Biology subjects

Hall, E.

Publications and source records attributed to Hall, E..

At least 19 recordsLinked to original sources

bioRxiv2026

Spatially-resolved multimodal profiling identifies functionally-heterogeneous cancer-associated fibroblasts associated with poor radiotherapy outcomes in muscle-invasive bladder cancer.

Background: Cancer-associated fibroblasts (CAFs) contribute to systemic therapy resistance in muscle-invasive bladder cancer (MIBC), but their functional heterogeneity and relevance to curative bladder-preserving radiotherapy, are poorly understood. Methods: Transcriptomic analysis was performed on 279 tumours from BC2001, a phase 3 radiotherapy clinical trial. To study CAF heterogeneity, we integrated bulk RNA-seq, single-cell spatial analysis of multiplex immunofluorescence images and quantification of extracellular matrix (ECM) features in 155 MIBC biopsies. The functional heterogeneity of distinct CAF populations was evaluated by single-nuclear RNA-seq. Spatial interactions between CAF populations and CD8+ T-cells was assessed and the relevance of lymphocytes to radiation responses was evaluated in a CAF-enriched murine bladder cancer model (BBN963). Results: BC2001 patients with CAF-enriched tumours had worse overall survival (HR=1.671, 95% CI 1.221-2.287, Log-rank p=0.0012). CAF abundance and antigen expression was highly heterogenous. Podoplanin (PDPN) was expressed on the majority of CAFs and was associated with inflammatory pathways. Enrichment of CAF gene signatures was associated with a significant increase in CAFs expressing fibroblast activation protein (FAP) (p=0.004) and dense ECM features (p=0.0004). Fifty-three percent of tumours exhibited stromal CD8+ T-cell exclusion with significant enrichment in FAP-dominant neighbourhoods (p<0.001). In vivo, lymphocytes were critical for radiation-induced tumour control, indicating that immune cold or excluded tumours may have limited radiotherapy responses. Conclusion: In MIBC, CAFs are associated with poor radiotherapy outcomes. Multiple mechanisms are deployed by functionally-heterogeneous CAFs, including promotion of chronic inflammation by PDPN+ CAFs and ECM remodelling by FAP+ CAFs which impact CD8+ T-cell distribution and radiation responses.

cancer biology↗

bioRxiv2026

Engineered OAA lectins as selective and sensitive high mannose glycan targeting tools

The Oscillatoria agardhii agglutinin (OAA) lectin interacts with N-glycans through a pentamannose core shared among all high mannose N-glycans (HMGs). Because HMGs only differ by number of mannose sugars, there is a scarcity of tools sensitive enough to resolve each specific HMG structure in their biological context. Here, we investigate the sequence space of OAA to tune the binding properties towards selectivity of Man5GlcNAc2, thus generating a structure-specific detection tool. Using phage display to screen a diverse library of OAA variants, we identify a variant with high selectivity for Man5GlcNAc2 that we further dissect to reveal four mutations necessary for selectivity and two mutations responsible for enhanced affinity for all HMGs. Coupling a crystal structure of the selective variant with binding analysis of specific point mutations, we reveal how co-dependent mutations achieve selectivity. We then demonstrate how variants can be valency-modulated on a single beta-barrel scaffold to improve their binding properties by orders of magnitude. Finally, we showcase the applicability of engineered OAA variants as improved HMG profiling tools and tunable antiviral agents.

biophysics↗

bioRxiv2026

Distinct sensing of BCAAs by mTOR and c-Myc governs T cell proliferation, independent of catabolism

The branched chain amino acids (BCAAs: leucine, isoleucine, valine) are essential amino acids that function as catabolic substrates and signaling molecules via mTORC1. While individual BCAAs have unique roles in organismal and cellular physiology, the mechanisms underlying their individual effects remain poorly understood. We demonstrate that the three BCAAs have distinct roles in T cell biology. We find that isoleucine and valine are necessary and sufficient for quiescence exit and cell division, whereas leucine is dispensable. Mechanistically, these effects are independent of their diverging catabolic fates and instead due to differential sensing of leucine and isoleucine/valine by mTOR and c-Myc. While isoleucine and valine are necessary and sufficient for c-Myc expression, mTORC1 leucine-sensing represses c-Myc and proliferation during BCAA restriction. Together, we find that the discrete sensing of the BCAAs uncouples two major anabolic regulators, mTORC1 and c-Myc, in cell growth. This provides mechanistic insight into the distinct roles of the BCAAs in cell physiology, highlighting divergent BCAA sensing rather than catabolism, and offering a new lens to appreciate their impact on immunity and pathophysiology.

immunology↗

bioRxiv2025

Glutamine Availability Impacts Lymphatic Endothelial Cell Glycolysis and Lymphangiogenesis in Hypoxic Environments

Dysfunctional lymphangiogenesis is a component of several diseases, including secondary lymphedema, allergic asthma, and solid malignancies. These vessels are ineffective at draining interstitial fluid, resulting in secondary complications such as edema, increased inflammation, slowed wound healing, and, for cancer patients, increased risk of metastasis. Hypoxic microenvironments drive dysfunctional vessel growth by upregulating vascular endothelial growth factor receptors, migratory signaling pathways, and glycolytic metabolism. Despite the potential benefits of normalizing lymphatic vasculature, most treatments for vascular normalization focus on blood vasculature, ignoring the unique properties of lymphatic endothelial cells (LECs). Moreover, previous targets for vascular normalization center around vascular endothelial growth factors, which risk adverse side effects. To address these issues, we approached lymphatic normalization from a metabolic perspective. In this study, we investigated the impact of glutamine availability on factors critical to lymphangiogenesis, including glycolysis, cell proliferation, and migration. We found that increasing the concentration of glutamine in media results in increased lactate production and the expression of glycolytic genes HK2, GLUT1, and GLUT3 under hypoxia. The presence of glutamine also encouraged LEC proliferation, while blocking glutamine transport reduced lactate production, HK2 expression, and slowed collective LEC migration. In a 2D vessel formation assay, we found that glutamine increased vessel formation in normoxic conditions but lowered vessel connectivity in hypoxic conditions, reflecting the dysfunction seen in hypoxic diseases. However, attenuating glycolysis by blocking glutamine transport caused LECs to form longer, interconnected vascular networks. This study reveals that glutamine availability can modulate LEC glycolysis, and therefore lymphangiogenesis, in a hypoxia-dependent manner.

cell biology↗

bioRxiv2025

Tunable multivalent Fe(II)-based glycoassemblies as mimetics for native high-mannose glycans.

High Mannose Glycans (HMGs) play key roles in eukaryotic biology, regulating processes ranging from protein folding to host pathogen defense. Lectins have evolved to interact with these glycans through multivalent interactions facilitated by the multiple sugars displayed on glycans and via multiple binding sites on each lectin. Using Fe(II) iminopyridine complexes, we generated chemically defined multivalent glycan displays where the valency, arm length, and spatial display of mannose residues can be controlled via subcomponent synthesis. Due to its sensitivity towards the geometric display of mannose residues, monomeric Griffithsin (mGRFT) was utilized as a model lectin. Interactions between the Fe(II) glycan assemblies and mGRFT were characterized using biolayer interferometry (BLI), isothermal titration calorimetry (ITC), and NMR spectroscopy. Our results display a >1000-fold range in KD for Fe(II) iminopyridine complexes that can be tuned by factors such as saccharide tether length and number of sugars displayed. Through leveraging systematic molecular-level modifications, we demonstrate that tunable Fe(II) glycan assemblies can be used both as mimetics for high mannose glycans as well as competitive inhibitors for native glycan binding.

biophysics↗

bioRxiv2025

POLQ variants with aberrant DNA polymerase activity protect against UV-induced cell death.

DNA polymerases are important for maintaining genomic stability by protecting against mutagenic lesions caused by external and internal factors. If left unrepaired, DNA damage can lead to replication errors resulting in changes in DNA sequences that could impact peptide sequences and gene expression, as well as lead to chromosomal breaks. Highlighting the importance of DNA polymerase repair function, variant DNA polymerases and cofactors of DNA repair pathways have been identified in many different cancer types. Recently, variant forms of DNA polymerase Q (POLQ) have been identified in patient isolated melanoma tumors. Previous work identifying biochemical characteristics of these variants has shown that they display aberrant DNA polymerase activity compared to wild-type (WT). To better understand the role these variants have in DNA repair, genomic stability and cell survival, we tested their ability to bypass and extend DNA past cyclobutane pyrimidine dimers (CPD) as well as prevent cell death when exposed to ultra-violet (UV) radiation. Biochemically we show that the patient derived variants of POLQ tested here display decreased efficiency during DNA bypass and extension of CPD lesions and prefer to incorporate purines. In addition, two of the three variants protect against UV induced cell death. Together these data suggest that POLQ variants can support cell survival and further supports that POLQ variants can act as both protectors of cell viability as well as drivers of genomic instability, characteristics important in cancer cells. HIGHLIGHTS[vrecto] POLQ variants have decreased efficiency during bypass and extension of CPD damaged DNA compared to WT POLQ [vrecto]POLQ is able to bypass and extend cyclobutane pyrimidine dimers, but prefers purine over pyrimidine incorporation [vrecto]POLQ variants can protect against UV induced cell death.

biochemistry↗

bioRxiv2025

Modulation of Filopodial Myosin Function

MyTH-FERM (MF) myosins are essential for filopodia formation in both Metazoa and Amoebozoa (Dictyostelium DdMyo7; mammalian Myo10) but their roles in filopodia formation are not fully understood. Taking advantage of a mutation in the highly conserved actin binding interface of another MF myosin, the jordan (jd) mutation of Myo15A that reduces its function, the impact of altering the myosin-F-actin interaction on filopodia formation was investigated. The jd mutation, a D to G substitution (Gong et al, 2022, Sci Adv), was introduced into DdMyo7 or Myo10 and filopodia formation assessed by quantitative analysis of number, length and myosin tip enrichment. The mutation significantly decreased filopodia initiation and tip intensity of both DdMyo7 and Myo10. This is consistent with impairment of myosin-based reorganization of cortical actin at the cortex, with myosins in the initiation complex remaining at the tip as the filopodium extends. The DdMyo7 jd mutation did not affect filopodia length. In the case of Myo10, while the jd mutation reduced Myo10 velocity of intrafilopodial motility by 40%, resulting in reduced levels of myosin at the filopodia tip, the jd mutant did not consistently affect the length of filopodia in different cell lines. This indicates that the major role of Myo10 may not be to promote filopodia elongation by reducing membrane tension, as has been proposed. Rather, the phenotype of the DdMyo7 and Myo10 jd mutants indicates that the major role of evolutionarily diverse filopodial myosins is to re-organize actin filaments at the membrane-cortex interface during the critical initiation step.

cell biology↗

bioRxiv2025

The Effect of Previously Encountered Sensory Information on Neural Representations of Predictability: Evidence from Human EEG

Accumulating evidence suggests that the brain continuously monitors the predictability of rapidly evolving sound sequences, even when they are not behaviorally relevant. An increasing body of empirical evidence links sustained tonic M/EEG activity to evidence accumulation and tracking the predictability, or inferred precision, of the auditory stimulus. However, it remains unclear whether, and how, this process depends on auditory contextual memory. In the present EEG study, we examined neural responses to sound sequences across two experiments, and compared them to predictions from ideal observer models with varying memory spans. Stimuli were sequences of 50 ms long tone-pips. In Experiment 1 (N=26; both sexes), a regularly repeating sequence of 10 tones (REG) transitioned directly to a different regular sequence (REGxREGy). In Experiment 2 (N=28; both sexes), the same regular sequence was repeated after an intervening random segment (REGxINTREGx). Results from Experiment 2 revealed that the inferred predictability of the resumed REGx pattern was influenced by the preceding INT tones, even several seconds after they ended, indicating that the brain retains contextual memory over time. In contrast, neural responses in Experiment 1 were best explained by models with minimal memory. This dissociation implies that the brain can dynamically adjust its strategy based on inferred environmental structure--resetting context when interruptions signal change, and preserving context when patterns are likely to resume.

neuroscience↗

bioRxiv2025

Fractionated radiation alters the extracellular matrix produced by muscle-invasive bladder cancer cells

Muscle-invasive bladder cancer (MIBC) is a prevalent disease that can be treated with radiotherapy, but has a poor prognosis. Radiation-induced extracellular matrix (ECM) remodelling and fibrosis can induce tumour resistance and recurrence, but has not been studied in MIBC. Here we aimed to characterise the impact of radiation on the ECM composition of MIBC. Materials and MethodsThree MIBC cell lines (T24, UMUC3, J82) were treated with fractionated radiation. We used proteomics to analyse the ECM composition produced by surviving cancer cells and immunofluorescence to investigate changes in the morphology and number of ECM fibres. We evaluated the RNA expression of identified ECM proteins (FN1, COL5A1, COL1A1, TNF6AIP6, FLG) in one cystectomy (TCGA-BLCA, n=397) and two radiotherapy (BC2001, n=313; BCON, n=151) cohorts. ResultsThere were 613 proteins affected by radiation (padj<0.05, fold change >2 or <-2), 68 of which were ECM-associated proteins. There was a general increase in proteases and protease regulators but heterogeneity across cell lines. Enrichment analysis showed ECM organisation was the primary pathway affected. Immunofluorescence confirmed radiation affected ECM structure, generally, reducing the number, length and width of fibres. High FN1, COL5A1, COL1A1, TNF6AIP6 and FLG mRNA levels were adverse prognostic markers in clinical cohorts. ConclusionRadiation alters the composition and structure of the ECM produced by MIBC. As a proof-of-concept, we showed the expression of radiation-affected ECM genes are prognostic markers in MIBC. Future studies should validate these radiation-induced ECM changes in clinical samples.

cancer biology↗

bioRxiv2025

Hypoxia-induced extracellular matrix changes are conserved in cancer and directly impact radiotherapy benefit

Our overarching aim was to determine how hypoxia affects the extracellular matrix (ECM). Transcriptomic analysis (21,941 patients; 10 cancer types) identified ECM remodelling as the predominant pathway affected by hypoxia. Multi-omics confirmed that hypoxia impacts ECM organisation and collagen degradation; 53 ECM genes were affected, of which 74% were HIF1/HIF2-regulated. Spatial transcriptomics highlighted different hypoxia remodelling processes in tumour and stroma. Five ECM genes commonly affected in tumour and in vitro constituted a signature. This signature was independently prognostic and independently predictive of radiotherapy benefit in multiple malignancies. Patients within either high or low hypoxic-ECM score tertiles benefited from radiotherapy versus surgery. Hypoxic ECMs generated in vitro increased adhesion and decreased migration of cancer cells, an effect enhanced by irradiation. Immunofluorescence demonstrated that hypoxia decreased collagen fibre number, and irradiation decreased cell-ECM interactions. Taken together, these findings demonstrate hypoxia induces pan-cancer ECM changes, directly impacting radiotherapy benefit.

cancer biology↗

bioRxiv2025

Biosynthetic plasticity enables CD8+ T cell functional resilience under nutrient stress

Summary / AbstractTo maintain lineage-specific functions, cells must acquire and allocate nutrients across diverse cellular processes, even in metabolically-dysregulated environments. The mechanisms allowing CD8+ T cells to maintain immune function in perturbed environments are poorly understood. We find that CD8+ T cells adapt to nutrient stresses over time, reconfiguring gene-regulatory and metabolic networks to license functional recovery. Under acute stress, T cells reorient translational programming, limiting nutrient demand while prioritizing stress-sensitive metabolic and transcriptional responses. Within these responses, the transcription factors ATF4 and CEBPG jointly establish an adaptive metabolic program, promoting amino acid synthesis and uptake while maintaining mitochondrial anaplerosis. Despite diminished energetic capacity under environmental stress, this program prevents failure of central carbon metabolism, mitigating stress amplification and cellular dysfunction to potentiate anti-tumor immunity. Altogether, we demonstrate that biosynthetic plasticity via translational and metabolic reprioritization confers functional resilience to immune cells in unfavorable environments, offering novel strategies to enhance immunotherapies.

immunology↗

bioRxiv2024

Malnutrition drives infection susceptibility and dysregulated myelopoiesis that persists after refeeding intervention

Undernutrition is one of the largest persistent global health crises, with nearly 1 billion people facing severe food insecurity. Infectious disease represents the main underlying cause of morbidity and mortality for malnourished individuals, with infection during malnutrition representing the leading cause of childhood mortality worldwide. In the face of this complex challenge, simple refeeding protocols have remained the primary treatment strategy. Although an association between undernutrition and infection susceptibility has been appreciated for over a century, the underlying mechanisms remain poorly understood and the extent to which refeeding intervention is sufficient to reverse nutritionally acquired immunodeficiency is unclear. Here we investigate how malnutrition leads to immune dysfunction and the ability of refeeding to repair it. We find that chronic malnutrition induced through prolonged dietary restriction (40% reduction in food intake) severely impairs the ability of mice to control a sub-lethal Listeria monocytogenes infection. Malnourished mice exhibit blunted immune cell expansion, impaired immune function, and accelerated contraction prior to pathogen clearance. While this defect is global, we find that myelopoiesis is uniquely impacted, resulting in reduced neutrophil and monocyte numbers prior to and post-infection. Upon refeeding, we observe that mice recover body mass, size, cellularity across all major immune organs, the capacity to undergo normal immune cell expansion in response to infection, and a restoration in T cell responses. Despite this broad improvement, refed mice remain susceptible to Listeria infection, uncoupling global lymphoid atrophy from immunodeficiency. We find peripheral neutrophil and monocyte numbers fail to fully recover and refed mice are unable to undergo normal emergency myelopoiesis. Altogether, this work identifies dysregulated myelopoiesis as a link between prior nutritional state and immunocompetency. We believe these findings raise the possibility that exposure to food scarcity should be treated as an immunologic variable, even post-recovery, with considerations for how patient medical history and public health policy.

immunology↗

bioRxiv2024

Nutrient loading from a sustainably certified aquaculture operation dwarfs annual nutrient inputs from a large multi-use watershed, Lake Yojoa, Honduras

Net-pen aquaculture is a popular and increasingly prevalent method for producing large quantities of low-fat protein in freshwater ecosystems across the tropics. While there are numerable social and economic advantages associated with aquaculture, there are also challenges related to the environmental sustainability of aquaculture. Chief among these risks is excessive nutrient loading which can drive rapid eutrophication in aquatic ecosystems. In this study, we compare the estimated annual nutrient loads from the six principal tributaries that contribute to Lake Yojoa to the estimated nutrient load of a large net-pen Tilapia operation located in the central region of the lake. We estimated that the Tilapia farm was responsible for ~ 86% of nitrogen (N) and ~95% of the phosphorus (P) annual contributions to Lake Yojoa. This disproportionate nutrient loading of both N and P suggests that this single aquaculture operation, more so than changes in nutrients inputs from the watershed, was responsible for the previously documented deterioration of the Lake Yojoa ecosystem. This study shows the potential for net-pen aquaculture to have disproportionately negative impacts on freshwater ecosystems, even when operations meet all standards for the current state-of-the-art sustainability certifications. We suggest shifts in metrics that could improve the impact of the certification process so that best practices can reduce the impact of net-pen aquaculture on freshwater ecosystems and arrive at the intended goal of long-term environmental sustainability.

ecology↗

bioRxiv2024

Measuring the Stoichiometry of Microbial Parasite Infrapopulations One Cell at a Time using Energy Dispersive Spectroscopy

O_LIProgress in the field of ecological stoichiometry has demonstrated that the outcome of ecological interactions can often be predicted a priori based on the nutrient ratios (e.g., carbon: nitrogen: phosphorus, C:N:P) of interacting organisms. However, the challenges of accurately measuring the nutrient content of active parasites within hosts has limited our ability to rigorously apply ecological stoichiometry to host-parasite systems. Traditional nutrient analyses require high parasite biomasses, often preventing individual-level analyses. This prevents researchers from estimating variation in the nutrient content of individual parasites within a single host infrapopulation, a critical factor that could define how the ecology of the parasite affects the host-parasite interaction. C_LIO_LIHere, we explain how energy dispersive technology, a technique currently used to measure the elemental content of free-living microbes, can be adapted for parasitic microbial infrapopulations. We demonstrate the power of accurately quantifying the biomass stoichiometry of individual microbial parasites sampled directly from individual hosts. C_LIO_LIUsing this approach we show that the stoichiometric composition of two microbial parasites capable of infecting the same host are stoichiometrically distinct and respond to host diet quality differently. We also demonstrate that characteristics of the stoichiometric trait distributions of these infrapopulations were important predictors of host fecundity, a proxy for virulence in this system, and better predictors of parasite load than the mean parasite stoichiometry or our parasite and diet treatments alone. C_LIO_LIEDS provides a rigorous tool for applying ecological stoichiometry to host-parasite systems and enables researchers to explore the nutritional physiology of host-parasite interactions at a scale that is more relevant to the ecology and evolution of the system than traditional nutrient analyses. Here we demonstrate that this level of resolution provides useful insights into the diet-dependent physiology of microbial parasites and their hosts. We anticipate that this improved level of resolution has the potential to elucidate a range of eco-evo interactions in host-parasite systems that were previously unobservable. C_LI

ecology↗

bioRxiv2023

The Effects of Preeclamptic Milieu on Cord Blood Derived Endothelial Colony-Forming Cells

Preeclampsia is one of the leading causes of infant and maternal mortality worldwide. Many infants born from preeclamptic pregnancies are born prematurely with higher risk of developing cardiovascular later in their life. A key mechanism by which these complications occur is through stress-induced dysfunction of endothelial progenitor cells (EPCs), including endothelial colony-forming cells (ECFCs). To gain insight into this, cord blood derived ECFCs isolated from preeclamptic pregnancies (PRECs) were analyzed and compared to their healthy counterparts. While PRECs preserve key endothelial markers, they upregulate several markers associated with oxidative stress and inflammatory response. Compared to ECFCs, PRECs also exhibit lower migratory behaviors and impaired angiogenic potential. Interestingly, treatment of neuropilin-1 can improve tube formation in vitro. Collectively, this study reports that preeclamptic milieu influence phenotypes and functionality of PRECs, which can be rejuvenated using exogenous molecules. Promising results from this study warrant future investigations on the prospect of the rejuvenated PRECs to improve lung function of infants born from preeclamptic pregnancies.

bioengineering↗

bioRxiv2023

A functional microbiome catalog crowdsourced from North American rivers

Predicting elemental cycles and maintaining water quality under increasing anthropogenic influence requires understanding the spatial drivers of river microbiomes. However, the unifying microbial processes governing river biogeochemistry are hindered by a lack of genome-resolved functional insights and sampling across multiple rivers. Here we employed a community science effort to accelerate the sampling, sequencing, and genome-resolved analyses of river microbiomes to create the Genome Resolved Open Watersheds database (GROWdb). This resource profiled the identity, distribution, function, and expression of thousands of microbial genomes across rivers covering 90% of United States watersheds. Specifically, GROWdb encompasses 1,469 microbial species from 27 phyla, including novel lineages from 10 families and 128 genera, and defines the core river microbiome for the first time at genome level. GROWdb analyses coupled to extensive geospatial information revealed local and regional drivers of microbial community structuring, while also presenting a myriad of foundational hypotheses about ecosystem function. Building upon the previously conceived River Continuum Concept1, we layer on microbial functional trait expression, which suggests the structure and function of river microbiomes is predictable. We make GROWdb available through various collaborative cyberinfrastructures2, 3 so that it can be widely accessed across disciplines for watershed predictive modeling and microbiome-based management practices.

microbiology↗

bioRxiv2022

Inhibition of advanced glycation end product formation and serum protein infiltration in bioprosthetic heart valve leaflets: Investigations of anti-glycation agents.

Bioprosthetic heart valves (BHV) fabricated from glutaraldehyde pretreated heterograft tissue, such as bovine pericardium (BP) are the most commonly used heart valve replacements. However, BHV durability is limited by structural valve degeneration (SVD) resulting from both calcification and advanced glycation end product (AGE) deposition together with serum protein infiltration. In the present study we investigated the hypothesis that anti-AGE agents, Aminoguanidine (AG), Pyridoxamine (PYR), and N-Acetylcysteine (NAC) could mitigate AGE-serum protein mechanisms in model studies, both in vitro and in vivo, using rat subdermal implants of BP. In vitro studies demonstrated that each of these agents could significantly inhibit AGE formation in BP. However, in rat 28 days BP subdermal implants, only PYR demonstrated both significant inhibition of AGE and serum albumin accumulation per immunostaining. BHV calcification was not mitigated by PYR. It is concluded that AGE-serum protein pathophysiology contributing to SVD can be ameliorated by PYR.

biochemistry↗

bioRxiv2021

Eukaryotic translation initiation factor eIF4E-5 is required for spermiogenesis in Drosophila melanogaster

Drosophila sperm development is characterized by extensive post-transcriptional regulation whereby thousands of transcripts are preserved for translation during later stages. A key step in translation initiation is the binding of eukaryotic initiation factor 4E (eIF4E) to the 5 mRNA cap. Drosophila has multiple paralogs of eIF4E, including four (eIF4E-3, -4, -5, and -7) that are highly expressed in the testis. Other than eIF4E-3, none of these has been characterized genetically. Here, using CRISPR/Cas9 mutagenesis, we determined that eIF4E-5 is essential for male fertility. eIF4E-5 mutants exhibit defects during post-meiotic stages, including a fully penetrant defect in individualization, resulting in failure to produce mature sperm. eIF4E-5 protein localizes to the distal ends of elongated spermatid cysts, where it regulates non-apoptotic caspase activity during individualization by promoting local accumulation of the E3 ubiquitin ligase inhibitor Soti. eIF4E-5 mutants also have mild defects in spermatid cyst polarization, similar to mutants affecting the cytoplasmic polyadenylation-element binding protein Orb2 and atypical protein kinase C (aPKC). Our results further extend the diversity of non-canonical eIF4Es that carry out distinct spatiotemporal roles during spermatogenesis. Summary StatementThe testis-enriched translation initiation factor eIF4E-5 is needed for spermatid cyst polarization, individualization of mature sperm and male fertility in Drosophila.

developmental biology↗