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The infection cycle of the haloarchaeal virus HFTV1 is tightly regulated and strongly inhibits motility of its host

Although viruses have been shown to infect all domains of life, our understanding of the genetic program behind the exploitation of host resources to produce progeny virions is thus far limited to several bacterial viruses. Therefore, to elucidate the transcriptome of euryarchaeal viruses and their hosts, we employed RNAseq analysis of samples taken at different time points from Haloferax gibbonsii LR2-5 cultures infected with the lytic model virus Haloferax Tailed Virus 1 (HFTV1). While following the transcription of viral genes throughout the infective life cycle, we observed a tight temporal regulation of viral transcripts as well as differential expression from within viral gene clusters. Furthermore, anti-sense RNAs (asRNAs) appear to play an important role in support of the timing of late-expressed viral genes. Therefore, with many differentially expressed transcripts, including intragenic transcripts and asRNAs, the regulatory machinery employed by HFTV1 contrasts with viral model systems (based on phages), in which antitermination and/or alternative polymerases (seemingly lacking in HFTV1) are more widespread. When looking into differentially expressed host genes, we observed a strong downregulation of genes involved in motility, such as the archaellum and chemotaxis machinery, which was confirmed with swimming assays of HFTV1 infected cells. This might be a strategy of the virus to redirect energy flowing into movement towards the production of virions. In conclusion, this work thus provides a stepping stone for further exploration of the intriguing strategies of viral transcriptional regulation of their infection cycle across the domains of life. IMPORTANCEViruses infect members of all three domains of life, including Archaea. Euryarchaea are widespread microorganisms found in various environments such as the human gut and solar salterns. Due to the exceptional availability of cell biology and genetic tools of some salt-loving archaea, they are a model system to extrapolate from. Insights into the regulation of viral infections are of particular importance, especially since HFTV1, has been adopted as a model virus by the archaeal viral community. We found that, while harboring parallels with bacterial viruses, such as tight temporal regulation, HFTV1 harbors an impressive number of differentially expressed transcriptional units. Furthermore, anti-sense RNAs and intragenic regulatory elements seem to play a much more prominent role in HFTV1 gene expression. Thus, this work challenges current models and provides valuable new insights into the gene regulation of viral infection of archaea, which mark similarities and differences with viruses from other domains of life.

microbiology↗

Multiplexed, scalable analog recording of gene regulation dynamics over weeks using intracellular protein tapes

Gene expression is constantly regulated by gene regulatory networks that consist of multiple regulatory components to mediate cellular functions. An ideal tool for analyzing gene regulation processes would provide simultaneous measurements of the dynamics of many components in the gene regulatory network, but existing methodologies fall short of simultaneously tracking the dynamics of components over long periods of time. Here, we present CytoTape--a genetically encoded, modular, and scalable analog recorder for continuous, multiplexed in situ recording of gene regulation dynamics over multiple days and weeks at single-cell resolution. CytoTape consists of a flexible, thread-like, elongating intracellular protein self-assembly engineered via AI-guided rational design. Gene regulation dynamics, together with timestamps for reconstruction of the continuous time axis, are directly encoded via distinct molecular tags distributed along single CytoTape assemblies in live cells, to be readout at scale after fixation via standard immunofluorescence imaging. CytoTape recorders are modularly designed to record gene expression driven by a variety of activity-dependent promoters. We demonstrated the utility of CytoTape in mammalian embryonic kidney cells, cancer cells, glial cells, and neurons, achieving simultaneous recording of five cell plasticity-associated transcription factor activities and immediate early gene expression levels, namely CREB, c-fos, Arc, Egr1, and Npas4 activities, within single cells in a spatiotemporally scalable manner. CytoTape revealed complex waveforms and nonlinear temporal couplings among these cellular activities, enabling investigations of how gene regulation histories and intrinsic signaling states shape transcriptional logics. We envision CytoTape to have broad applications in both basic and disease-related cell biology research.

synthetic biology↗

Chip-Based 3D Interferometric Nanoscopy

Ultra-high resolution 3D single-molecule localization microscopy (SMLM) traditionally requires complex dual-objective lenses (4Pi) configurations to enhance axial (z) precision through interferometry. Here we present a streamlined chip-based alternative, Silicon-assisted interferometric Localization Microscopy (SiLM), which achieves comparable performance using a single-objective lens design. By combining tunable axial structured illumination field, arising from surface-generated excitation interference, with asynchronous interferometry, SiLM enhances axial localization precision to approximately twice that of the lateral (xy), comparable to 4Pi-based methods. Additionally, SiLM provides intrinsic axial self-referencing, offering dramatically improved robustness against mechanical drift. Our method is readily implementable on standard SMLM-capable microscopes and supports a broad range of applications including dual-color imaging, extended-depth imaging, and live-cell 3D single-molecule tracking. Using SiLM, we demonstrate accurate mapping of the stratified nanoscale architecture of integrin-based focal adhesions, establishing it as a powerful and accessible method for high-precision 3D structural cell biology.

biophysics↗

A Guanine-quadruplex located on the negative strand of the hepatitis C virus facilitates efficient genomic RNA synthesis

Guanine-rich nucleic acids can form a unique secondary structure called Guanine-quadruplexes (G4s). These G4s are conserved across all domains of life and in viruses, where they can play important regulatory roles in the viral lifecycle. In flaviviruses, the (-) strand 3 untranslated region (UTR) is essential for initiating genomic RNA synthesis. Within the (-) strand 3 UTR of hepatitis C virus (HCV), a highly conserved G4 is located within stem-loop IIy (SLIIy), spanning nucleotides 110-131--a region that is essential for efficient replication. Using bioinformatics, we demonstrate that nucleotides 110-131 are highly conserved across HCV genotypes 1-7, and this region within SLIIy can likely adopt a more energetically favorable G4 conformation rather than the predicted hairpin. From biophysical and cell biology experiments, we show that stabilizing the predicted hairpin structure in SLIIy disrupts RNA synthesis, while successive guanine-to-adenine mutations within the G4 sequence impair G4 formation and hinder replication. Combining our findings with prior studies, we propose that the SLIIy G4 plays a crucial role in recruiting NS3 helicase, which is necessary for unwinding RNA structures and facilitating access for NS5B polymerase. The G4 may serve as a regulatory switch that modulates helicase binding and replication efficiency. We propose that in the absence of a stable G4, NS3 recruitment or unwinding is impaired, leading to inefficient RNA unwinding and reduced polymerase activity, ultimately hindering viral replication. These results reveal a previously unrecognized role for G4 structures in the HCV replication cycle, highlighting their importance in regulating (+) strand RNA synthesis. ImportanceHepatitis C virus is a positive-sense single-stranded RNA virus that relies on an intermediate negative strand to generate new genomic RNA. While the last 157 nucleotides of the negative-strand 3 untranslated region are known to be essential for replication, the underlying regulatory mechanisms have remained poorly understood. Our study demonstrates that a highly conserved guanine-rich sequence (107-131 nt) located on the negative strand SLIIy forms a guanine-quadruplex secondary structure that plays a pivotal role in orchestrating viral RNA synthesis. This G4 not only promotes efficient replication but likely acts as a molecular target to recruit the NS3 helicase, thereby allowing access for the NS5B polymerase. Disruption of the G4 structure and stabilization of the stem-loop severely impairs viral replication. These findings reveal an unrecognized layer of post-transcriptional regulation in the HCV lifecycle and establish G4 RNA structures as a critical element in HCV genome replication.

biophysics↗

Desmoplakin loss leads to PKC-dependent insertion of series sarcomeres and contractile dysfunction in cardiomyocytes

BackgroundMutations in DSP, which encodes the protein desmoplakin, lead to cardiomyopathy with unusually high penetrance. Clinical features include ventricular tachyarrhythmias, fibro-fatty infiltration of both ventricles, and ultimately dilated cardiomyopathy. While some data have been gathered to explain the electrophysiological and contractile consequences of desmoplakin cardiomyopathy, a comprehensive mechanism linking DSP mutations to ventricular dilation and heart failure remains elusive. MethodsWe use iPSC-derived engineered heart tissue (EHT) bearing a functional desmoplakin haploinsufficiency to model the heart failure phenotype that occurs in desmoplakin cardiomyopathy. Functional haploinsufficiency is secondary to a missense mutation, R451G, that results in proteolytic degradation of desmoplakin with no detectable protein. We complement functional data obtained in tissue-engineered constructs with cell biology assays in 2D cardiomyocytes to glean insights into the mechanism and mechanobiology of desmoplakin cardiomyopathy. ResultsEngineered heart tissues harboring a desmoplakin insufficiency recapitulate a patient phenotype notable for hypocontractility and ventricular dilation. Surprisingly, DSP-mutant tissues exhibited a shortened resting sarcomere length that was dependent on protein kinase C activity. Concurrently, mechanical load on -catenin was increased, suggesting a mechanism by which desmosomal insufficiency redistributes force to adherens junctions. Excessive loading on adherens junctions may act as a stimulus for avid insertion of series sarcomeres, shortening the length per sarcomere, and resulting in a contractile deficit. PKC inhibition rescues shortened sarcomere length in DSP- mutant tissues, suggesting that it could be a target for future molecular therapies. ConclusionsOur study uncovers a novel mechanism underlying systolic dysfunction in desmoplakin cardiomyopathy. We not only recapitulate the disease phenotype, but we identify sarcomere length regulation through altered force transmission at the intercalated disc as a previously-unrecognized mechanism.

physiology↗

Novel infection by Mucor hiemalis kills Caenorhabditis hosts through intestinal perforation

The nematode Caenorhabditis elegans has emerged as a popular model system to investigate cell biology and host-pathogen interactions. Presently, C. elegans is studied as a natural host of intracellular pathogens such as microsporidia and Orsay virus along with extracellular bacterial and fungal pathogens. The use of C. elegans as a model in host-pathogen research is limited by the number of naturally occurring pathogens to the organism. Through a sampling project to identify new pathogens of C. elegans, we identified the fungus Mucor hiemalis as a pathogen of Caenorhabditis species. We observed the fungus in the intestinal lumen of wild-caught Caenorhabditis briggsae, and co-culturing the wild-caught species with infection reporter C. elegans confirmed infection by M. hiemalis. This study characterizes the fungal infection by M. hiemalis in Caenorhabditis nematodes. Fluorescence microscopy with fungal staining revealed the life cycle of M. hiemalis within multiple Caenorhabditis species at varying growth stages. We observed the killing of nematodes by M. hiemalis via intestinal perforation and assessed its host range through a series of lifespan assays. We investigated the food preference of C. elegans and determined that nematodes show a preference towards food that contains M. hiemalis spores. Lastly, we evaluated common C. elegans transcriptional immune responses and found that M. hiemalis does not induce genes associated with the intracellular pathogen response or other responses seen with previously studied bacterial and fungal pathogens. Characterization of this fungal infection in Caenorhabditis nematodes will provide new insights into the biology of pathogenic fungi and host immune responses.

microbiology↗

A Tiered Approach to Human Synapse Proteomics: Optimized LC-MS/MS Analysis of Whole-Tissue and Synaptosome Preparations from Frozen Post-mortem Brain Samples

Recent advancements in neuroproteomics have enabled detailed analysis of protein expression and function in the human brain. Post-mortem human brain studies have significantly advanced our understanding of the relationship between genetics, cell biology of neurological and psychiatric disorders and their clinical diagnosis. Synaptic dysfunction often has a central role in these disorders. Therefore, we specifically evaluated the sensitivity of liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect synaptic proteins in whole-tissue lysates versus synaptosome preparations. First, we optimized sample preparation protocols for frozen human gray matter (GM), refining the suspension TRAPping (sTRAP) digestion method to improve protein solubilization and Cysteine reduction and alkylation using thin human tissue sections, and to accomplish low technical variation by minimizing sample handling. We achieved a highly reproducible sample preparation workflow by rigorously applying standardization and randomization across dissection, processing, and LC-MS/MS runs. Second, comparative LC-MS/MS analysis showed that cortical whole-tissue lysates are a practical solution for large-scale studies and broadly detected synaptic proteins focusing on excitatory neurons. However, enrichment by synaptosome isolation offered improved resolution of synapse-specific proteins. Because synapse-proteomics enables insight into spatial regulation--i.e., alterations at the synapse that are not reflected in the soma-we recommend a tiered approach: initial whole-tissue analysis for broad disease-associated changes, followed by targeted synaptosome proteomics to deepen insight into synaptic alterations. This strategy optimally balances throughput, reproducibility, and biological relevance, and enhances the study of brain disorders through proteomics. Moreover, analyzing synaptic proteins first at the tissue level improves insight into overall regulation of synaptic proteins induced by synapse loss or gain.

neuroscience↗

Developmental coordination of mitochondrial dynamics and membrane remodeling drives organelle morphogenesis

How complex organelles remodel their architecture during development remains a fundamental question in cell biology. Using the Drosophila nebenkern, a giant multilamellar mitochondrion that transforms into two elongated mitochondrial derivatives during spermiogenesis, we dissect the challenges underlying large organelle morphogenesis. We show that this transformation is achieved through coordinated control of fusogenicity, membrane geometry, and membrane redistribution. PINK1/Parkin-dependent downregulation of the mitofusin Marf reduces mitochondrial fusogenicity and stabilizes organelle separation following division. Concurrently, membrane tubulation establishes a geometric template for DRP1 recruitment, specifying a deterministic fission plane within a large mitochondrial sheet structure. Mitochondrial elongation is accompanied by the formation of mitochondria-derived inverted vesicles (MDiVs), previously undescribed double-membraned structures that may redistribute membrane through inward budding independently of canonical fission and fusion. More broadly, our work establishes a framework for understanding how membrane geometry, regulated fusogenicity, and vesicle-mediated remodeling cooperate to generate complex organelle architectures during development. Significance statementLarge organelles pose unique challenges for scaling and division because canonical membrane dynamics must be coordinated across extensive membrane architectures. Using a giant multilamellar mitochondrial structure as a model, we show that organelle morphogenesis is achieved through independent yet coordinately regulated mechanisms that spatially pattern division, stabilize partitioning, and redistribute membranes through intraorganellar vesicles with an inverted topology. These findings reveal principles of organelle morphogenesis that extend beyond conventional fission-fusion models.

developmental biology↗

Ultrasonic rewarming of cryopreserved alginate encapsulated liver spheroids

Rapid volumetric rewarming methods are needed to enable the effective cryopreservation and recovery of large volumes of biological cells for therapy and banking of tissues and organs. Ultrasonic rewarming is currently under development, but its effect on cells and their post-rewarming viability has not yet been established. Here, we compare ultrasonic rewarming with the gold-standard 37{degrees}C water bath using cryovials containing cryopreserved alginate encapsulated liver spheroids. Mean rewarming rates are used to establish the exposure time to rewarm to 5{degrees}C for higher power (100 W) and lower power (20 W) ultrasonic rewarming. These electrical powers correspond to free-field pressures along the central cryovial axis of 2.8 MPa and 1.3 MPa, respectively. Ultrasonic rewarming is faster than the gold-standard (120 {+/-}5 s), taking 88 s (36% faster) and 34 s (350% faster) to rewarm to 5{degrees}C with the lower and higher powers. We measure post-rewarming liver spheroid viability and viable cell number across the 96-hour recovery period. The lower power improves viability by 1% and the higher power reduces viability by 2% on average, relative to the gold-standard. There were no significant differences in viable cell number between rewarming methods. Our findings will serve as a foundation for ultrasonic cryovial rewarming and demonstrates potential for scaling to larger volumes.

biophysics↗

Identification of novel exosomal miRNAs and their role in diagnosis and prognosis of Triple Negative Breast Cancer

Triple-negative breast cancer (TNBC) is a clinically aggressive subtype with poor prognosis and limited treatment options. Exosomal microRNAs (miRNAs), encapsulated within secretory vesicles, have emerged as promising biomarkers for cancer detection and monitoring. In this study, we identify five novel exosomal miRNAs--hsa-miR-6803, hsa-miR-1180, hsa-miR-4728, hsa-miR-1915, and hsa-miR-940--that are consistently overexpressed in TNBC cells, stem-like subpopulations, and patient tumor tissues. Integrated analysis of public datasets and in vitro validation revealed that elevated expression of these miRNAs correlates with poor overall survival. Functional assays demonstrated that miR-1180 and miR-4728 significantly promote TNBC cell migration and invasion. These miRNAs also target critical oncogenic pathways, including Wnt, Notch, and EGFR. Their enrichment in exosomes highlights their translational potential as liquid biopsy-based biomarkers and therapeutic targets. This work is the first to link this miRNA panel to both TNBC tumorigenesis and stem-like cell biology, offering new insights into disease progression and potential strategies for personalized care.

cancer biology↗

A mouse model of PTEN Hamartoma Tumour Syndrome reveals that loss of the nuclear function of PTEN drives macrocephaly, lymphoid overgrowth, and late-onset cancer

PTEN Hamartoma Tumour Syndrome (PHTS) is a rare disorder characterized by germline heterozygous mutations in the PTEN tumour suppressor gene, leading to multi-organ/tissue overgrowth, autism spectrum disorder and increased cancer risk. PHTS individuals display heterogeneity in phenotypes, which has been linked in part to the diverse genetic alterations in the PTEN gene and the multifaceted functions of this protein. Indeed, while PTEN primarily functions as a PIP3 lipid phosphatase in the cytosol, regulating PI3K/AKT signalling, a pathway commonly deregulated in cancer, it also plays crucial roles in maintaining chromosomal stability through nuclear activities such as double strand (ds) DNA damage repair. Recent studies have identified a subset of missense PHTS variants that cause nuclear exclusion of PTEN, impairing its nuclear functions. Here, we present our findings from one such pathogenic variant, PTEN-R173C, frequently found in PHTS and somatic cancers. Using cell biological and mouse modelling approaches, we show that PTEN-R173C has higher PIP3 phosphatase activity than wild-type PTEN, resulting in effective regulation of canonical PI3K/AKT signalling. However, PTEN-R173C is unstable and excluded from the nucleus. Aligning with their near normal PI3K/AKT signalling, Pten+/R173Cmice display a low incidence of solid tumours compared to Pten+/-mice. Pten+/R173C mice also exhibit lymphoid hyperplasia and macrocephaly which correlates with compromised nuclear functions of PTEN-R173C. That nuclear functions are compromised is demonstrated by reduced dsDNA damage repair in Pten+/R173Cmice. Integrating PHTS patient data with findings from our mouse model, our study indicates that nuclear dysfunction of pathogenic PTEN variants is a key factor in predicting the onset of the different PHTS-associated phenotypes. We speculate that late-onset cancer in individuals with nuclear-excluded PTEN results from genetic alterations unrelated to PTEN itself, facilitated by impaired PTEN-mediated dsDNA damage repair.

genetics↗

Dendritic Polyglycerol Amine Substrate Extends the Viability of Mixed Glial Cultures for Repeated Isolation of Immature Oligodendrocyte Lineage Cells

Primary mixed glial cultures are key tools to isolate and study astrocytes, microglia and oligodendrocytes. Cell-substrate adhesion is critical for neural cell survival and differentiation. Cationic polymers like poly-D-lysine (PDL) are widely used to promote cell adhesion to cell culture substrates, however, PDL is not stable long-term, with cultured cells often detaching (peeling) after 2-3 weeks. Dendritic polyglycerol amine (dPGA) is a synthetic polycationic non-protein polymer biomimetic of poly-lysine that is highly resistant to degradation by cellular proteases. Substrates coated with dPGA promote cell adhesion and improve survival in long-term neuronal cultures. Here we assessed dPGA as a substrate coating to provide long-term support for mixed glial cultures. Oligodendrocyte precursor cells (OPCs) were isolated weekly by differential adhesion from cultures grown in T75 flasks with PDL or dPGA-coated substrates. Following two "shake-off" isolations, the cell layer in most PDL-coated flasks fully detached, rendering these flasks unusable for further culture. In contrast, dPGA-coated flasks consistently yielded cells for six or more sequential isolations over seven weeks in culture. dPGA-coated flasks produced more cells, a greater percentage of O4+ cells, and maintained similar proportions of OPCs and MBP-positive cells as when isolated from a PDL-coated substrate. dPGA is cyto-compatible, functionally superior, easy to use, low cost and a stable alternative to conventional cell substrate coatings. The enhanced long-term stability of mixed glial cultures grown on a dPGA substrate has the capacity to increase cellular yield, reduce animal use, and facilitate studies of oligodendrocyte cell biology.

neuroscience↗

Growth Mechanics and the emergence of metabolic oscillations in growing cells

Unicellular organisms exhibit oscillations in metabolic activity and gene expression, even when growing in static media with no external dynamic stimuli. The cause and possible function of these endogenous oscillations are still not fully understood, with different hypotheses including its origin as a byproduct of misguided regulatory processes. To investigate whether such oscillations could be functional rather than incidental, we introduce Growth Mechanics (GM) as a general mathematical framework to study the dynamic resource allocation in models of whole self-replicating cells, built exclusively on the first principles of fitness maximization, mass conservation, nonlinear reaction kinetics, and constant cell density. Inspired by the physical theory of classical mechanics, we first find the simplest mathematical description the problem in terms of generalized coordinates, and then solve the optimal dynamical resource allocation for cells growing in any given medium using the Euler-Lagrange equation, resulting in analytical "equations of optimal motion" (EOM) that apply for growing cells in general. We then solve these equations numerically for simple growing cell models and show that, in general, oscillations are necessary to maximize cell fitness by getting more saturated reactions at the time they are most needed. We also show how the EOM predict the emergence of quasi-linear dependencies of the metabolic oscillation frequency and the ribosome protein allocation with the cell growth rate at different growth media, in line with the "growth laws" observed experimentally in microbes. This work contributes to the foundations of theoretical cell biology with general quantitative principles and a bridge to theoretical tools of physics.

biophysics↗

Quantifying distribution shifts in single-cell data with scXMatch

A basic task that frequently arises when analyzing single-cell data is to assess if there is a global distribution shift between the data profiles of cells from two different conditions. Widely used approaches to address this task such as visual inspection of two-dimensional representations or clustering-based workflows lack a solid statistical underpinning and are notoriously unstable and prone to confirmation bias. To promote more rigorous analysis, we here present the scverse-compatible Python tool scXMatch. scXMatch is based on a non-parametric graph-based test to quantify distribution shifts in arbitrary data spaces for which a suitable distance measure is available. We evaluated scXMatch on single-cell gene expression, chromatin accessibility, and imaging-derived cell morphology data, showing that it can robustly detect distribution shifts for different types of single-cell data. scXMatch thus aims to set a new standard in the single-cell biology field, replacing easy-to-manipulate semi-manual distribution shift quantification workflows by principled statistical testing.

bioinformatics↗

Multiparametric Correlative Topographical and Volumetric Fluorescence Microscopy

Live-cell imaging of cell surface topography and intracellular architecture is essential for understanding cellular function. However, conventional approaches often involve trade-offs between resolution, invasiveness, and volumetric coverage. Here, we present an integrated Scanning Ion Conductance Microscope and single-objective Oblique Plane Microscope (SICM-OPM) system that enables simultaneous non-contact topographical imaging and volumetric fluorescence imaging within the same live cell. Beyond correlative live imaging, the platform supports nanomechanical mapping with tens-of-nanometres resolution, fluorescence-guided localised molecular delivery via the SICM, and benefits from reduced photobleaching due to light-sheet excitation. We demonstrate this platforms capabilities by visualising imipramine-induced T-tubule remodelling in live cardiomyocytes, revealing subsurface detubulation while surface morphology remains preserved. Additionally, we show precision delivery of fluorescent cargos--including dextrans and -synuclein--into diatom and mammalian cells, alongside localised stiffness mapping to evaluate mechanical responses. We believe this technique opens new avenues for correlative structural, functional, and biophysical studies in live cells, with broad relevance to cell biology, neurodegeneration, and mechanobiology.

biophysics↗

DIA-PASEF Proteomic Profiling Reveals MpkA-Dependent Iron Stress Responses and Siderophore Biosynthesis in Aspergillus nidulans

Filamentous faungi play essential roles in biotechnology as producers of valuable bioproducts and, conversely, as opportunistic pathogens. Aspergillus nidulans is a widely used model organism for fungal genetics and cell biology; however, comprehensive proteomic references for this species remain limited. In this study, we applied a data-independent acquisition-parallel accumulation serial fragmentation (DIA-PASEF) approach to enable efficient and in-depth proteome profiling of A. nidulans. Leveraging ion mobility-based ion cloud information from DDA-PASEF experiments, we developed DIA-PASEF methods that identified 3,904 proteins across biological triplicates grown in rich medium. Compared to prior studies, this represents an increase in protein identifications of more than 140% and was achieved with more than five-fold reduction in analysis time. We employed this newly developed DIA-PASEF methodology to conduct both proteomic and phosphoproteomic analyses under iron-depleted conditions in an MpkA protein-kinase deficient mutant ({Delta}mpkA). The {Delta}mpkA strain exhibited expression of approximately 500 additional proteins and occupancy of over 1,800 additional phosphosites relative to a control. Differentially expressed and phosphorylated proteins increased by more than an order of magnitude in the {Delta}mpkA mutant across both iron-replete and iron-deplete conditions. Gene Ontology (GO) enrichment analysis revealed broader and distinct biological processes under iron-depleted conditions, highlighting adaptive responses specific to iron limitation and MAPK pathway disruption. This work establishes a high-coverage proteomic resource for A. nidulans and provides novel insights into fungal stress responses and signaling network perturbation. Importantly, high-throughput proteomic profiling reveals that limited iron availability and MAPK pathway disruption increases siderophore biosynthesis.

systems biology↗

The RXLR-EER Motif Determines an Unconventional Secretion Pathway Associated with Extracellular Vesicle Production

Phytophthora infestans, the cause of potato late blight disease, delivers a suite of RXLR effectors into host plant cells to subvert immunity, whereas apoplastic effectors act extracellularly. Although the RXLR-EER motif in these effectors is critical for host translocation and is cleaved prior to secretion, the relevance of this processing is poorly understood. Prior evidence suggests RXLR effectors utilize a distinct, unconventional secretion pathway, raising the question of whether the RXLR-EER motif influences selection of the secretion route. Here, we combined genetic, molecular and cell biology approaches to investigate the secretion pathway of RXLR effectors. Confocal microscopy revealed that RXLR and apoplastic effectors localize to distinct vesicular compartments in cultured hyphae. Moreover, fusing the ER retention signal KDEL to RXLR effectors did not impair their secretion, in contrast to apoplastic effectors, which were retained in the endomembrane system, indicating that RXLR effectors bypass the canonical ER-to-Golgi pathway. Importantly, RXLR effectors associate with extracellular vesicles (EVs), whereas RXLR-EER motif mutants show reduced EV association and are rerouted through the ER-to-Golgi secretion pathway. These findings demonstrate that the RXLR-EER motif governs effector sorting into an unconventional, EV-linked secretion route. This study sheds light on the molecular basis of effector trafficking in P. infestans and underscores the potential role of EVs in delivering virulence factors during host colonization.

microbiology↗

Is Language a Mechanical Signal? Cytoskeletal Responses to Speech in Yeast

What if vocal language were not only a medium for human communication but a vibrational force that leaves structural traces in living cells? This study explores how audible sound, particularly the structured elements of human speech, affects the cytoskeleton of Saccharomyces cerevisiae. Using a direct-contact acoustic system, we exposed yeast to distinct sound types: tonal vibrations, broadband noise, and consonant phonemes. Fluorescence microscopy revealed that tonal stimuli with coherent low-frequency patterns enhanced actin polymerization and shmoo formation, both markers of polarity and mating. In contrast, broadband noise disrupted actin integrity, while consonants produced no measurable effects. These results suggest that rhythmic continuity and spectral coherence, key features of speech, can modulate cytoskeletal organization in non-auditory cells. By reframing vocal language as mechanical input rather than semantic content, this study bridges microbial cell biology with acoustic ecology and proposes a new lens for exploring how human-generated soundscapes physically influence living systems.

microbiology↗