Biology subjects

Lange, J. J.

Publications and source records attributed to Lange, J. J..

At least 19 recordsLinked to original sources

bioRxiv2026

Fss1, a CTNS/PQ domain protein, has dual roles in promoting fertility in Schizosaccharomyces pombe

Sexual reproduction is critical in most eukaryotes, but many genes required for the process remain to be identified and characterized. A previous saturation mutagenesis study in Schizosaccharomyces pombe implicated several unnamed genes in having roles in sexual reproduction. In this work, we provide an initial characterization of 13 of these genes and an in-depth characterization of one gene, fss1 (facilitates starvation sex). Fss1 has high structural homology to the human PQ-loop repeat family PQLC2 transceptor (amino acid transporter plus membrane receptor), but likely lacks the ability to transport amino acids due to loss of a critical PQ motif. fss1{Delta} mutants have no defects in meiosis but have pronounced defects in packaging the products of meiosis into spores, particularly in nutrient-poor environments. The spore formation phenotypes are recessive as fss1{Delta} spores produced by a fss1+/fss1{Delta} heterozygote develop normally. Immuno-gold electron microscopy shows Fss1 localizes to vesicles that fuse to the ephemeral outer spore membrane as the spore wall matures below. Fss1 protein is also produced within spores and this population is required for normal vacuole morphology and timely germination. We propose a model in which Fss1 is a vesicle receptor that facilitates spore wall formation from diploid-expressed protein, while spore-expressed protein acts at the vacuole to promote an internal cellular environment that facilitates germination. This work expands our catalog of genes that contribute to sexual reproduction and identifies two previously unknown tasks in spore biology that are both facilitated by Fss1.

genetics↗

bioRxiv2026

Nascent protein retention at polysomes reduces kinetic barriers to self-assembly

Living proteomes are necessarily far from equilibrium. It is paradoxical, then, that reducing the translation of new proteins -- which should promote equilibration -- instead prolongs life. We investigated the impact of translational flux to nucleation barriers that preserve the solubility of proteins destined to form amyloids or other assemblies. By manipulating translation initiation rates directly or indirectly, across yeast and human cells, and across a variety of supersaturable proteins, we find that accelerating translation initiation broadly accelerates nucleation irrespective of their global concentrations. We showed that this effect was confined to polysomes and was enhanced by N-terminal placement or other features that retained the nascent aggregating domain at polysomes. Finally, we show that intrinsically disordered regions with high tendencies to self-associate are specifically positioned to do so co-translationally, providing evidence that cotranslational nucleation has shaped proteome evolution.

cell biology↗

bioRxiv2026

Pericentromeric repeat copy number tunes heterochromatin dosage to control chromosome segregation and gene expression in fission yeast

Centromeres are essential for chromosome segregation, yet in many genomes they are composed entirely of rapidly evolving repetitive DNA, embedded in other repetitive DNA that forms pericentromeric heterochromatin. Due to the difficulties of manipulating these repeat-rich regions, how the relative size of pericentromeric repeat regions influences chromosome segregation remains an open question. Here, we take advantage of the tractable Schizosaccharomyces pombe system by combining population-level analysis, complete long-read assemblies, and engineered near-isogenic strains to test how pericentromeric repeat copy number affects chromosome biology in its native context. We find that pericentromeric dh/dg arrays on chromosome 3 vary almost tenfold in size among natural S. pombe isolates, ranging from 35 to 265 kb. We converted this natural diversity into an experimental system of nearly isogenic strains that primarily differ in pericentromere size (35 to >350 kb). We found that pericentromere size does not alter baseline growth under standard conditions. However, larger pericentromeres alter transcriptional output and sensitize cells to spindle stress. We show that this spindle-stress phenotype depends on heterochromatin: loss of the H3K9 methyltransferase Clr4 abolishes size-dependent differences, whereas artificial targeting of the Chromosomal Passenger Complex to heterochromatin partially rescues the defect. Thus, we find that larger pericentromeres act as sinks for limiting regulatory factors, weakening their effective concentration at centromeres and compromising faithful chromosome segregation under stress. These results establish that naturally occurring copy-number variation within repetitive pericentromeric DNA is not merely noise, but a functional source of variation in chromosome segregation and gene regulation. Our work provides an experimentally tractable framework for understanding how repeat expansion in centromere-proximal heterochromatin influences chromosome behavior across eukaryotes.

genetics↗

bioRxiv2026

Eukaryotic domestication of a bacterial immune protein following horizontal transfer

Many components of eukaryotic innate immunity originated from bacterial immune systems. However, it is unclear how eukaryotes acquire these genes and how the components are domesticated into eukaryotic physiology. Here, we discovered a recent instance of bacteria-eukaryote horizontal transfer and used it to characterize the genetic and biochemical changes that accompanied eukaryotic acquisition. We focus on Toll/interleukin-1 receptor (TIR) domains, which are widespread yet potentially costly immune modules associated with inflammation and/or cell death. By generating an atlas of TIR diversity across the tree of life, we phylogenetically categorized the domains and uncovered highly diverged, eukaryotic TIR families. This analysis revealed a horizontal transfer event that created the TirBCD protein family of Dictyostelium amoebae, which is closely related to the bacterial immune protein TIR-STING. While the TIR domain was transferred into amoebae, the genomic locus did not contain known regulatory domains nor other components of a bacterial operon. Nevertheless, TirC retained biochemical and physiological similarities to TIR-STING. TirC is a highly potent NADase, capable of oligomerizing into large complexes and depleting NAD+ even at very low protein concentrations. When expressed in heterologous systems, TirC was spontaneously active and highly toxic. In contrast, the natural amoeba host tolerated expression of full length TirC, showing that the cells can typically regulate its activity and avoid autoimmunity. However, a truncated form of TirC induced rapid rounding and cell lysis. These results suggest that amoebae have used TirC to retool a form of bacterial cell death for use in eukaryotic cells. Overall, this study uncovers recent eukaryotic TIR evolution that captures features of both bacterial and eukaryotic immunity. We also expect that the TIR domain atlas will be useful to researchers across model systems as they explore the vast diversity of TIR molecular and cellular functions.

microbiology↗

bioRxiv2026

Intracellular TDP-43 amyloid nucleates from arrested nascent condensates

TDP-43 is a model protein for pathophysiological phase transitions, forming a multitude of intracellular assemblies with different physical properties. Physiological condensation is widely presumed to precede pathological aggregation, but the causal relationships between different modes of assembly in vivo are still unclear. Here we use Distributed Amphifluoric FRET (DAmFRET) and complementary approaches to map the phase space of TDP-43 self-assembly in yeast cells. We discovered that the low-complexity C-terminal domain (CTD) on its own partitions into soluble clusters that dynamically arrest en route to liquid-liquid phase separation. These clusters uniquely supported amyloid formation, and only when templated by pre-existing amyloids of other proteins. Self-interacting modules outside the CTD, whether in the full-length TDP-43, pathological C-terminal fragments, or fusion partners, all suppressed amyloid nucleation. They did this by promoting CTD condensation beyond the arrested state. Stress and cotranslational self-association had the same effect. We leveraged this property of condensation to stop CTD amyloid formation by co-expressing an oligomeric binder in cells. Our findings reveal that TDP-43 amyloid formation occurs only under very specific physical and biological circumstances that present new opportunities for therapeutic control.

biophysics↗

bioRxiv2025

Impacts of stress and aging on spore health in Schizosaccharomyces pombe

Most fungi can produce dormant, long-lived cells known as spores. Spores play a critical role in fungal biology and human health, but much about spores is unknown. Here, we investigate factors affecting spore fitness using the fission yeast Schizosaccharomyces pombe as a model. We found that storage conditions affect spore longevity, and that spore health declines over time. We identified a delay in dormancy breaking (germination), decreased asymmetry during cell division, and reduced stress tolerance as aging phenotypes. These results support that S. pombe spores are affected by both time and experiences during dormancy, highlighting critical features of spore biology and revealing parallels between aging in spores and aging in animal cells. ImportanceFungi are integral to human health and well-being. Fungi also offer scientists easy experimental systems to study facets of life shared with other eukaryotes, including humans. Most studies of fungal cell biology focus on actively growing cells, but fungi can also produce dormant cells known as spores. Spores promote fungal survival and dispersal and are often the agents of infection in pathogenic fungi. In this work, we characterize the spores produced by an easy to study model fungus, Schizosaccharomyces pombe. We find that the longevity and the health of S. pombe spores declines over time and in response to heat stress. We characterize several traits associated with stressed and aged spores and identify parallels to aging cells in animals. This study expands the foundation for using S. pombe spores as a model system for fungal spore biology and as a model for aging of non-dividing cells.

cell biology↗

bioRxiv2025

A tunable affinity fusion tag for protein self-assembly

The concentrations of individual proteins vary between cells, both developmentally and stochastically. The functional consequences of this variation remain largely unexplored due to limited experimental tools to manipulate the relationship of protein concentration to activity. Here, we introduce a genetically encoded tool based on a tunable amyloid that enables precise control of protein concentration thresholds in cells. By systematically screening dipeptide repeats, we identified poly-threonine alanine (poly-TA) as an ideal candidate due to its unique ability to form amyloid-like assemblies with a negligible nucleation barrier at arbitrarily chosen concentration thresholds. We demonstrate that the saturating concentration (Csat) of poly-TA can be finely tuned by adjusting the length of uninterrupted TA repeats, even while maintaining length and composition, providing a modular system for manipulating protein solubility. This tool offers a powerful approach to investigate the relationship between protein concentration, phase separation, and cellular function, with potential applications in cell-, developmental-, and synthetic biology.

cell biology↗

bioRxiv2024

Common AAV gene therapy vectors show indiscriminate transduction of living human brain cell types

The development of cell-type-specific gene therapy vectors for treating neurological diseases holds great promise, but has relied on animal models with limited translational utility. We have adapted an ex vivo organotypic model to evaluate adeno-associated virus (AAV) transduction properties in living slices of human brain tissue. Using fluorescent reporter expression and single-nucleus RNA sequencing, we found that common AAV vectors show broad transduction of normal cell types, with protein expression most apparent in astrocytes; this work introduces a pipeline for identifying and optimizing AAV gene therapy vectors in human brain samples.

bioengineering↗

bioRxiv2024

Functional and evolutionary constraints of wtf killer meiotic drivers

Killer meiotic drivers are selfish DNA loci that sabotage the gametes that do not inherit them from a driver+/driver- heterozygote. These drivers often employ toxic proteins that target essential cellular functions to cause the destruction of driver- gametes. Identifying the mechanisms of drivers can expand our understanding of infertility and reveal novel insights about the cellular functions targeted by drivers. In this work, we explore the molecular mechanisms underlying the wtf family of killer meiotic drivers found in fission yeasts. Each wtf killer acts using a toxic Wtfpoison protein that can be neutralized by a corresponding Wtfantidote protein. The wtf genes are rapidly evolving and extremely diverse. Here we found that self-assembly of Wtfpoison proteins is broadly conserved and associated with toxicity across the gene family, despite minimal amino acid conservation. In addition, we found the toxicity of Wtfpoison assemblies can be modulated by protein tags designed to increase or decrease the extent of the Wtfpoison assembly, implicating assembly size in toxicity. We also identified a conserved, critical role for the specific co-assembly of the Wtfpoison and Wtfantidote proteins in promoting effective neutralization of Wtfpoison toxicity. Finally, we engineered wtf alleles that encode toxic Wtfpoison proteins that are not effectively neutralized by their corresponding Wtfantidote proteins. The possibility of such self-destructive alleles reveals functional constraints on wtf evolution and suggests similar alleles could be cryptic contributors to infertility in fission yeast populations. As rapidly evolving killer meiotic drivers are widespread in eukaryotes, analogous self-killing drive alleles could contribute to sporadic infertility in many lineages. Author SummaryDiploid organisms, such as humans, have two copies of most genes. Only one copy, however, is transmitted through gametes (e.g., sperm and egg) to any given offspring. Alternate copies of the same gene are expected to be equally represented in the gametes, resulting in random transmission to the next generation. However, some genes can "cheat" to be transmitted to more than half of the gametes, often at a cost to the host organism. Killer meiotic drivers are one such class of cheater genes that act by eliminating gametes lacking the driver. In this work, we studied the wtf family of killer meiotic drivers found in fission yeasts. Each wtf driver encodes a poison and an antidote protein to specifically kill gametes that do not inherit the driver. Through analyzing a large suite of diverse natural and engineered mutant wtf genes, we identified multiple properties--such as poison self-assembly and poison-antidote co-assembly--that can constrain poison toxicity and antidote rescue. These constraints could influence the evolution of wtf genes. Additionally, we discovered several incompatible wtf poison-antidote pairs, demonstrating expanded potential for self-killing wtf alleles. Such alleles could potentially arise spontaneously in populations cause infertility.

genetics↗

bioRxiv2023

Pathologic polyglutamine aggregation begins with a self-poisoning polymer crystal

A long-standing goal of amyloid research has been to characterize the structural basis of the rate-determining nucleating event. However, the ephemeral nature of nucleation has made this goal unachievable with existing biochemistry, structural biology, and computational approaches. Here, we addressed that limitation for polyglutamine (polyQ), a polypeptide sequence that causes Huntingtons and other amyloid-associated neurodegenerative diseases when its length exceeds a characteristic threshold. To identify essential features of the polyQ amyloid nucleus, we used a direct intracellular reporter of self-association to quantify frequencies of amyloid appearance as a function of concentration, conformational templates, and rational polyQ sequence permutations. We found that nucleation of pathologically expanded polyQ involves segments of three glutamine (Q) residues at every other position. We demonstrate using molecular simulations that this pattern encodes a four-stranded steric zipper with interdigitated Q side chains. Once formed, the zipper poisoned its own growth by engaging naive polypeptides on orthogonal faces, in a fashion characteristic of polymer crystals with intramolecular nuclei. We further show that self-poisoning can be exploited to block amyloid formation, by genetically oligomerizing polyQ prior to nucleation. By uncovering the physical nature of the rate-limiting event for polyQ aggregation in cells, our findings elucidate the molecular etiology of polyQ diseases.

biophysics↗

bioRxiv2023

Protein supersaturation powers innate immune signaling

How minute pathogenic signals trigger decisive immune responses is a fundamental question in biology. Classical signaling often relies on ATP-driven enzymatic cascades, but innate immunity frequently employs death fold domain (DFD) self-assembly. The energetic basis of this assembly is unknown. Here, we show that specific DFDs function as energy reservoirs through metastable supersaturation. Characterizing all 109 human DFDs, we identified sequence-encoded nucleation barriers specifically in the central adaptors of inflammatory signalosomes, allowing them to accumulate far above their saturation concentration while remaining soluble and poised for activation. We demonstrate that the inflammasome adaptor ASC is constitutively supersaturated in vivo, retaining energy that powers on-demand cell death. Swapping a non-supersaturable DFD in the apoptosome with a supersaturable one sensitized cells to sublethal stimuli. Mapping all DFD nucleating interactions revealed that supersaturated adaptors are triggered to polymerize specifically by other DFDs in their respective pathways, limiting potentially deleterious crosstalk. Across human cell types, adaptor supersaturation strongly correlates with cell turnover, implicating this thermodynamic principle in the trade-off between immunity and longevity. Profiling homologues from fish and sponge, we find nucleation barriers to be conserved across metazoa. These findings reveal DFD adaptors as biological phase change materials in thermal batteries to power cellular life-or-death decisions on demand.

immunology↗

bioRxiv2023

Serial Capture Affinity Purification and Integrated Structural Modeling of the H3K4me3 Binding and DNA Damage Related WDR76:SPIN1 Complex

WDR76 is a multifunctional protein involved in many cellular functions. With a diverse and complicated protein interaction network, dissecting the structure and function of specific WDR76 complexes is needed. We previously demonstrated the ability of the Serial Capture Affinity Purification (SCAP) method to isolate specific complexes by introducing two proteins of interest as baits at the same time. Here, we applied SCAP to dissect a subpopulation of WDR76 in complex with SPIN1, a histone marker reader that specifically recognizes trimethylated histone H3 lysine4 (H3K4me3). In contrast to the SCAP analysis of the SPIN1:SPINDOC complex, H3K4me3 was copurified with the WDR76:SPIN1 complex. In combination with crosslinking mass spectrometry, we built an integrated structural model of the complex which revealed that SPIN1 recognized the H3K4me3 epigenetic mark while interacting with WDR76. Lastly, interaction network analysis of copurifying proteins revealed the potential role of the WDR76:SPIN1 complex in the DNA damage response. TeaserIn contrast to the SPINDOC/SPIN1 complex, analyses reveal that the WDR76/SPIN1 complex interacts with core histones and is involved in DNA damage.

biochemistry↗

bioRxiv2022

rRNA transcription is integral to liquid-liquid phase separation and maintenance of nucleolar structure

Beginning with transcription of ribosomal RNA (rRNA) by RNA Polymerase (Pol) I in the nucleolus, ribosome biogenesis is intimately tied to cell growth and proliferation. Perturbation of ribosome biogenesis has been previously shown to affect nucleolar structure, yet the underlying mechanism is unknown. We generated loss-of-function mouse mutants of Pol I subunits, Polr1a, Polr1b, Polr1c and Polr1d, thereby genetically inhibiting rRNA transcription and ribosome biogenesis. Pol I mutant embryos are preimplantation lethal and have fewer nucleoli. Using hiPSCs triple labeled for the three nucleolar compartments, we observe two phenotypes upon Pol I inhibition: a single condensed nucleolus, and fragmented nucleoli. We find that when rRNA transcription is inhibited, the viscosity of the granular compartment of the nucleolus is increased disrupting its liquid-liquid phase separation properties, which results in a condensed nucleolus. Taken together, our data suggests that Pol I function and rRNA transcription are required for maintaining nucleolar structure and integrity.

developmental biology↗

bioRxiv2022

The wtf meiotic driver gene family has unexpectedly persisted for over 100 million years

Meiotic drivers are selfish elements that bias their own transmission into more than half of the viable progeny produced by a driver+/driver- heterozygote. Meiotic drivers are thought to exist for relatively short evolutionary timespans because a driver gene or gene family is often found in a single species or in a group of very closely related species. Additionally, drivers are generally considered doomed to extinction when they spread to fixation or when suppressors arise. In this study, we examine the evolutionary history of the wtf meiotic drivers first discovered in the fission yeast Schizosaccharomyces pombe. We identify homologous genes in three other fission yeast species S. octosporus, S. osmophilus, and S. cryophilus, which are estimated to have diverged over 100 million years ago from the S. pombe lineage. Synteny evidence supports that wtf genes were present in the common ancestor of these four species. Moreover, the ancestral genes were likely drivers as wtf genes in S. octosporus cause meiotic drive. Our findings indicate that active meiotic drive systems can be maintained for long evolutionary timespans.

evolutionary biology↗

bioRxiv2022

Lola-I is a developmentally regulated promoter pioneer factor

While enhancers are often regulated at the level of accessibility by pioneer factors, promoters tend to be constitutively accessible and poised for activation by paused Pol II -- thus are often not considered as sites of developmental regulation. Here we show that the accessibility of promoters and the acquisition of paused Pol II can be subject to developmental regulation by pioneer factors. We show that Lola-I, a Drosophila zinc finger transcription factor, is ubiquitously expressed at the end of embryogenesis and causes its target promoters to become accessible and acquire paused Pol II throughout the embryo. This promoter transition is required but not sufficient for tissue-specific target gene expression. Lola-I mediates this function by binding to the edges of the promoter nucleosomes, which leads to their depletion, similar to the action of pioneer factors at enhancers. These results uncover a level of regulation for promoters that is normally found at enhancers, providing further evidence that promoters and enhancers display unexpectedly similar characteristics.

genomics↗

bioRxiv2022

A nucleation barrier spring-loads the CBM signalosome for binary activation

Immune cells activate in a binary, switch-like fashion that involves proteins polymerizing into large complexes known as signalosomes. The switch-like nature of signalosome formation has been proposed to result from large energy barriers to polymer nucleation. Whether such nucleation barriers indeed drive binary immune responses has not yet been shown. Here, we employed an in-cell biophysical approach to dissect the assembly mechanism of the CARD-BCL10-MALT1 (CBM) signalosome, a key determinant of transcription factor NF-{kappa}B activation in both innate and adaptive immunity. We found that the adaptor protein BCL10 encodes an intrinsic nucleation barrier, and that this barrier has been conserved from cnidaria to humans. Using optogenetic tools and a single-cell transcriptional reporter of NF-{kappa}B activity, we further revealed that endogenous human BCL10 is supersaturated even in unstimulated cells, indicating that the nucleation barrier operationally stores energy for subsequent activation. We found that upon stimulation, BCL10 nucleation by CARD9 multimers triggers self-templated polymerization that saturates NF-{kappa}B activation to produce a binary response. Pathogenic mutants of CARD9 that cause human immunodeficiencies eliminated nucleating activity. Conversely, a hyperactive cancer-causing mutation in BCL10 increased its spontaneous nucleation. Our results indicate that unassembled CBM signalosome components function analogously to a spring-loaded mousetrap, constitutively poised to activate NF-{kappa}B through irrevocable polymerization. This finding may inform our understanding of the root causes and progressive nature of pathogenic and age-associated inflammation.

cell biology↗

bioRxiv2021

S. pombe wtf genes use dual transcriptional regulation and selective protein exclusion from spores to cause meiotic drive

Meiotic drivers bias gametogenesis to ensure their transmission into more than half the offspring of a heterozygote. In Schizosaccharomyces pombe, wtf meiotic drivers destroy the meiotic products (spores) that do not inherit the driver from a heterozygote, thereby reducing fertility. wtf drivers encode both a Wtfpoison protein and a Wtfantidote protein using alternative transcriptional start sites. Here, we analyze how the expression and localization of the Wtf proteins are regulated to achieve drive. We show that transcriptional timing and selective protein exclusion from developing spores ensure that all spores are exposed to Wtf4poison, but only the spores that inherit wtf4 receive a dose of Wtf4antidote sufficient for survival. In addition, we show that the Mei4 transcription factor, a master regulator of meiosis, controls the expression of the wtf4poison transcript. This dual transcriptional regulation, which includes the use of a critical meiotic transcription factor, likely complicates the universal suppression of wtf genes without concomitantly disrupting spore viability. We propose that these features contribute to the evolutionary success of the wtf drivers. Author SummaryKiller meiotic drivers are one type of selfish DNA sequence. When only one copy of a killer meiotic driver is found in a genome, the driver is expected to be transmitted to only half of the gametes (e.g. eggs or sperm). Killer meiotic drivers, however, kill developing gametes that do not inherit them, giving the driver a transmission advantage into the next generation. The molecular mechanisms used by these killers are not well understood. In this work, we analyzed how one killer meiotic driver, wtf4 from fission yeast, ensures targeted gamete (spore) killing. Previous work showed that wtf meiotic drivers encode a poison protein that is transmitted to all spores and an antidote protein that rescues only spores that inherit the locus. Here, we show that different timing of the expression of the two proteins, combined with differential inclusion of the proteins in developing spores, both contribute to targeted spore killing. We also demonstrate that wtf4 exploits an essential gene expression pathway, making it difficult for the genome to prevent this locus from being expressed and killing. This extends our knowledge both of how these genetic parasites act and how they are equipped to evade host suppression mechanisms.

genetics↗

bioRxiv2021

Genome-wide identification of sexual-reproduction genes in fission yeast via transposon-insertion sequencing

Many genes required for sexual reproduction remain to be identified. Moreover, many of the genes that are known have been characterized in distinct experiments using different conditions, which complicates understanding the relative contributions of genes to sex. To address these challenges, we developed an assay in Schizosaccharomyces pombe that couples transposon mutagenesis with high-throughput sequencing (TN-seq) to quantitatively measure the fitness contribution of nonessential genes across the genome to sexual reproduction. This approach identified 532 genes that contribute to sex, including more than 200 that were not previously annotated to be involved in the process, of which more than 150 have orthologs in vertebrates. Among our verified hits was an uncharacterized gene, ifs1 (important for sex), that is required for spore viability. In two other hits, plb1 and alg9, we observed a novel mutant phenotype of poor spore health wherein viable spores are produced, but the spores exhibit low fitness and are rapidly outcompeted by wildtype. Finally, we fortuitously discovered that a gene previously thought to be essential, sdg1 (social distancing gene), is instead required for growth at low cell densities. Our assay will be valuable in further studies of sexual reproduction in S. pombe and identifies multiple candidate genes that could contribute to sexual reproduction in other eukaryotes, including humans.

genetics↗