Biology subjects

Walczak, M.

Publications and source records attributed to Walczak, M..

17 recordsLinked to original sources

bioRxiv2026

Solvent-Isotope Effects in Biomolecular Phase Separation and Fibrillation of Disordered Proteins and Peptides

Heavy water (D2O) is widely used in biomolecular spectroscopy and imaging, often under the assumption that it is an inert replacement for H2O. However, D2O differs subtly in hydrogen-bonding, viscosity, and dielectric properties, which can alter biomolecular interactions and self-assembly. Here, we test how solvent isotope substitution modulates protein/peptide phase separation and amyloid formation in multiple intrinsically disordered systems. Using turbidity-based phase diagrams and microscopy, we quantify how D2O shifts protein-RNA complex coacervation boundaries and alters condensate morphology. Droplet recovery measurements indicate significant solvent-dependent changes in condensate material properties. We further evaluate amyloid formation kinetics, in the presence or absence of a cofactor, supported by orthogonal structural characterization, and assess the functional consequences of tau fibrils using a tau biosensor seeding assay with explicitly defined seed delivery conditions. Together, these results show that D2O can systematically bias liquid-liquid phase separation and aggregation readouts and should be treated as an active experimental variable rather than a neutral solvent substitute.

biophysics↗

bioRxiv2026

Deciphering underground decarboxylase activity towards Nε-modified lysine derivatives in enterobacteria.

Thermal food processing generates diverse compounds interacting with the gut microbiota. Despite their abundance, the microbial turnover of diet-borne N{varepsilon}-modified lysine derivatives remains largely unexplored. We demonstrate that the enterobacterial ornithine decarboxylase SpeC degrades the prevalent advanced glycation end product N{varepsilon}-carboxymethyllysine (CML) to carboxymethylcadaverine via an underground activity ([~]4 molecules/enzyme/min). This promiscuity extends to additional N{varepsilon}-modified lysine derivatives - namely fomylated (FmL), monomethylated (MML) and dimethylated (DML) lysine - yielding previously unknown biogenic amines (mono- and dimethylcadaverine, formylcadaverine). Functionally, SpeC enables Escherichia coli to utilize CML as a sole nitrogen source. In specific strains, this metabolism reinforces pH-stress responses, supporting survival under mild acidic conditions typical for the colon. Furthermore, SpeC orthologs are widespread across human gut genomes, correlating with geography, diet, and disease. Together, these findings suggest a potential diet-microbiome communication axis, linking the intake of modified dietary chemicals to microbial physiology and hypothesized host impacts.

microbiology↗

bioRxiv2026

Tunable Bessel beam two-photon fluorescence microscopy for high-speed volumetric imaging of brain dynamics

High-speed volumetric imaging of the brain is essential for linking diverse cellular events to tissue-level functions. However, the brains structural and dynamic heterogeneity--spanning microns to millimeters and milliseconds to hours--requires imaging techniques with tunable spatiotemporal resolution, flexible 3D sampling, and compatibility with targeted perturbations. Here, we present tunable Bessel beam two-photon fluorescence microscopy (tBessel-TPFM), a compact, low-cost, and versatile platform for intravital brain imaging across millimeter scale with subcellular resolution. tBessel-TPFM transforms slow 3D volume scans into fast 2D frame scans via an axially elongated Bessel focus, achieving acquisition rates [~]100-fold faster and reduced motion artifacts compared with conventional TPFM. Exploiting its full tunability of the Bessel focus, we applied tBessel-TPFM for quantitative mapping of cerebral blood flow and neurovascular coupling in normal and ischemic stroke mice. Unlike existing Bessel focus generation methods, the axial center of tBessel-TPFM remains fixed at the objective focal plane during profile tuning. Leveraging this advantage, we integrated tBessel-TPFM with simultaneous 3D targeted optogenetic stimulation for volumetric neuronal connectivity mapping. We also tracked microglial process dynamics following single-cell laser ablation, revealing diverse neuroimmune responses across spatial and temporal scales. By combining high speed, deep penetration, tunable sampling, and multimodal perturbation, tBessel-TPFM empowers a broad spectrum of neurobiological investigations--from vascular physiology and functional connectivity to neuroimmune interactions.

bioengineering↗

bioRxiv2025

N-Linked Glycosylation as a Driver of Tau Pathology and Neuronal Transmission

N-Linked glycosylation of Tau is implicated in Alzheimers disease, yet how it influences Tau propagation and neuronal uptake remains unclear. Here, we use chemically defined N-glycoforms of 2N4R Tau and the K18 repeat domain to map how glycan site and structure reprogram Tau-polyanion binding, seeding, and entry into human neurons. Site-specific GlcNAc or high-mannose glycans at Asn359 or Asn410 remodel heparin and RNA binding kinetics in biolayer interferometry assays, revealing avidity-dominated, slow-off heparin interactions that are dampened by N-glycans and faster RNA interactions that can be potentiated by small GlcNAc modifications. Cross-seeding experiments show that N-glycosylation selectively tunes nucleation compatibility between Tau proteoforms; GlcNAc at Asn410 in full-length Tau and small glycans at Asn359 in K18 most strongly accelerate aggregation. In HEK293T FRET biosensor cells and iPSC-derived cortical neurons, N-glycans enhance or suppress seeding and pHrodo-reported uptake in a site- and glycan-dependent manner, with Asn410 glycosylation markedly potentiating cellular seeding. Pharmacological inhibition with atropine, dynasore, and cytochalasin D further indicates that N-glycans rebalance muscarinic, dynamin-dependent, and actin-dependent uptake routes. Together, these data establish N-linked glycosylation as a tunable regulator of Tau trafficking and prion-like spread, coupling extracellular receptor usage to intracellular cofactor engagement, and reveals druggable nodes in uptake.

neuroscience↗

bioRxiv2025

tRNA thiolation defects disrupt cellular proteostasis and tissue homeostasis in mammals

Sulfur modification of tRNA wobble uridines is an evolutionarily conserved mechanism that ensures efficient protein synthesis. In humans, loss of this anticodon modification due to mutations in CTU2 (cytosolic thiouridylase 2) causes DREAM-PL syndrome, a severe congenital disorder often leading to early postnatal death. However, the mechanisms by which loss of tRNA thiolation drives pathology remain unclear. Here, we show that loss of CTU2 triggers significant cellular proteostasis defects in patient cells and model cell lines. Structural and biochemical analyses reveal that the pathogenic CTU2L63P mutation destabilizes the CTU1/CTU2 complex and abolishes tRNA binding and thiolation. Acute loss of CTU2 caused codon-specific ribosome pausing at A-ending codons decoded by thiolated tRNAs, and decreased ribosome occupancy of A-rich transcripts in a dosage-dependent manner. Codon-biased mRNAs transcribed from genes critical for ciliogenesis are predicted to be most affected, linking their reduced translation to DREAM-PL etiology in humans. Surprisingly, Ctu2L63P mice display severe thiolation defects, but develop normally, are viable and fertile. Our findings highlight the importance of functional tRNA thiolation for organismal health in humans and identify species-specific vulnerabilities during embryonic development in mammals.

cell biology↗

bioRxiv2025

Lipid droplets in felid kidneys: prevalence and composition by lipidomics

An accepted and common phenotypic curiosity of Felidae is the presence of intracytoplasmic lipid droplets in renal proximal tubule epithelial cells (RPTEC), also frequently in urine (lipuria). Both outcomes are currently considered, and taught, as incidental - without obvious pathophysiological consequence. This contrasts markedly with clinical (human) medicine where lipid vacuoles in RPTEC are usually associated with metabolic or chronic disease, such as CKD. Despite domestic felids having a high incidence of CKD as they age, no study has fully characterised feline RPTEC lipid droplets in the context of CKD. Here, we first characterised the incidence of RPTEC lipid in domestic cat (with/without CKD or chronic interstitial nephritis) versus domestic dog and Scottish Wildcat, across a wide age range. Felids (domestic, wildcat) consistently had greater renal lipid content than dogs at all ages studied. Intracytoplasmic lipid extraction with chromatography, fatty acid characterisation and mass spectrometry-based lipidomics revealed unusual presence of a panoply of novel lipids found only in domestic cat: lipids were primarily modified (i.e. less polar) ether-soluble triacylglycerols including monoalkyldiacylglycerols (MADAGs) and other branched-chain fatty acids. We suggest common presence of such rare lipid species in tubular lipid droplets in domestic cat reflects an aspect of felid biology that parallels age-related disease prevalence, in particular, being associated with the aetiopathogenesis of chronic renal interstitial nephritis (CIN) - a hallmark of CKD in felids.

zoology↗

bioRxiv2025

OPTRACE: Optical Imaging Guided Transplantation and Tracking of Cells in the Mouse Brain

Intracerebral cell transplantation holds great promise for treating stroke and other neurological disorders, but progress is hindered by challenges in precise cell delivery and limited methods for monitoring post-engraftment dynamics. To address these limitations, we introduce OPTRACE (OPtical imaging-guided Transplantation and tRAcking of CElls), a two-step framework that integrates precision delivery with longitudinal in vivo tracking to advance cell therapy. During transplantation, OPTRACE enables real-time visualization using cost-effective translucent glass micropipettes, guided by predictive modeling to optimize delivery parameters. A novel pulse-elevation injection technique further enhances the precision of superficial cortical engraftment. Following transplantation, multicolor labeling combined with two-photon fluorescence microscopy permits longitudinal single-cell tracking, revealing host microglial responses, and altered neuronal calcium signaling at the graft interface. By unifying high-resolution delivery and tracking strategies, OPTRACE provides a powerful platform for elucidating engraftment biology and accelerating the development of effective cell-based interventions for neurological diseases.

bioengineering↗

bioRxiv2024

Expression screen of TNFR1 R347A, MyD88, IRAK4 death domains in E. coli followed by purification and biophysical characterization of TNFR1 R347A death domain

Death domains play a crucial role in signaling pathways related to inflammation and programmed cell death, rendering them promising targets for therapeutic interventions. However, their expression as recombinant proteins often pose challenges. Here, we present expression screening of TNFR1, IRAK4, and MyD88 death domains in E. coli, followed by the biophysical characterization of TNFR1 death domain after subsequent construct optimization. The study also discusses the influence of pH and ionic strength on TNFR1R347A stability, providing statistical models to predict optimal conditions of the buffer to achieve the highest protein stability. HighlightsO_LIOptimization of expression conditions for TNFR1R347A, MyD88, IRAK4 death domains in E. coli BL21(DE3) cells. C_LIO_LIHigh-yield production of soluble monomeric TNFR1R347A death domain. C_LI

biochemistry↗

bioRxiv2024

Impact of soil moisture on microbial diversity and their enzyme activity on agricultural soil

In this study, the impact of a two-month of drought stress on the microbial abundance, their enzymes and functional diversity in four agricultural soil (Gniewkowo (G), Lulkowo (L), Nieszawa (N), and Suchatowka (S) sites from Poland) was investigated during summer season. The physicochemical parameters (pH, organic carbon (C), calcium carbonate (CaCO3), total nitrogen (N), nitrate (NO3-), ammonium (NH4+), total phosphorus (P), available phosphate (P2O5)), and specific biological parameters (microbial abundance, CLPP, and soil enzymes (phosphatases (acid; ACP and alkaline; AKP), dehydrogenase (DH), and urease (UR)) were conducted on the soil samples in this study. The physicochemical and biological data were compared between zero-week (T0) and 8th week (T8) time intervals. The microbial enumeration showed higher bacterial populations (496.63 x 104 CFU g-1 dry soil) compared to actinomycetes (13.43 x 104 CFU g-1 dry soil), and lowest were the fungal population (67.68 x 102 CFU g-1 dry soil) at T8. The functional diversity showed strong positive significance in the G, N and S sites at T8. On the contrary, most of the L sites showed negative significance with the utilization of amines only, by the end of the experiment. Overall, the microbial population, their enzymes and functional diversity showed positive correlation with soil moisture content in all four investigated sites. The findings of our study indicate that soil biological activities in agricultural regions can be modified by a mere two months of drought.

microbiology↗

bioRxiv2024

Extremely distinct microbial communities in closely related leafhopper subfamilies: Typhlocybinae and Eurymelinae (Cicadellidae, Hemiptera)

AO_SCPLOWBSTRACTC_SCPLOWAmong the Hemiptera insects, a widespread way of feeding is sucking sap from host plants. Due to diet poor in nutrients, these insects enter into obligate symbiosis with their microorganisms. However, within the Cicadellidae family, there is a relatively large group of mesophyll feeders - Typhlocybinae that is considered to be devoid of symbiotic companions. In this work, we examine the composition of microorganisms in this subfamily and compare the results with their close relatives - the Eurymelinae subfamily. To study the microbiome, we used high-throughput next-generation sequencing (NGS, Illumina) and advanced microscopic techniques such as transmission electron microscopy (TEM) and fluorescence in situ hybridization (FISH) in a confocal microscope. The Typhlocybinae insects have very poor microbial communities in their bodies, these are mainly facultative microorganisms, such as alphaproteobacteria of the genus Wolbachia or Rickettsia. We detected also the presence of bacteria that can be considered as facultative symbionts e.g. Spiroplasma, Acidocella, Arsenophonus, Sodalis, Lariskella, Serratia, Cardinium and Asaia. On the other hand, the Eurymelinae group is characterized by a large diversity of the microbial communities, similar to those described in other Cicadomorpha. We find obligate co-symbionts involved in the synthesis of essential amino acids such as Sulcia, betaproteobacteria related to genus Nasuia or gammaproteobacteria Sodalis. In other representatives, we observed symbiotic yeast-like fungi from the family Ophiocordycipitaceae and within some genera we discovered Arsenophonus bacteria inhabiting the interior of Sulcia bacteria. Additionally, we investigated the transovarial transmission of obligate symbionts, which occurs via infection of the ovaries of females. IO_SCPLOWMPORTANCEC_SCPLOWThe Typhlocybinae and Eurymelinae leafhoppers differ significantly in their symbiotic communities. This is undoubtedly due to their different diets, as Typhlocybinae insects feed on parenchyma, richer in nutrients, while Eurymelinae, like most representatives of Auchenorrhyncha, consume sap from the phloem fibers of plants. Our work presents comprehensive studies of 42 species belonging to two above-mentioned, so far poorly known Cicadomorpha subfamilies. Phylogenetic studies we conducted confirm that the insects from the groups studied have a common ancestor. Since obligate symbionts, having a reduced genome, may affect the reduction of their hosts adaptation to changing environmental conditions, e.g. temperature, and facultative microbiomes may influence the increase in such adaptation and expansion of host niche space. Therefore, Typhlocybinae species may show greater resistance to future climate change than representatives of the Eurymelinae. The research that considers the role of ecological niches in microbiome composition is essential in the era of climate change.

microbiology↗

bioRxiv2024

Post-Translational Modifications Control Phase Transitions of Tau

The self-assembly of Tau(297-391) into filaments, which mirror the structures observed in Alzheimers disease (AD) brains, raises questions about the role of AD-specific post-translational modifications (PTMs) in the formation of paired helical filaments (PHFs). To investigate this, we developed a synthetic approach to produce Tau(291-391) featuring N-acetyllysine, phosphoserine, phosphotyrosine, and N-glycosylation at positions commonly modified in post-mortem AD brains, thus facilitating the study of their roles in Tau pathology. Using transmission electron microscopy (TEM), cryo-electron microscopy (cryo-EM), and a range of optical microscopy techniques, we discovered that these modifications generally hinder the in vitro assembly of Tau into PHFs. Interestingly, while acetylations effect on Tau assembly displayed variability, either promoting or inhibiting phase transitions in the context of cofactor free aggregation, heparin-induced aggregation, and RNA-mediated liquid-liquid phase separation (LLPS), phosphorylation uniformly mitigated these processes. Our observations suggest that PTMs, particularly those situated outside the fibrils rigid core are pivotal in the nucleation of PHFs. Moreover, in scenarios involving heparin-induced aggregation leading to the formation of heterogeneous aggregates, most AD-specific PTMs, except for K311, appeared to decelerate the aggregation process. The impact of acetylation on RNA-induced LLPS was notably site-dependent, exhibiting both facilitative and inhibitory effects, whereas phosphorylation consistently reduced LLPS across all proteoforms examined. These insights underscore the complex interplay between site-specific PTMs and environmental factors in modulating Tau aggregation kinetics, enhancing our understanding of the molecular underpinnings of Tau pathology in AD and highlighting the critical role of PTMs located outside the ordered filament core in driving the self-assembly of Tau into PHF structures.

biophysics↗

bioRxiv2023

A synthetic signalling network imitating the action of immune cells in response to bacterial metabolism

State-of-the-art bottom-up synthetic biology allows us to replicate many basic biological functions in artificial cell-like devices. To mimic more complex behaviours, however, artificial cells would need to perform many of these functions in a synergistic and coordinated fashion, which remains elusive. Here we considered a sophisticated biological response, namely the capture and deactivation of pathogens by neutrophil immune cells, through the process of netosis. We designed a consortium consisting of two synthetic agents - responsive DNA-based particles and antibiotic-loaded lipid vesicles - whose coordinated action mimics the sought immune-like response when triggered by bacterial metabolism. The artificial netosis-like response emerges from a series of interlinked sensing and communication pathways between the live and synthetic agents, and translates into both physical and chemical antimicrobial actions, namely bacteria immobilisation and exposure to antibiotics. Our results demonstrate how advanced life-like responses can be prescribed with a relatively small number of synthetic molecular components, and outlines a new strategy for artificial-cell-based antimicrobial solutions.

synthetic biology↗

bioRxiv2022

Reaction-diffusion patterning of DNA-based artificial cells

Biological cells display complex internal architectures, with distinct micro environments that establish the chemical heterogeneity needed to sustain cellular functions. The continued efforts to create advanced cell mimics - artificial cells - demands strategies to construct similarly heterogeneous structures with localized functionalities. Here, we introduce a platform for constructing membrane-less artificial cells from the self-assembly of synthetic DNA nanostructures, in which internal domains can be established thanks to prescribed reaction-diffusion waves. The method, rationalized through numerical modeling, enables the formation of up to five distinct, concentric environments, in which functional moieties can be localized. As a proof-of-concept, we apply this platform to build DNA-based artificial cells in which a prototypical nucleus synthesizes fluorescent RNA aptamers, which then accumulate in a surrounding storage shell, thus demonstrating spatial segregation of functionalities reminiscent of that observed in biological cells.

synthetic biology↗

bioRxiv2021

Structure-function relationship for a divergent Atg8 protein required for a non-autophagic function in malaria parasites

Atg8 family proteins are highly-conserved eukaryotic proteins with diverse autophagy and non-autophagic functions in eukaryotes. While the structural features required for conserved autophagy functions of Atg8 are well-established, little is known about the molecular changes that facilitated acquisition of divergent, non-autophagic functions of Atg8. The malaria parasite Plasmodium falciparum offers a unique opportunity to study non-autophagic functions of Atg8 family proteins because it encodes a single Atg8 homolog whose only essential function is in the inheritance of an unusual secondary plastid called the apicoplast. Here we used functional complementation to investigate the structure-function relationship for this divergent Atg8 protein. We showed that the LC3-interacting region (LIR) docking site (LDS), the major interaction interface of Atg8 protein family, is not sufficient for PfAtg8 apicoplast function. Other regions previously implicated in canonical Atg8 interactions, the ubiquitin-interacting motif (UIM) docking site (UDS) and the N-terminal helix are not required for PfAtg8 function. Finally, the unique Apicomplexan-specific loop previously implicated in interaction with membrane conjugation machinery in vitro, is not required in vivo neither for membrane conjugation nor for the effector function of PfAtg8. These results suggest that the effector function of PfAtg8 is mediated by structural features distinct from those previously identified for macroautophagy and selective autophagy functions. ImportanceThe most extensively studied role of Atg8 proteins is in autophagy. However, it is clear that they have other non-autophagic functions critical to cell function and disease pathogenesis yet understudied compared to their canonical role in autophagy. Mammalian cells contain multiple Atg8 paralogs that have diverse, specialized functions. Gaining molecular insight into their non-autophagic functions is difficult because of redundancy between the homologs and their role in both autophagy and non-autophagic pathways. Malaria parasites such as Plasmodium falciparum are a unique system to study a novel, non-autophagic function of Atg8 separate from its role in autophagy: They have only one Atg8 protein whose only essential function is in the inheritance of the apicoplast, a unique secondary plastid organelle. Insights into the molecular basis of PfAtg8s function in apicoplast biogenesis will have important implications for the evolution of diverse non-autophagic functions of the Atg8 protein family.

biochemistry↗

bioRxiv2021

A modular, dynamic, DNA-based platform for regulating cargo distribution and transport between lipid domains

Cell membranes regulate the distribution of biological machinery between phase-separated lipid domains to facilitate key processes including signalling and transport, which are among the life-like functionalities that bottom-up synthetic biology aims to replicate in artificial-cellular systems. Here, we introduce a modular approach to program partitioning of amphiphilic DNA nanostructures in co-existing lipid domains. Exploiting the tendency of different hydrophobic "anchors" to enrich different phases, we modulate the lateral distribution of our devices by rationally combining hydrophobes, and by changing nanostructure size and its topology. We demonstrate the functionality of our strategy with a bio-inspired DNA architecture, which dynamically undergoes ligand-induced reconfiguration to mediate cargo transport between domains via lateral re-distribution. Our findings pave the way to next-generation biomimetic platforms for sensing, transduction, and communication in synthetic cellular systems.

biophysics↗

bioRxiv2018

A mutagenesis screen for essential plastid biogenesis genes in human malaria parasites

Endosymbiosis has driven major molecular and cellular innovations. Plasmodium spp. parasites that cause malaria contain an essential, non-photosynthetic plastid, the apicoplast, which originated from a secondary (eukaryote-eukaryote) endosymbiosis. To discover organellar pathways with evolutionary and biomedical significance, we performed a mutagenesis screen for essential genes required for apicoplast biogenesis in P. falciparum. Apicoplast-minus mutants were isolated using a chemical rescue that permits conditional disruption of the apicoplast and a new fluorescent reporter for organelle loss. Five candidate genes were validated (out of 12 identified), including a TIM-barrel protein that likely derived from a core metabolic enzyme but evolved a new activity. Our results demonstrate the first forward genetic screen to assign essential cellular functions to unannotated P. falciparum genes. A putative TIM-barrel enzyme and other newly-identified apicoplast biogenesis proteins open opportunities to discover new mechanisms of organelle biogenesis, molecular evolution underlying eukaryotic diversity, and drug targets against multiple parasitic diseases.

cell biology↗

bioRxiv2017

Atg8 is essential specifically for an autophagy-independent function in apicoplast biogenesis in blood-stage malaria parasites

Plasmodium parasites and related pathogens contain an essential non-photosynthetic plastid organelle, the apicoplast, derived from secondary endosymbiosis. Intriguingly, a highly conserved eukaryotic protein, autophagy-related protein 8 (Atg8), has an autophagy-independent function in the apicoplast. Little is known about the novel apicoplast function of Atg8 and its importance in blood-stage P. falciparum. Using a P. falciparum strain in which Atg8 expression was conditionally regulated, we showed that PfAtg8 is essential for parasite replication. Significantly, growth inhibition caused by the loss of PfAtg8 was reversed by addition of isopentenyl pyrophosphate (IPP), which was previously shown to rescue apicoplast defects in P. falciparum. Parasites deficient in PfAtg8, but growth rescued by IPP, had lost their apicoplast. We designed a suite of functional assays, including a new fluorescence in situ hybridization (FISH) method for detection of the low-copy apicoplast genome, to interrogate specific steps in apicoplast biogenesis and detect apicoplast defects which preceded the block in parasite replication. Though protein import and membrane expansion of the apicoplast were unaffected, the apicoplast was not inherited by daughter parasites. Our findings demonstrate that, though multiple autophagy-dependent and independent functions have been proposed for PfAtg8, only its role in apicoplast biogenesis is essential. We propose that PfAtg8 is required for fission or segregation of the apicoplast during parasite replication.

cell biology↗