Biology subjects

Schmidt, C. A.

Publications and source records attributed to Schmidt, C. A..

16 recordsLinked to original sources

bioRxiv2025

Compositional Analysis of Mouse Sperm Subpopulations During Capacitation In Vitro

Mammalian sperm must undergo capacitation to become competent for fertilization, yet this process is marked by substantial phenotypic heterogeneity among sperm cells. How such variability emerges and how it relates to fertilizing potential remain unresolved, in part because sperm subpopulations are typically analyzed independently despite being intrinsically interdependent. Here, we combine large-scale single-cell spectral flow cytometry with compositional statistical modeling to quantify how sperm population structure responds to controlled capacitation signals in vitro. Using cauda epididymal mouse sperm, we implemented a two-dimensional assay that systematically varies extracellular bicarbonate and free calcium--key regulators of capacitation--and classified millions of individual cells into four irreversible physiological states defined by cell viability and acrosome reaction status. We show that bicarbonate and calcium interact nonlinearly to redistribute sperm across these subpopulations, revealing structured responses that would be otherwise obscured in measurements lacking single-cell resolution. Elevated intracellular calcium was associated with increased cell death, while the highest proportions of live, acrosome-reacted sperm occurred under relatively low extracellular calcium conditions. To enable subpopulation-level analysis, we applied a hierarchical Dirichlet-multinomial regression model that accounts for multinomial sampling noise and between-male variability, yielding posterior probability surfaces that describe how sperm subpopulations reallocate across functional states as microenvironmental signaling conditions change. Together, these results demonstrate that capacitation is a stochastic, cell-population level process shaped by structured phenotypic heterogeneity. This framework provides a quantitative foundation for linking sperm subpopulation composition to measures of fertility competence and for improving existing interpretation of flow cytometry-based assessments of male fertility.

cell biology↗

bioRxiv2025

A framework for evaluating predicted sperm trajectories in crowded microscopy videos

Since the 1980s, semi-automated sperm motility analysis of phase contrast microscopy videos has been used to measure and categorize sperm motility patterns. Motility categories are determined from various kinematic parameters such as Curvilinear Velocity (VCL) and Beat Cross Frequency (BCF). These measures ultimately rely on the quality of the tracking for each individual sperm in the microscopy video. However, common approaches to sperm tracking require sample dilution and shortening the time window of observation (less than 1 to 2 seconds) to avoid tracking errors that occur when sperm cross paths. The post-ejaculatory lifespan of sperm can exceed several hours to days in some species, and long-term adaptive changes in motility pattern may be an important distinguishing factor for predictive modeling of sperm fertilizing competence. Improving the predictive value of computer assisted semen analysis will require accurate tracking of sperm trajectories over physiologically-relevant time scales and at the high cell densities typically found in semen. In this work, we identify a framework for accurately assessing the quality of sperm trajectory tracking that is independent of standard motility measures. We utilize cell tracking metrics adapted from the more common task of tracking adherent somatic cells and propose modifications based on the unique challenges of sperm video-microscopy. We also provide a small dataset of labeled microscopy videos to allow for future comparisons and developments. Author summaryThis report develops a computer vision framework to track individual sperm cells in crowded (high cell-density) microscopy videos, with the goal of improving automated analysis of sperm motility patterns. The task of accurately analyzing sperm movement is deceptively challenging due to the high rate of cell crossovers, an issue that has long impeded long-term tracking of sperm trajectories. Here, we introduce new evaluation metrics and provide a labeled dataset to serve as a baseline for future improvements.

bioinformatics↗

bioRxiv2024

Computational Analysis of Human Cysteine Redox Proteoforms Reveals Novel Insights

Since cysteine redox proteoforms (i) are virtually unstudied, we derived novel insights by computationally analysing the human proteome. Our analysis revealed a vast, effectively infinite, theoretical i space housing 3.02 x 10169 unique cysteine redox proteoforms. For >80% and 99% of the human proteome, the i space comprises 6.83 x 108 and 1.76 x 1031 unique proteoforms, respectively. The heterogenous distribution of the i space by gene ontology terms, suggests, but does not prove, functional speciation. To theoretically limit the number of cysteine redox proteoforms that can be "downloaded" from the abstract i "cloud", we implement novel equations. Protein copy numbers limit the i space by 161-logs to 4.04 x 107 unique cysteine redox proteoforms per HeLa cell. An immutable law: the number of cysteine redox proteoform molecules (Ni) must equal the number of cysteine-containing protein molecules. We compute an Ni value of 1.70 x 109 per HeLa cell. While Ni will be displaced from thermodynamic equilibrium towards the reduced state (e.g., {approx}90%-reduced), it is possible that the number of partially oxidised cysteine redox proteoform molecules is in the order of 106-8 per HeLa cell. Consistent with this, 100%-oxidised forms were observed in 60% of the proteins studied to date. Our analysis advances understanding of redox biology at the proteoform level.

biochemistry↗

bioRxiv2024

The Drosophila maternal-effect gene abnormal oocyte (ao) does not repress histone gene expression

The abnormal oocyte (ao) gene of Drosophila melanogaster is a maternal-effect lethal gene previously identified as encoding a transcriptional regulator of core histones. However, background genetic mutations in existing ao mutant strains could compromise their utility in manipulating histone levels. To distinguish the true ao phenotype from background effects, we created two new ao reagents: a CRISPR/Cas9-mediated knockout of the ao allele for genetic and molecular analyses and an epitope-tagged ao allele for cytological experiments. Using these reagents, we confirm previous findings that loss of ao causes maternal-effect lethality, which can be rescued by either a decrease in the histone gene copy number or by Y chromosome heterochromatin. Our data indicate that ao genetically interacts with the heterochromatin, as previously suggested. However, contrary to a prior study, we find neither Ao localization to histone genes nor ao repression of core histone transcript levels. Thus, the molecular basis for ao-associated maternal-effect lethality remains unknown. Article SummaryA series of foundational papers established that abnormal oocyte (ao), a euchromatic maternal-effect lethal gene, interacts with heterochromatin and the histone multigene cluster to dictate embryonic viability in D. melanogaster. An earlier report argued that ao encodes a protein that localizes to and represses histone gene expression, thereby connecting histone gene overexpression with ao mutant maternal-effect lethality. Using new reagents for genetics and cytology, we recapitulate findings that ao encodes a maternal-effect lethal gene, whose loss is ameliorated by excess heterochromatin or loss of histone genes. However, we find that ao does not affect histone gene expression. Thus, how ao loss causes maternal-effect lethality remains unknown.

genetics↗

bioRxiv2024

Modeling Diffusive Search by Non-Adaptive Sperm: Empirical and Computational Insights

During fertilization, mammalian sperm undergo a winnowing selection process that reduces the candidate pool of potential fertilizers from [~]106-1011 cells to 101-102 cells (depending on the species). Classical sperm competition theory addresses the positive or stabilizing selection acting on sperm phenotypes within populations of organisms but does not strictly address the developmental consequences of sperm traits among individual organisms that are under purifying selection during fertilization. It is the latter that is of utmost concern for improving assisted reproductive technologies (ART) because low fitness sperm may be inadvertently used for fertilization during interventions that rely heavily on artificial sperm selection, such as intracytoplasmic sperm injection (ICSI). Importantly, some form of sperm selection is used in nearly all forms of ART (e.g., differential centrifugation, swim-up, or hyaluronan binding assays, etc.). To date, there is no unifying quantitative framework (i.e., theory of sperm selection) that synthesizes causal mechanisms of selection with observed natural variation in individual sperm traits. In this report, we reframe the physiological function of sperm as a collective diffusive search process and develop multi-scale computational models to explore the causal dynamics that constrain sperm fitness during fertilization. Several experimentally useful concepts are developed, including a probabilistic measure of sperm fitness as well as an information theoretic measure of the magnitude of sperm selection, each of which are assessed under systematic increases in microenvironmental selective pressure acting on sperm motility patterns. Author SummaryFertilization in mammals requires many orders of magnitude more sperm than eggs to be fertilized. This study models collective sperm movement as a diffusive search process, combining experiments and simulations to explore how heterogeneity in motility patterns and microenvironmental complexity shape fertilization success. We introduce simple metrics to quantify sperm fitness and the magnitude of selection pressure imposed by the microenvironment, revealing that sperm phenotype distributions interact with environmental constraints to determine the range of sperm traits that ultimately support successful egg contact. These insights improve our understanding of sperm population dynamics and offer practical tools for optimizing assisted reproductive technologies in clinical and agricultural settings.

cell biology↗

bioRxiv2024

A Low-Cost Microfluidics System for Light Microscopy Experiments

Microfluidics devices are powerful tools for studying dynamic processes in live cells, especially when used in conjunction with light microscopy. There are many applications of microfluidics devices including recording dynamic cellular responses to small molecules or other chemical conditions in perfused media, monitoring cell migration in constrained spaces, or collecting media perfusate for the study of secreted compounds in response to experimental inputs/manipulations. Here we describe a configurable low-cost (channel-based) microfluidics platform for live-cell microscopy, intended to be useful for experiments that require more precision/flexibility than simple rubber spacers, but less precision than molded elastomer-based platforms. The materials are widely commercially available, low-cost, and device assembly takes only minutes.

bioengineering↗

bioRxiv2024

Mitochondria inside acute myeloid leukemia cells hydrolyze ATP to resist chemotherapy

Despite early optimism, therapeutics targeting oxidative phosphorylation (OxPhos) have faced clinical setbacks, stemming from their inability to distinguish healthy from cancerous mitochondria. Herein, we describe an actionable bioenergetic mechanism unique to cancerous mitochondria inside acute myeloid leukemia (AML) cells. Unlike healthy cells which couple respiration to the synthesis of ATP, AML mitochondria were discovered to support inner membrane polarization by consuming ATP. Because matrix ATP consumption allows cells to survive bioenergetic stress, we hypothesized that AML cells may resist cell death induced by OxPhos damaging chemotherapy by reversing the ATP synthase reaction. In support of this, targeted inhibition of BCL-2 with venetoclax abolished OxPhos flux without impacting mitochondrial membrane potential. In surviving AML cells, sustained polarization of the mitochondrial inner membrane was dependent on matrix ATP consumption. Mitochondrial ATP consumption was further enhanced in AML cells made refractory to venetoclax, consequential to downregulations in both the proton-pumping respiratory complexes, as well as the endogenous F1-ATPase inhibitor ATP5IF1. In treatment-naive AML, ATP5IF1 knockdown was sufficient to drive venetoclax resistance, while ATP5IF1 overexpression impaired F1-ATPase activity and heightened sensitivity to venetoclax. Collectively, our data identify matrix ATP consumption as a cancer-cell intrinsic bioenergetic vulnerability actionable in the context of mitochondrial damaging chemotherapy.

cancer biology↗

bioRxiv2023

Sequence reliance of a Drosophila context-dependent transcription factor

Despite binding similar cis elements in multiple locations, a single transcription factor often performs context-dependent functions at different loci. How factors integrate cis sequence and genomic context is still poorly understood and has implications for off-target effects in genetic engineering. The Drosophila context-dependent transcription factor CLAMP targets similar GA-rich cis elements on the X-chromosome and at the histone gene locus but recruits very different, loci-specific factors. We discover that CLAMP leverages information from both cis element and local sequence to perform context-specific functions. Our observations imply the importance of other cues, including protein-protein interactions and the presence of additional cofactors.

genetics↗

bioRxiv2023

A bioinformatics screen reveals Hox and chromatin remodeling factors at the Drosophila histone locus

Cells orchestrate histone biogenesis with strict temporal and quantitative control. To efficiently regulate histone biogenesis, the repetitive Drosophila melanogaster replication-dependent histone genes are arrayed and clustered at a single locus. Regulatory factors concentrate in a nuclear body known as the histone locus body (HLB), which forms around the locus. Historically, HLB factors are largely discovered by chance, and few are known to interact directly with DNA. It is therefore unclear how the histone genes are specifically targeted for unique and coordinated regulation. To expand the list of known HLB factors, we performed a candidate-based screen by mapping 30 publicly available ChIP datasets and 27 factors to the Drosophila histone gene array. We identified novel transcription factor candidates, including the Drosophila Hox proteins Ultrabithorax, Abdominal-A and Abdominal-B, suggesting a new pathway for these factors in influencing body plan morphogenesis. Additionally, we identified six other transcription factors that target the histone gene array: JIL-1, Hr78, the long isoform of fs(1)h as well as the generalized transcription factors TAF-1, TFIIB, and TFIIF. Our foundational screen provides several candidates for future studies into factors that may influence histone biogenesis. Further, our study emphasizes the powerful reservoir of publicly available datasets, which can be mined as a primary screening technique.

molecular biology↗

bioRxiv2022

A cost-free CURE: Using bioinformatics to identify DNA-binding factors at a specific genomic locus

Research experiences provide diverse benefits for undergraduates. Many academic institutions have adopted course-based undergraduate research experiences (CUREs) to improve student access to research opportunities. However, potential instructors of a CURE might still face financial or practical hurdles that prevent implementation. Bioinformatics research offers an alternative that is free, safe, compatible with remote learning, and may be more accessible for students with disabilities. Here, we describe a bioinformatics CURE that leverages publicly available datasets to discover novel proteins that target an instructor-determined genomic locus of interest. We use the free, user-friendly bioinformatics platform Galaxy to map ChIP-seq datasets to a genome, which removes the computing burden from students. Both faculty and students directly benefit from this CURE, as faculty can perform candidate screens and publish CURE results. Students gain not only basic bioinformatics knowledge, but also transferable skills, including scientific communication, database navigation, and primary literature experience. The CURE is flexible and can be expanded to analyze different types of high-throughput data or to investigate different genomic loci in any species.

scientific communication and education↗

bioRxiv2021

Mutations in Drosophila tRNA processing factors cause phenotypes similar to Pontocerebellar Hypoplasia

Mature tRNAs are generated by multiple RNA processing events, which can include the excision of intervening sequences. The tRNA splicing endonuclease (TSEN) complex is responsible for cleaving these intron-containing pre-tRNA transcripts. In humans, TSEN copurifies with CLP1, an RNA kinase. Despite extensive work on CLP1, its in vivo connection to tRNA splicing remains unclear. Interestingly, mutations in CLP1 or TSEN genes cause neurological diseases in humans that are collectively termed Pontocerebellar Hypoplasia (PCH). In mice, loss of Clp1 kinase activity results in premature death, microcephaly and progressive loss of motor function. To determine if similar phenotypes are observed in Drosophila, we characterized mutations in crowded-by-cid (cbc), the CLP1 ortholog, as well as in the fly ortholog of human TSEN54. Analyses of organismal viability, larval locomotion and brain size revealed that mutations in both cbc and Tsen54 phenocopy those in mammals in several details. In addition to an overall reduction in brain lobe size, we also found increased cell death in mutant larval brains. Ubiquitous or tissue-specific knockdown of cbc in neurons and muscles reduced viability and locomotor function. These findings indicate that we can successfully model PCH in a genetically-tractable invertebrate.

genetics↗

bioRxiv2020

Muscle progenitor cells are required for the regenerative response and prevention of adipogenesis after limb ischemia

Peripheral artery disease (PAD) is nearly as common as coronary artery disease, but few effective treatments exist, and it is associated with significant morbidity and mortality. Although PAD studies have focused on the vascular response to ischemia, skeletal muscle cells play a critically important role in determining the phenotypic manifestation of PAD. Here, we demonstrate that genetic ablation of Pax7+ muscle progenitor cells (MPCs, or satellite cells) in a murine model of hind limb ischemia (HLI) resulted in a complete absence of normal muscle regeneration following ischemic injury, despite a lack of morphological or physiological changes in resting muscle. Compared to ischemic muscle of control mice (Pax7WT), the ischemic limb of Pax7-deficient mice (Pax7{Delta}) was unable to generate significant force 7- or 28-days after HLI in ex vivo force measurement studies. A dramatic increase in adipose infiltration was observed 28 days after HLI in Pax7{Delta} mice, which replaced functional muscle. To investigate the mechanism of this adipogenic change, mice with inhibition of fibro/adipogenic precursors (FAPs), another pool of MPCs, were subjected to HLI. Inhibition of FAPs decreased muscle adipose fat but increased fibrosis. MPCs cultured from mouse muscle tissue failed to form myotubes in vitro following depletion of satellite cells in vivo, and they displayed an increased propensity to differentiate into fat in adipogenic medium. Importantly, this phenotype was recapitulated in patients with critical limb ischemia (CLI), the most severe form of PAD. Skeletal muscle samples from CLI patients demonstrated an increase in adipose deposition in more ischemic regions of muscle, which corresponded with a decrease in the number of satellite cells in those regions. Collectively, these data demonstrate that Pax7+ MPCs are required for normal muscle regeneration after ischemic injury, and they suggest that targeting muscle regeneration may be an important therapeutic approach to prevent muscle degeneration in PAD.

pathology↗

bioRxiv2019

Reconstitution of the Human tRNA Splicing Endonuclease Complex: insight into the regulation of pre-tRNA cleavage.

The splicing of tRNA introns is a critical step in pre-tRNA maturation. In archaea and eukaryotes, tRNA intron removal is catalyzed by the tRNA splicing endonuclease (TSEN) complex. Eukaryotic TSEN is comprised of four core subunits (TSEN54, TSEN2, TSEN34, and TSEN15). The human TSEN complex additionally co-purifies with the polynucleotide kinase CLP1; however, CLP1s role in tRNA splicing remains unclear. Mutations in genes encoding all four TSEN subunits, as well as CLP1, are known to cause neurodegenerative disorders, yet the mechanisms underlying the pathogenesis of these disorders are unknown. Here, we developed a recombinant system that produces active TSEN complex. Co-expression of all four TSEN subunits is required for efficient formation and function of the complex. We show that human CLP1 associates with the active TSEN complex, but is not required for tRNA intron cleavage in vitro. Moreover, RNAi knockdown of the Drosophila CLP1 orthologue, cbc, promotes biogenesis of mature tRNAs and circularized tRNA introns (tricRNAs) in vivo. Collectively, these and other findings suggest that CLP1/cbc plays a regulatory role in tRNA splicing by serving as a negative modulator of the direct tRNA ligation pathway in animal cells.

molecular biology↗

bioRxiv2019

Effects of fasting induced carbohydrate depletion on murine ischemic skeletal muscle function.

Stored muscle carbohydrate supply and energetic efficiency constrain muscle functional capacity during exercise and are influenced by common physiological variables (e.g. age, diet, and physical activity level). Whether these constraints affect overall functional capacity or the timing of muscle energetic failure during acute ischemia is not known. We interrogated skeletal muscle contractile properties in two anatomically distinct hindlimb muscles that have well characterized differences in energetic efficiency (locomotory-extensor digitorum longus (EDL) and postural-soleus muscles) under conditions of reduced carbohydrate supply. 180 mins of acute ischemia resulted in complete energetic failure in all muscles tested, indicated by: loss of force production, substantial reductions in total adenosine nucleotide pool intermediates, and increased adenosine nucleotide degradation product - inosine monophosphate (IMP). These changes occurred in the absence of apparent myofiber structural damage assessed histologically by both transverse section and whole mount. Restriction of the available intracellular carbohydrate pool by fasting (~50% decrease in skeletal muscle) did not significantly alter the timing to muscle functional impairment or affect the overall force/work capacities of either muscle type. Fasting did cause rapid development of passive tension in both muscle types, which may have implications for optimal timing of reperfusion or administration of precision therapeutics.

physiology↗

bioRxiv2018

Molecular determinants of metazoan tricRNA biogenesis

Mature tRNAs are generated by multiple post-transcriptional processing steps, which can include intron removal. Recently, our laboratory discovered a new class of metazoan circular RNAs formed by ligation of excised tRNA introns; we termed these molecules tRNA intronic circular (tric)RNAs. To investigate the mechanism of tricRNA biogenesis, we generated constructs that replace the native introns of two Drosophila tRNA genes with the Broccoli fluorescent RNA aptamer. Using these reporters, we identified cis-acting elements required for tricRNA formation in both human and fly cells. We observed that disrupting the conserved anticodon-intron base pair dramatically reduces tricRNA levels. Although the integrity of this base pair is necessary for proper splicing, it is not sufficient. Furthermore, we found that strengthening weak base pairs in the pre-tRNA also impairs tricRNA production. We also used the reporters to identify trans-acting tricRNA processing factors. We found that several known tRNA processing factors, such as RtcB ligase and components of the TSEN endonuclease complex, are involved in tricRNA biogenesis. Depletion of these factors inhibits tRNA intron circularization. Furthermore, we observed that depletion of Clipper endonuclease results in increased tricRNA levels. In summary, our work characterizes the major players in Drosophila tricRNA biogenesis.

molecular biology↗

bioRxiv2018

Composition of the Survival Motor Neuron (SMN) complex in Drosophila melanogaster

Spinal Muscular Atrophy (SMA) is caused by homozygous mutations in the human survival motor neuron 1 (SMN1) gene. SMN protein has a well-characterized role in the biogenesis of small nuclear ribonucleoproteins (snRNPs), core components of the spliceosome. SMN is part of an oligomeric complex with core binding partners, collectively called Gemins. Biochemical and cell biological studies demonstrate that certain Gemins are required for proper snRNP assembly and transport. However, the precise functions of most Gemins are unknown. To gain a deeper understanding of the SMN complex in the context of metazoan evolution, we investigated the composition of the SMN complex in Drosophila melanogaster. Using a stable transgenic line that exclusively expresses Flag-tagged SMN from its native promoter, we previously found that Gemin2, Gemin3, Gemin5, and all nine classical Sm proteins, including Lsm10 and Lsm11, co-purify with SMN. Here, we show that CG2941 is also highly enriched in the pulldown. Reciprocal co-immunoprecipitation reveals that epitope-tagged CG2941 interacts with endogenous SMN in Schneider2 cells. Bioinformatic comparisons show that CG2941 shares sequence and structural similarity with metazoan Gemin4. Additional analysis shows that three other genes (CG14164, CG31950 and CG2371) are not orthologous to Gemins 6-7-8, respectively, as previously suggested. In D.melanogaster, CG2941 is located within an evolutionarily recent genomic triplication with two other nearly identical paralogous genes (CG32783 and CG32786). RNAi-mediated knockdown of CG2941 and its two close paralogs reveals that Gemin4 is essential for organismal viability.

genetics↗