Biology subjects

Podbilewicz, B.

Publications and source records attributed to Podbilewicz, B..

10 recordsLinked to original sources

bioRxiv2026

Human Sperm-Induced Cell-Cell Fusion Requiring JUNO (hSPICER): A paradigm shift to test sperm fertilizing potential

Current evaluation of male fertility is largely based on indirect sperm parameters such as viability, concentration, morphology, and motility; however, each of these parameters, alone or combined, has been shown to have limited predictive value for successful fertilization. To address this problem, we introduce hSPICER (human SPerm-Induced CEll-cell fusion Requiring JUNO), an assay that evaluates sperm function based on their ability to induce fusion of somatic cells expressing human JUNO (hJUNO), the egg-specific sperm receptor. Similarly to our previous discovery in mice, we found that human sperm can fuse with somatic cells expressing hJUNO on their surface (pseudo-eggs) and promote content mixing between cells in culture, as measured using a split GFP system. The assay is sensitive, specific, and species-dependent, requiring hJUNO for optimal signal. We generated a stable cell line expressing hJUNO, enhancing reproducibility and sensitivity. We also show that hSPICER is compatible with cryopreserved sperm and consistent over different days. Importantly, hSPICER values correlate with fertilization outcomes of patients during fertility treatments, indicating its potential as a functional diagnostic tool. Beyond diagnostic uses, hSPICER establishes a platform to explore sperm fusion mechanisms and to screen for therapeutic compounds and interventions to treat low fertility, enhance fertilization, and develop non-hormonal contraceptives for males and females, as well as quality assessment of semen samples in fertility clinics and sperm banks.

developmental biology↗

bioRxiv2024

The PVD neuron has male-specific structure and mating function in C. elegans

Neurons display unique shapes and establish intricate networks, which may differ between sexes. In complex organisms, studying sex differences in structure and function of individual neurons is difficult. The nematode Caenorhabditis elegans hermaphrodites and males present an exceptional model for studying neuronal morphogenesis in a simple, sexually-dimorphic system. We focus on the polymodal sensory bilateral neuron pair PVD, which forms a complex but stereotypic dendritic tree composed of multiple subunits that resemble candelabra. PVD is well studied in hermaphrodites, but not in males. We show here that during larval development, male PVDs extend a similar architecture to the hermaphrodite utilizing the sexually-shared Menorin patterning mechanism. In early adulthood, however, male PVD develops a unique extension into the copulatory tail structure. Alongside established tail ray neurons RnA and RnB, we show PVD is a third, previously unrecognized, neuron within the tail rays. Unlike RnA and RnB, PVD extends anterogradely, branches and turns within the ray hypodermis, and is non-ciliated. This PVD sexually-dimorphic arborization is absent in mutant backgrounds which perturb the Menorin guidance complex. SAX-7/L1CAM, a hypodermal component of this complex, shows a male-specific expression pattern which precedes PVD extension, and its presence allows PVD to enter the tail rays. Further, our results reveal that genetically altered arborization or ablation of the PVD result in male mating behavioral defects, particularly as males turn around the hermaphrodite. These results uncover an adult-stage sexual dimorphism of dendritic branching and uncover a function for PVD in male sexual behavior. Significance StatementNeurons form intricate shapes and networks, which may display sexual differences. Pinpointing these changes at the single cell level, however, is challenging. In C. elegans, the PVD neuron is a powerful model for stereotypical neuron shaping, yet most studies concern only one of two sexes. Our research focuses on the understudied male, where we show an adult-stage extension of PVD into the male copulatory tail organ. We further show how this stems from a sex-shared patterning complex, progressing independently and in a different environment compared with nearby male-specific neurons. We further find PVD has a role in the male adult-specific mating behavior. PVD thus presents a unique example of a highly arborized neuron showing sexually dimorphic behavioral function and structure.

neuroscience↗

bioRxiv2023

Sperm induction of somatic cell-cell fusion as a novel functional test

The fusion of mammalian gametes requires the interaction between IZUMO1 on the sperm and JUNO on the oocyte. We have recently shown that ectopic expression of mouse IZUMO1 induces cell-cell fusion and that sperm can fuse to fibroblasts expressing JUNO. Here, we found that the incubation of mouse sperm with hamster fibroblasts or human epithelial cells in culture induces the fusion between these somatic cells and formation of syncytia, a pattern previously observed with some animal viruses. This sperm-induced cell-cell fusion requires a species-matching JUNO on both fusing cells, can be blocked by an antibody against IZUMO1, and does not rely on the synthesis of new proteins. The fusion is dependent on the sperms fusogenic capacity, making this a reliable, fast and simple method for predicting sperm function during the diagnosis of male infertility. HighlightsO_LISperm induces viral-like fusion of somatic cells expressing JUNO C_LIO_LIWe developed a new technique to determine the fertilization potential of sperm C_LIO_LIThe test measures the capacity of sperm to induce somatic cell fusion C_LIO_LIThe degree of somatic cell fusion correlates with the fertilizing ability of sperm C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/549574v2_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@7f1d7corg.highwire.dtl.DTLVardef@168d0c3org.highwire.dtl.DTLVardef@61e845org.highwire.dtl.DTLVardef@1d83b72_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2023

Enveloped viruses pseudotyped with mammalian myogenic cell fusogens target skeletal muscle for gene delivery

Entry of enveloped viruses into cells is mediated by fusogenic proteins that form a complex between membranes to drive rearrangements needed for fusion. Skeletal muscle development also requires membrane fusion events between progenitor cells to form multinucleated myofibers. Myomaker and Myomerger are muscle-specific cell fusogens, but do not structurally or functionally resemble classical viral fusogens. We asked if the muscle fusogens could functionally substitute for viral fusogens, despite their structural distinctiveness, and fuse viruses to cells. We report that engineering of Myomaker and Myomerger on the membrane of enveloped viruses leads to specific transduction of skeletal muscle. We also demonstrate that locally and systemically injected virions pseudotyped with the muscle fusogens can deliver micro-Dystrophin (Dys) to skeletal muscle of a mouse model of Duchenne muscular dystrophy. Through harnessing the intrinsic properties of myogenic membranes, we establish a platform for delivery of therapeutic material to skeletal muscle.

cell biology↗

bioRxiv2022

IZUMO1 is a sperm fusogen

Mammalian sperm-egg adhesion depends on the trans-interaction between the sperm-specific type I glycoprotein IZUMO1 and its oocyte-specific GPI-anchored receptor JUNO. However, the mechanisms and proteins (fusogens) which mediate the following step of gamete fusion remain unknown. Using live imaging and content mixing assays in a heterologous system and structure-guided mutagenesis, we unveil an unexpected function for IZUMO1 in cell-to-cell fusion. We show that IZUMO1 alone is sufficient to induce fusion, and that this ability is retained in a mutant unable to bind JUNO. On the other hand, a triple mutation in exposed aromatic residues prevents this fusogenic activity without impairing JUNO interaction. Our findings suggest a second, crucial function for IZUMO1 as a unilateral mouse gamete fusogen. HighlightsO_LIIZUMO1 expression in somatic cells in culture induces cell-to-cell fusion C_LIO_LIThe fusogenic activity of IZUMO1 is unilateral C_LIO_LICell fusion is independent of the binding of IZUMO1 to JUNO C_LIO_LIIZUMO1-mediated cell merger depends on its transmembrane domain, and three solvent-exposed aromatic residues C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/478669v2_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1f06f47org.highwire.dtl.DTLVardef@19d5adorg.highwire.dtl.DTLVardef@1b8d6eorg.highwire.dtl.DTLVardef@e23e26_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

Archaeal origins of gamete fusion

Sexual reproduction consists of genome reduction by meiosis and subsequent gamete fusion. Presence of meiotic genes in prokaryotes suggests that DNA repair mechanisms evolved toward meiotic recombination; however, fusogenic proteins resembling those found in eukaryotes were not identified in prokaryotes. Here, we identify archaeal proteins that are homologs of fusexins, a superfamily of fusogens that mediate eukaryotic gamete and somatic cell fusion, as well as virus entry. The crystal structure of a trimeric archaeal Fusexin1 reveals novel features such as a six-helix bundle and an additional globular domain. Ectopically expressed Fusexin1 can fuse mammalian cells, and this process involves the additional domain and a conserved fusion loop. Archaeal fusexin genes exist within integrated mobile elements, potentially linking ancient archaeal gene exchanges and eukaryotic sex. One-Sentence SummaryCell membrane fusion proteins of viruses and eukaryotes are also present in archaea.

evolutionary biology↗

bioRxiv2021

Neuron tracing and quantitative analyses of dendritic architecture reveal symmetrical three-way-junctions and phenotypes of git-1 in C. elegans

Complex dendritic trees are a distinctive feature of neurons. Alterations to dendritic morphology are associated with developmental, behavioral and neurodegenerative changes. The highly-arborized PVD neuron of C. elegans serves as a model to study dendritic patterning; however, quantitative, objective and automated analyses of PVD morphology are missing. Here, we present a method for neuronal feature extraction, based on deep-learning and fitting algorithms. The extracted neuronal architecture is represented by a database of structural elements for abstracted analysis. We obtain excellent automatic tracing of PVD trees and uncover that dendritic junctions are unevenly distributed. Surprisingly, these junctions are three-way-symmetrical on average, while dendritic processes are arranged orthogonally. We quantify the effect of mutation in git-1, a regulator of dendritic spine formation, on PVD morphology and discover a localized reduction in junctions. Our findings shed new light on PVD architecture, demonstrating the effectiveness of our objective analyses of dendritic morphology and suggest molecular control mechanisms. Author SummaryNerve cells (neurons) collect input signals via branched cellular projections called dendrites. A major aspect of the study of neurons, dating back over a century, involves the characterization of neuronal shapes and of their dendritic processes. Here, we present an algorithmic approach for detection and classification of the tree-like dendrites of the PVD neuron in C. elegans worms. A key feature of our approach is to represent dendritic trees by a set of fundamental shapes, such as junctions and linear elements. By analyzing this dataset, we discovered several novel structural features. We have found that the junctions connecting branched dendrites have a three-way-symmetry, although the dendrites are arranged in a crosshatch pattern; and that the distribution of junctions varies across distinct sub-classes of the PVDs dendritic tree. We further quantified subtle morphological effects due to mutation in the git-1 gene, a known regulator of dendritic spines. Our findings suggest molecular mechanisms for dendritic shape regulation and may help direct new avenues of research.

systems biology↗

bioRxiv2020

Tissue-specific delivery system via AFF-1-coated pseudotyped Vesicular Stomatitis Virus in C. elegans

A hallmark of muscle development is that myoblasts fuse to form myofibers. However, smooth muscles and cardiomyocytes do not generally fuse. In C. elegans, the body wall muscles (BWMs), the physiological equivalents of skeletal muscles, are mononuclear. Here, to determine what would be the consequences of fusing BWMs, we express the cell-cell fusogen EFF-1 in these cells. We find that EFF-1 induces paralysis and dumpy phenotypes. To determine whether EFF-1-induced muscle fusion results in these pathologies we injected viruses pseudotyped with AFF-1, a paralog of EFF-1, into the pseudocoelom of C. elegans. When these engineered viruses encounter cells expressing EFF-1 or AFF-1 they are able to infect them as revealed by GFP expression from the viral genome. We find that AFF-1 viruses can fuse to EFF-1-expressing muscles revealing multinucleated fibers that cause paralysis and abnormal muscle morphogenesis. Thus, aberrant fusion of otherwise non-syncytial muscle cells may lead to pathological conditions. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/099622v2_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@ebdf7dorg.highwire.dtl.DTLVardef@19d9246org.highwire.dtl.DTLVardef@1ee3698org.highwire.dtl.DTLVardef@425a0c_HPS_FORMAT_FIGEXP M_FIG C_FIG Significance statementMost cells are individual units that do not mix their cytoplasms. However, some cells fuse to become multinucleated in placenta, bones and muscles. In most animals, muscles are formed by myofibers that originate by cell-cell fusion. In contrast, in C. elegans the body wall muscles are mononucleated cells that mediate worm-like movement. EFF-1 and AFF-1 fusogens mediate physiological cell fusion in C. elegans. By ectopically expressing EFF-1 in body wall muscles we induce their fusion resulting in behavioral and morphological deleterious effects, revealing possible causes of congenital myopathies in humans. Using AFF-1-coated pseudoviruses we infect EFF-1-expressing muscle cells retargeting viral infection into these cells. We suggest that virus retargeting can be utilized to study myogenesis, neuronal regeneration, gamete fusion and screens for new fusogens in different organisms. In addition, our virus retargeting system can be used in gene-therapy, viral-based oncolysis and to study viral-host interactions.

cell biology↗

bioRxiv2018

Sensory experience controls dendritic structure and behavior by distinct pathways involving degenerins

Dendrites are crucial for receiving information into neurons. Sensory experience affects the structure of these tree-like neurites, which, it is assumed, modifies neuronal function, yet the evidence is scarce, and the mechanisms are unknown. To study whether sensory experience affects dendritic morphology, we use the Caenorhabditis elegans arborized nociceptor PVD neurons, under natural mechanical stimulation induced by physical contacts between individuals. We found that mechanosensory signals induced by conspecifics and by glass beads affect the dendritic structure of the PVD. Moreover, developmentally isolated animals show a decrease in their ability to respond to harsh touch. The structural and behavioral plasticity following sensory deprivation are functionally independent of each other and are mediated by an array of evolutionarily conserved mechanosensory amiloride-sensitive epithelial sodium channels (degenerins). Calcium imaging of the PVD neurons in a micromechanical device revealed that controlled mechanical stimulation of the body wall produces similar calcium dynamics in both isolated and crowded animals. Our genetic results, supported by optogenetic, behavioral, and pharmacological evidence, suggest an activity- dependent homeostatic mechanism for dendritic structural plasticity, that in parallel controls escape response to noxious mechanosensory stimuli. Significance StatementLittle is known about how neuronal activity and sensory experience influence the structure and function of dendritic trees. Furthermore, how dendritic structures affect neuronal functions remains to a large extent a mystery despite their fundamental functions in neuronal sensory and synaptic activities in health and diseases. Here we show that complex dendritic trees of the nociceptive and proprioceptive PVD neuron in C. elegans display a dynamic nature where they undergo pronounced dendritic modifications during adulthood. These modifications are determined by the received sensory signals generated by conspecific worms on the plate or by inert glass beads. We revealed functions for degenerins/Epithelial sodium channels in this phenomenon, using genetic and optogenetic approaches. We found that the degenerin MEC-10 acts cell autonomously to translate environmental mechanical signals into structural and functional modifications in the PVD.

neuroscience↗

bioRxiv2016

Arabidopsis HAP2/GCS1 is a gamete fusion protein homologous to somatic and viral fusogens

Cell-cell fusion is inherent to any form of sexual reproduction. Loss of HAPLESS 2/GENERATIVE CELL SPECIFIC 1 (HAP2/GCS1) proteins results in gamete fusion failure in different organisms but their exact role is unclear. Here we show that Arabidopsis HAP2/GCS1 expression in mammalian cells is sufficient to promote cell-cell fusion. Hemifusion and complete fusion depend on HAP2/GCS1 presence in both fusing cells. Furthermore, expression of HAP2 on the surface of pseudotyped vesicular stomatitis virus and on the target cells results in HAP2-dependent virus-cell fusion. This bilateral requirement can be bypassed by replacing the plant gene with C. elegans EFF-1 somatic cell fusogen in one of the fusing cells or the virus, indicating that HAP2/GCS1 and EFF-1 share a similar fusion mechanism. Structural modeling of the HAP2/GCS1 protein family predicts that they are homologous to EFF-1 and class II fusion proteins from enveloped viruses (e.g. dengue and Zika viruses). We name this superfamily FUSEXINS: FUSion proteins essential for sexual reproduction and EXoplasmic merger of plasma membranes. Thus, Fusexins unify the origin and evolution of sexual reproduction, enveloped virus entry into cells and somatic cell fusion.

cell biology↗