Biology subjects

Courtemanche, N.

Publications and source records attributed to Courtemanche, N..

7 recordsLinked to original sources

bioRxiv2026

Translation of a small upstream open reading frame functions as a rheostat for the regulation of lin-41 by the Let-7 microRNA in Caenorhabditis elegans

MicroRNAs have been likened to the "dark matter" of eukaryotic genomes, reflecting their pervasive regulatory influence. MicroRNAs were first identified through genetic studies of developmental timing in the nematode Caenorhabditis elegans. Let-7 was the first microRNA recognized to be broadly conserved. The principal target of Let-7 in the developmental timing pathway is the TRIM-NHL RNA-binding protein LIN-41. During the L4 larval stage, Let-7 represses lin-41 translation by binding to two Let-7 complementary sites in the lin-41 3'UTR. Despite the importance of microRNA-based translational regulation, the underlying molecular mechanisms are incompletely understood. Through genetic analysis, we discovered an unrecognized feature of the mechanism by which Let-7 controls lin-41 translation. This mechanism requires a 5'-regulatory exon containing a seven-amino acid upstream open reading frame (uORF) and conserved sequence elements. Genome editing indicates that the specific uORF amino acid sequence itself is not important. Our data suggest that uORF translation and 5'UTR structure limit initiation at the downstream lin-41 start codon, enabling tight control by Let-7. Without this mechanism, the Let-7 microRNA is unable to properly regulate lin-41 to enable proper development.

developmental biology↗

bioRxiv2025

Mechanism of formin-mediated filament nucleation from profilin-actin

Formins direct the assembly of unbranched actin filaments through dynamic interactions with the abundant, actin-binding protein profilin. Although the process of formin-mediated filament elongation is well characterized, the mechanism by which formins nucleate actin filaments is poorly understood. In this study, we used in vitro reconstitution assays and kinetic modeling to dissect filament nucleation mediated by the S. cerevisiae formin Bni1p. We found that formins assemble filament nuclei by sequentially binding three actin monomers in a process that takes place in parallel to spontaneous actin assembly. Whereas the formin FH2 domain preferentially binds actin monomers, the flexible FH1 domain facilitates de novo filament formation by binding and transferring profilin-actin complexes to a free FH2 dimer. FH1-mediated delivery stimulates filament nucleation in a distance- and sequence-dependent manner. The architectures of FH1 domains therefore maximize the efficiency of formin-mediated polymerization through optimized engagement of each of their profilin-binding sites.

biophysics↗

bioRxiv2025

A sperm-oocyte protein partnership required for egg activation in Caenorhabditis elegans

ABSTRACTFertilization triggers the completion of female meiosis and launches the oocyte-to-embryo transition. C. elegans spe-11 is one of the few known paternal-effect embryonic lethal genes. We report that the sperm protein, SPE-11, forms a complex with an oocyte protein, OOPS-1 (Oocyte Partner of SPE-11), and that the protein complex is required for the completion of meiosis, the block to polyspermy, and eggshell formation. Consistent with the molecular interaction of their encoded proteins, oops-1 and spe-11 exhibit identical null phenotypes, displaying defects in meiotic progression and cytokinesis. We show that the complex binds F-actin in the absence of other proteins and inhibits the nucleation of actin filaments in vitro. Thus, the OOPS-1-SPE-11 complex may function to promote F-actin-mediated meiotic cytokinesis. Both OOPS-1 and SPE-11 are intrinsically disordered proteins that are highly phosphorylated. Biochemical and genetic experiments define interactions with the protein phosphatase 1 homologs GSP-3/4, which appear to promote OOPS-1-SPE-11 function. Genetic results support a model in which the OOPS-1-SPE-11 complex interacts with the cortical EGG complex to promote meiotic cytokinesis and to activate synthesis of the eggshell.

developmental biology↗

bioRxiv2022

Treatment with Tumor-Treating Fields (TTFields) Suppresses Intercellular Tunneling Nanotube Formation In Vitro and Upregulates Immuno-Oncologic Biomarkers In Vivo in Malignant Mesothelioma

Intercellular communication is critical for the development of invasive cancers. Multiple forms of intercellular communication have been well characterized, involving diffusible soluble factors or contact-dependent channels for immediately adjacent cells. Over the past 1-2 decades, the emergence of a unique form of F-actin-based cellular protrusion known as tunneling nanotubes (TNTs) has filled the niche of long-range cell-contact dependent intercellular communication that facilitates cell growth, differentiation, and in the case of invasive cancer phenotypes, a more chemoresistant phenotype. The cellular machinery of TNT-mediated transport is an area of active investigation, and microtubules have been implicated in this process as they are in other membranous protrusions. Tumor-Treating Fields (TTFields) therapy is a novel therapeutic strategy in clinical use for patients with advanced cancers, based on the principle of using low-intensity alternating electric fields to disrupt microtubules in cancer cells undergoing mitosis. Other mechanisms of action have also been demonstrated. In this study, we investigated the effects of TTFields on TNTs in malignant pleural mesothelioma (MPM) in vitro and also on the spatial transcriptomic landscape in vivo. We found that applying TTFields at 1.0 V/cm significantly suppressed TNT formation in a biphasic MPM cell line (MSTO-211H), but not in sarcomatoid MPM (VAMT). At these parameters, TTFields significantly reduced cell count in MSTO-211H, but did not significantly alter intercellular transport of mitochondria via intact TNTs. To understand how TTFields may impact expression of genes with known involvement to TNT formation and overall tumor growth, we performed spatial genomic assessment of TTFields-treated tumors from an in vivo animal model of MPM, and detected upregulation of immuno-oncologic biomarkers with simultaneous downregulation of pathways associated with cell hyperproliferation, invasion, and other critical regulators of oncogenic growth. Several molecular classes and pathways coincide with markers that we and others have found to be differentially expressed in cancer cell TNTs, including MPM specifically. In this study, we report novel cellular and molecular effects of TTFields in relation to tumor communication networks enabled by TNTs and related molecular pathways. These results position TNTs as potential therapeutic targets for TTFields-directed cancer treatment strategies; and also identify the ability of TTFields to potentially remodel the tumor microenvironment, thus enhancing response to immunotherapeutic drugs.

cancer biology↗

bioRxiv2021

Formin's nucleation activity influences actin filament length

Formins stimulate actin polymerization by promoting both filament nucleation and elongation. Because nucleation and elongation draw upon a common pool of actin monomers, the rate at which each reaction proceeds influences the other. This interdependent mechanism determines the number of filaments assembled over the course of a polymerization reaction, as well as their equilibrium lengths. In this study, we used kinetic modeling and in vitro polymerization reactions to dissect the contributions of filament nucleation and elongation to the process of formin-mediated actin assembly. We found that the rates of nucleation and elongation evolve over the course of a polymerization reaction. The period over which each process occurs is a key determinant of the total number of filaments that are assembled, as well as their average lengths at equilibrium. Inclusion of formin in polymerization reactions speeds filament nucleation, thus increasing the number and shortening the lengths of filaments that are assembled over the course of the reaction. Although variations in elongation rates produce modest changes in the equilibrium lengths of formin-bound filaments, nucleation constitutes the primary mode of monomer consumption over the course of assembly. Sustained elongation of small numbers of formin-bound filaments therefore requires inhibition of nucleation via monomer sequestration and a low concentration of activated formin. Our results underscore the mechanistic advantage for keeping formins nucleation efficiency relatively low in cells, where unregulated actin assembly would produce deleterious effects on cytoskeletal dynamics. Under these conditions, differences in the elongation rates mediated by formin isoforms are most likely to impact the kinetics of actin assembly.

biophysics↗

bioRxiv2019

Structural state recognition facilitates tip tracking of EB1 at growing microtubule ends in cells

The microtubule binding protein EB1 specifically targets the growing ends of microtubules in cells, where EB1 facilitates the interactions of cellular proteins with microtubule plus-ends. Microtubule end targeting of EB1 has been attributed to high affinity binding of EB1 to GTP-tubulin that is present at growing microtubule ends. However, our 3D single-molecule diffusion simulations predicted a [~]6000% increase in EB1 arrivals to open, tapered microtubule tip structures relative to closed lattice conformations. Using quantitative fluorescence, single-molecule, and electron microscopy experiments, we found that the binding of EB1 onto opened, structurally disrupted microtubules was dramatically increased relative to closed, intact microtubules, regardless of hydrolysis state. Correspondingly, in cells, the conversion of growing microtubule ends from a tapered into a blunt configuration resulted in reduced EB1 targeting. Together, our results suggest that microtubule structural recognition, based on a fundamental diffusion-limited binding model, facilitates the tip tracking of EB1 at growing microtubule ends.

cell biology↗

bioRxiv2018

Gating Mechanisms during Actin Filament Elongation by Formins

Formins play an important role in the polymerization of unbranched actin filaments, and particular formins slow elongation by 5-95%. We studied the interactions between actin and the FH2 domains of formins Cdc12, Bni1 and mDia1 to understand the factors underlying their different rates of polymerization. All-atom molecular dynamics simulations revealed two factors that influence actin filament elongation and correlate with the rates of elongation. First, FH2 domains can sterically block the addition of new actin subunits. Second, FH2 domains flatten the helical twist of the terminal actin subunits, making the end less favorable for subunit addition. Coarse-grained simulations over longer time scales support these conclusions. The simulations show that filaments spend time in states that either allow or block elongation. The rate of elongation is a time-average of the degree to which the formin compromises subunit addition rather than the formin-actin complex literally being in open or closed states.

biophysics↗