influence measures and their corresponding cut-off values. In this work, we are specifically concerned with the presence of influential data points that directly impact hypothesis testing of linear regressions, which none of the established measures describe. Interestingly, the highly overlooked influence measure dfstat and its derived leave-one-out p-value exists exactly for this purpose, unmentioned in the majority of statistical text books as well as absent from all available statistical software packages. Its application for identifying these data points seems pivotal, as scientific reasoning in publications is almost exclusively based on the p-value of the fit, commonly adhering to the = 0.05 threshold to state significance or not. With this metric, we found for 29 of 100 digitizable papers published in Science, Nature and PNAS in 2016, a time when the "reproducibility crisis" was a growing concern, that stated significances (or their absence) are based on the presence of a single influential data point.

scientific communication and education↗

Arf GTPase activates the WAVE Regulatory Complex through a novel binding site

Crosstalk between Rho- and Arf-family GTPases plays an important role in linking actin cytoskeletal remodeling to membrane protrusion, organelle structure, and vesicle trafficking. The central actin regulator, WAVE Regulatory Complex (WRC), is a converging point of Rac1 (a Rho-family GTPase) and Arf signaling in many processes, but how Arf promotes WRC activation is unknown. Here we reconstituted a direct interaction between Arf and WRC. This interaction can be greatly enhanced by Rac1 binding to the D site of the WRC. Arf1 binds to a newly identified conserved surface on Sra1 located between the D site and the WH2 helix of WAVE1, which can drive WRC activation using a mechanism distinct from that of Rac1. Mutating Arf binding site abolishes Arf1-WRC interaction, disrupts Arf1-mediated WRC activation, and impairs lamellipodia morphology. This work uncovers a new mechanism underlying WRC activation and provides a mechanistic foundation for studying how WRC-mediated actin polymerization links Arf and Rac signaling in the cell.

biochemistry↗

Structures reveal a key mechanism of WAVE Regulatory Complex activation by Rac1 GTPase

Rho-family GTPase Rac1 activates the WAVE regulatory complex (WRC) to drive Arp2/3-mediated actin polymerization in many essential processes. Rac1 binds to WRC at two distinct sites--the A and D sites. Precisely how Rac1 binds and how the binding triggers WRC activation remain unknown. Here we report WRC structures by itself, and when bound to single or double Rac1 molecules, at [~]3 [A] resolutions by cryogenic-electron microscopy. The structures reveal that Rac1 binds to the two sites by distinct mechanisms, and binding to the A site, but not the D site, drives WRC activation. Activation involves a series of unique conformational changes leading to the release of sequestered WCA (WH2- central-acidic) polypeptide, which stimulates the Arp2/3 complex to polymerize actin. Together with biochemical and cellular analyses, the structures provide a novel mechanistic understanding of how the Rac1-WRC-Arp2/3-actin signaling axis is regulated in diverse biological processes and diseases.

biochemistry↗

Lamellipodia-like actin networks in cells lacking WAVE Regulatory Complex

Cell migration frequently involves the formation of lamellipodia induced by Rac GTPases mediating activation of WAVE Regulatory Complex (WRC) driving Arp2/3 complex-dependent actin assembly. Previous genome editing studies solidified the view of an essential, linear pathway employing aforementioned components. Using disruption of the WRC subunit Nap1 and its paralogue Hem1 followed by serum and growth factor stimulation or expression of active GTPases now revealed a pathway to formation of Arp2/3 complex-dependent, lamellipodia-like structures (LLS) that require both Rac and Cdc42, but not WRC. These observations were independent of WRC subunit eliminated and coincided with the lack of recruitment of Ena/VASP family actin polymerases. Moreover, aside from the latter, induced LLS contained all common lamellipodial regulators tested, including cortactin, the Ena/VASP ligand lamellipodin or FMNL subfamily formins. Our studies thus establish the existence of a signaling axis to Arp2/3 complex-dependent actin remodeling at the cell periphery operating without WRC and Ena/VASP.

cell biology↗

A structure-derived mechanism reveals how capping protein promotes nucleation in branched actin networks

Heterodimeric capping protein (CP/CapZ) is an essential factor for the assembly of branched actin networks, which push against cellular membranes to drive a large variety of cellular processes. Aside from terminating filament growth, CP stimulates the nucleation of actin filaments by the Arp2/3 complex in branched actin networks through an unclear mechanism. Here, we report the structure of capped actin filament barbed ends, which reveals how CP not only prevents filament elongation, but also controls access to both terminal filament subunits. In addition to its primary binding site that blocks the penultimate subunit, we find that the CP sterically occludes the central interaction site of the terminal actin protomer through one of its C-terminal "tentacle" extensions. Deletion of this {beta} tentacle only modestly impairs capping. However in the context of a growing branched actin network, its removal potently inhibits nucleation promoting factors (NPFs) by tethering them to capped filament ends. End tethering of NPFs prevents their loading with actin monomers required for activation of the Arp2/3 complex and thus strongly inhibits branched network assembly both in cells and reconstituted motility assays. Our results mechanistically explain how CP couples two opposed processes -capping and nucleation- in branched actin network assembly.

biochemistry↗

Amplitude Analysis of Polarization Modulation Data and 3D-Polarization Demodulation (3D-SPoD)

Super-resolution optical fluctuation imaging (SOFI) is a technique that uses the amplitude of fluorescence correlation data for improved resolution of fluorescence images. Here, we explore if also the amplitude of superresolution by polarisation demodulation (SPoD) data can be used to gain additional information about the underlying structures. Highly organized experimental as well a simulated actin filament data demonstrate a principle information gain from this approach. In addition, we explored theoretically the benefits of analyzing the entire 3D-polarization information instead of only 2D-projections thereof. Due to fundamental principles, the probability of finding parallel orientations is approaching zero in 3D-SPoD in contrast to 2D-approaches. Using the modulation-amplitude based analysis we explored systematically simulated 3D-single molecules data (for which the true structures are known) under different conditions that are typically observed in experiments. We found that this approach can significantly improve the distinction, reconstruction and localization. In addition, these approaches are less sensitive to uncertainties in the knowledge about the true experimental point-spread-function (PSF) used for reconstruction compared to approaches using non-modulated data. Finally, they can effectively remove higher levels of non-modulated back-ground intensity.

biophysics↗

Molecular dissection of neurodevelopmental disorder-causing mutations in CYFIP2

Actin remodelling is frequently regulated by antagonistic activities driving protrusion and contraction downstream of Rac and Rho small GTPases, respectively. WAVE regulatory complex (WRC), which primarily operates downstream of Rac, plays pivotal roles in neuronal morphogenesis. Recently, two independent studies described de novo mutations in the CYFIP2 subunit of WRC, which caused intellectual disability (ID) in humans. Although mutations had been proposed to effect WRC activation, no experimental evidence for this was provided. Here, we made use of CRISPR/Cas9-engineered B16-F1 cell lines that were reconstituted with ID-causing CYFIP variants in the context of compromised WRC activation with or without reduced Rac activities, which established that the majority of CYFIP2 mutations (5 out of 8) indeed cause constitutive WRC activation. Strikingly, activating mutations are positioned in a conserved WAVE- binding region mediating WRC transinhibition. As opposed to such gain-of-function mutations, a truncating mutant represented a loss-of-function variant, because it failed to interact with WRC components, and two mutants displayed no or at best a moderate increase of WRC activation. Collectively, our data show that CYFIP2 mutations frequently but not always coincide with WRC activation and suggest that normal brain development requires a delicate and precisely tuned balance of neuronal WRC activity.

cell biology↗

Lamellipodin tunes cell migration by stabilizing protrusions and promoting adhesion formation

Efficient migration on adhesive surfaces involves the protrusion of lamellipodial actin networks and their subsequent stabilization by nascent adhesions. The actin binding protein lamellipodin (Lpd) is thought to play a critical role in lamellipodium protrusion, by delivering Ena/VASP proteins onto the growing plus ends of actin filaments and by interacting with the WAVE regulatory complex (WRC), an activator of the Arp2/3 complex, at the leading edge. Using B16-F1 melanoma cell lines, we demonstrate that genetic ablation of Lpd compromises protrusion efficiency and coincident cell migration without altering essential parameters of lamellipodia, including their maximal rate of forward advancement and actin polymerization. We also confirmed lamellipodia and migration phenotypes with CRISPR/Cas9-mediated Lpd knockout Rat2 fibroblasts, excluding cell type-specific effects. Moreover, computer-aided analysis of cell edge morphodynamics on B16-F1 cell lamellipodia revealed that loss of Lpd correlates with reduced temporal protrusion maintenance as a prerequisite of nascent adhesion formation. We conclude that Lpd optimizes protrusion and nascent adhesion formation by counteracting frequent, chaotic retraction and membrane ruffling. Summary statementWe describe how genetic ablation of the prominent actin- and VASP-binding protein lamellipodin combined with software-aided protrusion analysis uncovers mechanistic insights into its cellular function during cell migration.

cell biology↗

RhoG and Cdc42 can contribute to Rac-dependent lamellipodia formation through WAVE Regulatory Complex-binding

Cell migration frequently involves the formation of lamellipodial protrusions, the initiation of which requires Rac GTPases signalling to heteropentameric WAVE regulatory complex (WRC). While Rac-related RhoG and Cdc42 can potently stimulate lamellipodium formation, so far presumed to occur by upstream signalling to Rac activation, we show here that the latter can be bypassed by RhoG and Cdc42 given that WRC has been artificially activated. This evidence arises from generation of B16-F1 cells simultaneously lacking both Rac GTPases and WRC, followed by reconstitution of lamellipodia formation with specific Rho-GTPase and differentially active WRC variant combinations. We conclude that formation of canonical lamellipodia requires WRC activation through Rac, but can possibly be tuned, in addition, by WRC interactions with RhoG and Cdc42.

cell biology↗