Biology subjects

Pelletier, L.

Publications and source records attributed to Pelletier, L..

12 recordsLinked to original sources

bioRxiv2026

Macrophage to Myocyte Mitochondrial Transfer in the Myometrium: A Novel Mechanism for the Initiation of Labor

Labor initiation involves influx of peripheral monocytes into uterine tissue, where they differentiate into macrophages (Macs) and polarize toward pro-inflammatory (M1) or anti-inflammatory (M2) phenotypes. Crosstalk between uterine myocytes (MYO) and Macs is implicated in myometrial activation, but the underlying mechanisms remain unclear. Using immunofluorescence and live-cell imaging, we show that M1-Macs (but not M2-Macs) form tunneling nanotubes (TNT) with MYOs, enabling direct, unidirectional mitochondrial transfer. TNT-mediated mitochondrial transfer enhances ATP production and 20-HSD expression in MYO. Functionally, M1-Macs induce intracellular and functional progesterone (P4) withdrawal in MYOs via 20-HSD mediated P4 metabolism and increased PR-A phosphorylation. In vivo, transfer of monocytes, carrying dendra-labelled mitochondria in pregnant mice confirm their myometrial recruitment, differentiation, polarization to M1-Macs, and M1-Mac/MYO mitochondrial transfer. Increased M1-Macs and mitochondrial transfer to MYOs are observed before labor onset, implicating M1-Macs/MYO interaction as key regulator of labor initiation and potentially a therapeutic target to prevent preterm birth.

immunology↗

bioRxiv2026

Physiological perfusion of human vasculature reveals a YAP/TAZ-Apelin switch linking intraluminal flow to endothelial state transitions and vessel remodeling

Vascular flow delivers nutrients and imposes hemodynamic forces that govern vessel behavior in health and disease, yet fully human systems that recapitulate and tune physiological intraluminal flow in three-dimensional (3D) tissues are lacking. We developed VIVOS (Vascularized In Vitro Organ Systems), a platform that couples perfused human vascular beds to tunable pumps, generating continuous intraluminal flow through millimetre-scale vessels and 3D tissues at physiological shear stresses and pressures. VIVOS supports integration and perfusion of diverse human organoids and tissues, including lung organoids, cerebral organoids, vascular organoids, breast spheroids, and human retinal explants, as well as enables direct measurement and control of pressure, shear stress, and perfusion-dominant compound transport over extended culture periods. By tuning intraluminal flow and applying single-cell transcriptomics, we uncover a remodeling program in which laminar shear stress acts through a YAP/TAZ-TEAD "switch" to rewire an Apelin ligand-receptor axis and bias tip-stalk endothelial states, reshaping human vascular networks and linking hemodynamic cues to cell state transitions. We further model fast-flow arteriovenous malformations (AVMs) from Hereditary Hemorrhagic Telangiectasia and show that BMP9 constrains vessel caliber and perfusion while antagonizing a VEGF-driven angiogenic program, generating flow-quantified AVM-like lesions in a fully human 3D context. Together, these findings establish VIVOS as a generalizable platform that links physiological intraluminal flow to endothelial state transitions and vessel remodeling, enabling preclinical testing and mechanistic dissection of flow-regulated vascular pathologies in perfused 3D human tissues under defined hemodynamic conditions.

bioengineering↗

bioRxiv2023

qTAG: An adaptable CRISPR-based endogenous tagging protocol using optimized repair cassettes

Endogenous tagging makes it possible to study a proteins localization, dynamics, and function within its native regulatory context. This is typically accomplished via CRISPR, which involves inserting a sequence encoding a functional tag into the reading frame of a gene. However, this process is often inefficient. Here, we introduce the "quickTAG," or qTAG system, a versatile collection of optimized repair cassettes designed to make CRISPR-mediated tagging more accessible. By including a desired tag sequence linked to a selectable marker in the cassette, integrations can be quickly isolated post-editing. The core sequence scaffold within these constructs incorporates several key features that enhance flexibility and ease of use, such as: specific cassette designs for N- and C-terminus tagging; standardized cloning sequences to simplify the incorporation of homology arms for HDR or MMEJ-based repairs; restriction sites next to each genetic element within the cassette for easy modification of tags and selectable markers; and the inclusion of lox sites flanking the selectable marker to allow for marker gene removal following integration. We showcase the versatility of these cassettes with a diverse range of tags, demonstrating their applications in fluorescence imaging, proximity-dependent biotinylation, epitope tagging, and targeted protein degradation. The adaptability of this scaffold is also exhibited by incorporating novel tags such as mStayGold, which offer enhanced brightness and photostability, reconciling prolonged live-cell imaging of proteins at their endogenous levels. Finally, by leveraging the restriction sites, entirely distinct cassette structures and editing schemes were developed. These enabled scenarios that included conditional expression tagging, selectable knockout tagging, and safe-harbor expression. Our existing and forthcoming collection of plasmids will be accessible through Addgene. It includes ready-to-use constructs targeting common subcellular marker genes, as well as an assortment of tagging cassettes for the tagging of genes of interest. The qTAG system offers an accessible framework to streamline endogenous tagging and will serve as an open resource for researchers to adapt and tailor for their own experiments.

molecular biology↗

bioRxiv2023

lower case Greek beta-catenin mediates growth defects induced my centrosome loss in APC mutant colorectal cancer independently of p53

Colorectal cancer is the third most common cancer and the second leading cause of cancer-related deaths worldwide. The centrosome is the main microtubule-organizing center in animal cells and centrosome amplification is a hallmark of cancer cells. To investigate the importance of centrosomes in colorectal cancer, we induced centrosome loss in normal and cancer human-derived colorectal organoids using centrinone B, a Polo-like kinase 4 (Plk4) inhibitor. We show that centrosome loss represses human normal colorectal organoid growth in a p53-dependent manner in accordance with previous studies in cell models [1]. However, cancer colorectal organoid lines exhibited different sensitivities to centrosome loss independently of p53. Centrinone-induced cancer organoid growth defect/death positively correlated with a loss of function mutation in the APC gene, suggesting a causal role of the hyperactive WNT pathway. Consistent with this notion, {beta}-catenin inhibition using XAV-939 or ICG-001 partially prevented centrinone-induced death and rescued the growth of APC-mutant organoid lines. Our study reveals a novel role for canonical WNT signaling in regulating centrosome loss-induced growth defect/death in APC-mutant colorectal cancer independently of the classical p53 pathway.

cancer biology↗

bioRxiv2022

Centriolar satellites expedite mother centriole remodeling to promote ciliogenesis.

Centrosomes are orbited by centriolar satellites, dynamic multiprotein assemblies nucleated by PCM1. To study the requirement for centriolar satellites, we generated mice lacking PCM1. Pcm1-/- mice display partially penetrant perinatal lethality with survivors exhibiting hydrocephalus, oligospermia and cerebellar hypoplasia, as well as variable expressivity of other ciliopathy features including cystic kidneys. Pcm1-/- multiciliated ependymal cells and PCM1-/- retinal pigmented epithelial 1 (RPE1) cells showed reduced ciliogenesis. PCM1-/- RPE1 cells displayed reduced docking of the mother centriole to the ciliary vesicle and removal of CP110 and CEP97 from the distal mother centriole, indicating compromized early ciliogenesis. We show these molecular cascades are maintained in vivo, and we suggest that the cellular threshold to trigger ciliogenesis varies between cell types. We propose that PCM1 and centriolar satellites facilitate efficient trafficking of proteins to and from centrioles, inducing the departure of CP110 and CEP97 to initiate ciliogenesis.

cell biology↗

bioRxiv2021

Global cellular response to chemical perturbation of PLK4 activity and abnormal centrosome number

Centrosomes act as the main microtubule organizing centre (MTOC) in metazoans. Centrosome number is tightly regulated by limiting centriole duplication to a single round per cell cycle. This control is achieved by multiple mechanisms, including the regulation of the protein kinase PLK4, the most upstream facilitator of centriole duplication. Altered centrosome numbers in mouse and human cells cause p53-dependent growth arrest through poorly defined mechanisms. Recent work has shown that the E3 ligase TRIM37 is required for cell cycle arrest in acentrosomal cells. To gain additional insights into this process, we undertook a series of genome-wide CRISPR/Cas9 screens to identify factors important for growth arrest triggered by treatment with centrinone B, a selective PLK4 inhibitor. We found that TRIM37 is a key mediator of growth arrest after partial or full PLK4 inhibition. Interestingly, PLK4 cellular mobility decreased in a dose-dependent manner after centrinone B treatment. In contrast to recent work, we found that growth arrest after PLK4 inhibition correlated better with PLK4 activity than with mitotic length or centrosome number. These data provide insights into the global response to changes in centrosome number and PLK4 activity and extend the role for TRIM37 in regulating the abundance, localization and function of centrosome proteins.

cell biology↗

bioRxiv2021

Saturation variant interpretation using CRISPR prime editing

Over the last decade, next generation sequencing has become widely implemented in clinical practice. However, as genetic variants of uncertain significance (VUS) are frequently identified, the need for scaled functional interpretation of such variants has become increasingly apparent. One method to address this is saturation genome editing (SGE), which allows for scaled multiplexed functional assessment of single nucleotide variants. The current applications of SGE, however, rely on homology-directed repair (HDR) to introduce variants of interest, which is limited by low editing efficiencies and low product purity. Here, we have adapted CRISPR prime editing for SGE and demonstrated its utility in understanding the functional significance of variants in the NPC1 gene underlying the lysosomal storage disorder Niemann-Pick disease type C1 (NPC). Additionally, we have designed a genome editing strategy that allows for the haploidization of gene loci, which permits isolated variant interpretation in virtually any cell type. By combining saturation prime editing (SPE) with a clinically relevant assay, we have functionally scored and interpreted 256 variants in NPC1 haploidized HEK293T cells. To further demonstrate the applicability of this strategy, we used SPE and cell model haploidization to functionally score 465 variants in the BRCA2 gene. We anticipate that our work will be translatable to any gene with an appropriate cellular assay, allowing for more rapid and accurate diagnosis and improved genetic counselling and ultimately precise patient care.

genetics↗

bioRxiv2020

The CP110-CEP97-CEP290 module orchestrates a centriolar satellite dependent response to proteotoxic stress

Protein degradation at the centrosome, the primary microtubule organizing centre of the cell, is critical to a myriad of cellular processes. Perturbation of the ubiquitin proteasome system causes the formation of an inclusion, or aggresome, at the centrosome. By systematic microscopy analysis, we have placed a subset of centrosomal proteins within the aggresome. Centriolar satellites, proteinaceous granules found in the vicinity of centrosomes, also became incorporated into this structure. Through high-resolution quantitative analysis, we have defined aggresome assembly at the centrosome, demonstrating a requirement for satellites in this process. Furthermore, a module consisting of CP110-CEP97-CEP290 was required to recruit aggresome components early in the pathway and senescent cells were defective in aggresome formation due to limiting amounts of CP110. Finally, satellites and the CP110-CEP97-CEP290 module were required for the aggregation of mutant huntingtin. The accumulation of protein aggregates is central to the pathology of a range of human disorders. These data thereby reveal new roles for CP110, its interactors, and centriolar satellites in controlling cellular proteostasis and the aggregation of disease relevant proteins.

cell biology↗

bioRxiv2020

In vivo RyR1 reduction in muscle triggers a core-like myopathy

Some mutations in the RYR1 gene lead to congenital myopathies, through reduction in this calcium channel expression level, but the functional whole organism consequences of reduction in RyR1 amount have never been studied. We have developed and characterized a mouse model with inducible muscle specific RYR1 deletion. Recombination in the RYR1 gene resulted in a progressive reduction in the protein amount and was associated with a progressive muscle weakness and atrophy. Calcium fluxes in isolated muscle fibers were accordingly reduced. Alterations in the muscle structure were observed, with fibers atrophy, abnormal mitochondria distribution, membrane remodeling, associated with increase in the expression level of many proteins and inhibition of the autophagy process. This model demonstrates that RyR1 reduction is sufficient to recapitulate most features of Central Core Disease, and accordingly similar alterations were observed in muscle biopsies from Central Core Disease patients, pointing to common pathophysiological mechanisms related to RyR1 reduction.

neuroscience↗

bioRxiv2020

Comparison of SARS-CoV-2 Indirect and Direct Detection Methods

The COVID-19 pandemic caused by the SARS-CoV-2 virus has placed extensive strain on RNA isolation and RT-qPCR reagents. Rapid development of new test kits has helped to alleviate these shortages. However, comparisons of these new detection systems are largely lacking. Here, we compare indirect methods that require RNA extraction, and direct RT-qPCR on patient samples. For RNA isolation we compared four different companies (Qiagen, Invitrogen, BGI and Norgen Biotek). For detection we compared two recently developed Taqman-based modules (BGI and Norgen Biotek), a SYBR green-based approach (NEB Luna Universal One-Step Kit) with published and newly-developed primers, and clinical results (Seegene STARMag RNA extraction system and Allplex 2019-nCoV RT-qPCR assay). Most RNA isolation procedures performed similarly, and while all RT-qPCR modules effectively detected purified viral RNA, the BGI system proved most sensitive, generating comparable results to clinical diagnostic data, and identifying samples ranging from 65 copies - 2.1x105 copies of viral Orf1ab/l. However, the BGI detection system is [~]4x more expensive than other options tested here. With direct RT-qPCR we found that simply adding RNase inhibitor greatly improved sensitivity, without need for any other treatments (e.g. lysis buffers or boiling). The best direct methods were [~]10 fold less sensitive than indirect methods, but reduce sample handling, as well as assay time and cost. These studies will help guide the selection of COVID-19 detection systems and provide a framework for the comparison of additional systems.

microbiology↗

bioRxiv2019

A proximity biotinylation map of a human cell

Compartmentalization is a defining characteristic of eukaryotic cells, partitioning cellular processes into discrete subcellular locations. High throughput microscopy1 and biochemical fractionation coupled with mass spectrometry2-6 have helped to define the proteomes of a variety of organelles and macromolecular structures. However, many other intracellular compartments have remained refractory to such approaches, due for example to difficulty in purifying non-membrane bound structures. Proximity-dependent biotinylation techniques such as BioID provide an alternative approach for defining the composition of cellular compartments in living cells7-10. Here we present a BioID-based map of a human cell based on 192 markers from 32 different subcellular compartments, comprising 35,902 high confidence proximity interactions, and defining the intracellular locations of 4,145 unique proteins in HEK 293 cells. Our localization predictions meet or exceed previous-approaches, with higher specificity, and enabled the discovery of proteins at the mitochondrial outer membrane-endoplasmic reticulum (ER) interface that are critical for mitochondrial homeostasis. Based on this dataset, we have established humancellmap.org as a community resource that provides online tools for localization analysis of user BioID data, and demonstrate how this resource can be used to better understand BioID datasets.

molecular biology↗

bioRxiv2019

LUZP1 and the tumour suppressor EPLIN are negative regulators of primary cilia formation

Cilia/flagella are microtubule-based cellular projections with important sensory and motility functions. Their absence or malfunction is associated with a growing number of human diseases collectively referred to as ciliopathies. However, the fundamental mechanisms underpinning cilia biogenesis and functions remain only partly understood. Here, we show that LUZP1, and its interacting protein EPLIN, are negative regulators of primary cilia formation. LUZP1 is an actin-associated protein that localizes to both actin filaments and the centrosome/basal body. Like EPLIN, LUZP1 is an actin-stabilizing protein and likely regulates actin dynamics at the centrosome. Both proteins interact with ciliogenesis and cilia length regulators, and are potential players in the actin-dependent processes involved in centrosome to basal body conversion. Ciliogenesis deregulation caused by LUZP1 or EPLIN loss may contribute to the pathology of their associated diseases.\n\nSUMMARYGoncalves et al. show that LUZP1 and its interactor EPLIN are negative regulators of ciliogenesis. LUZP1 is a novel actin-stabilizing protein localizing at the centrosome and basal body where it may regulate actin-associated processes.

cell biology↗