Biology subjects

Landoni, J. C.

Publications and source records attributed to Landoni, J. C..

9 recordsLinked to original sources

bioRxiv2026

MTERF1 loss buffers against pathogenic mtDNA deletions through transcriptional regulation

Large-scale mitochondrial DNA (mtDNA) deletions cripple oxidative phosphorylation once they exceed a critical heteroplasmy threshold, causing incurable mitochondrial pathologies. Using a genome-wide CRISPR/Cas9 screen in an engineered human cell line carrying a large-scale mtDNA deletion at high heteroplasmy, we identified mitochondrial transcription termination factor 1 (MTERF1) as a suppressor of the heteroplasmy burden. Loss of MTERF1 restored mitochondrial function and increased cellular proliferation in cells with a mtDNA deletion burden exceeding the pathogenic threshold, without altering heteroplasmy or mtDNA copy number. MTERF1 binds wild-type and deletion-bearing mitochondrial genomes indiscriminately at a site downstream of the ribosomal RNA genes and curbs transcription. Relieving this constraint broadly increased OXPHOS transcripts, thereby eliciting more respiratory output from the residual wild-type genomes. Notably, the buffering effect of MTERF1 loss extended beyond mtDNA deletions. In a counter-screen, MTERF1 loss could also restore respiratory growth in cells depleted of nuclear-encoded mitochondrial genes such as OPA1 and COX5A. Together, these findings indicate that by relieving a transcriptional constraint, MTERF1 loss compensates for reduced genome dosage, defining a strategy to enhance residual mitochondrial function in mtDNA deletion disorders and related conditions.

molecular biology↗

bioRxiv2026

Quantitative Expansion Microscopy for In Situ Estimation of Endogenous Target Abundance

Spatially mapping protein abundance in situ can offer important insights into molecular mechanisms and the physiological functions of protein complexes. This is typically achieved by combining super-resolution microscopy to image fluorescently-labeled protein locations with statistical estimators to retrieve abundances, where accuracy is strongly impacted by labeling efficiency. We introduce quantitative expansion microscopy (qExM) as a method to estimate endogenous protein abundance on ExM data, which offers improved antibody targeting through molecular decrowding. Using cryo-fixation, we preserved ultrastructure and enhanced labeling efficiency to improve accuracy in abundance estimations. We benchmark the effectiveness of qExM by quantifying the stoichiometry of well-characterized nuclear pore complex subunits, and find a mean percent error of 9.4%. We further apply qExM to investigate the abundance of mitochondrial respiratory chain complexes in functionally distinct organelle subpopulations, and of mitochondrial respiratory chain super-complexes in differentially activated human T-cells. qExM provides a robust methodological framework for quantifying endogenous protein abundance in expanded samples in situ.

cell biology↗

bioRxiv2025

Asymmetric mitochondrial trafficking maintains network morphology by balancing perinuclear biogenesis

In functionally polarized cells, mitochondria can form distinct subpopulations, positioned at sites of varying metabolic and energetic demands. Thus far, the potential presence of such subpopulations and implications of their intracellular trafficking in immobile and proliferative cells remains largely undescribed, despite such cells serving as key models. Here, we use substrate micropatterning to create reproducible morphologies of cultured immortalized cells, enabling us to define mitochondrial subpopulations and follow their trafficking by photoactivation. We discovered that mitochondrial material is dispersed asymmetrically throughout the cell via biased anterograde transport from the perinuclear area. Combining quantitative analysis and in silico modeling, we characterize the causes and consequences of unbalanced mitochondrial trafficking. Our findings indicate that this bias is required to distribute new material resulting from perinuclear mitochondrial biosynthesis to sustain mitochondrial mass distribution across the cell, and to maintain normal network connectivity.

cell biology↗

bioRxiv2025

Smart hybrid microscopy for cell-friendlydetection of rare events

Fluorescence microscopy offers unparalleled access to the spatial organization and dynamics of biological events in living samples, yet capturing rare processes over extended durations remains challenging due to tradeoffs between exposure to excitation light and sample health. Here, we introduce hybrid-EDA, an event-driven acquisition (EDA) framework that combines the gentleness and contextual richness of phase contrast with the functional specificity of fluorescence. We developed surveillance for events of interest in label-free microscopy by novel dynamics-informed neural networks that trigger smart acquisitions in fluorescence upon detection. This allows us to dramatically reduce phototoxic damage while obtaining specific and functional information from fluorescence when beneficial. We demonstrate how hybrid-EDA enables improved imaging acquisitions of organelle contacts and mitochondrial divisions. We envision that hybrid-EDA will enable new insights into a range of dynamic and rare biological processes, providing a powerful and general strategy for cell-friendly imaging.

cell biology↗

bioRxiv2025

An updated inventory of genes essential for oxidative phosphorylation identifies a mitochondrial origin in familial Meniere disease

Mitochondrial disorders (MDs) are among the most common inborn errors of metabolism and primarily arise from defects in oxidative phosphorylation (OXPHOS). Their complex mode of inheritance and diverse clinical presentations render the diagnosis of MDs challenging and, to date, most lack a cure. Here, we build on previous efforts to discover genes necessary for OXPHOS and report a highly complementary galactose-sensitized CRISPR-Cas9 "growth" screen, presenting an updated inventory now with 481 OXPHOS genes, including 157 linked to MDs. We further focus on FAM136A, a gene associated with Menieres disease, and show that it supports inter-membrane space protein homeostasis and OXPHOS in cell lines, mice, and patients. Our study identifies a mitochondrial basis in a familial form of Menieres disease (fMD), provides a comprehensive resource of OXPHOS-related genes, and sheds light on the pathways involved in mitochondrial disorders, with the potential to guide future diagnostics and treatments for MDs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/635272v1_ufig1.gif" ALT="Figure 1"> View larger version (79K): org.highwire.dtl.DTLVardef@1841a8forg.highwire.dtl.DTLVardef@1ebbaa8org.highwire.dtl.DTLVardef@12b70aeorg.highwire.dtl.DTLVardef@f2da2a_HPS_FORMAT_FIGEXP M_FIG C_FIG Bullet pointsO_LIGenome-wide CRISPR-Cas9 growth screening complements death screening C_LIO_LI481 genes essential for OXPHOS, including 157 mitochondrial disease genes C_LIO_LIFAM136A supports mitochondrial intermembrane space protein homeostasis C_LIO_LIDepletion of FAM136A in Menieres disease models causes OXPHOS defects C_LI

cell biology↗

bioRxiv2024

The biophysical mechanism of mitochondrial pearling

Mitochondrial networks exhibit remarkable dynamics that are driven in part by fission and fusion events. However, there are other reorganizations of the network that do not involve fission and fusion. One such exception is the elusive, "beads-on-a-string" morphological transition of mitochondria. During such transitions, the cylindrical tubes of the mitochondrial membrane transiently undergo shape changes to a string of "pearls" connected along thin tubes. These dynamics have been anecdotally observed in many contexts and given disparate explanations. Here we unify these observations by proposing a common underlying mechanism based on the biophysical properties of tubular fluid membranes for which it is known that, under particular regimes of tension and pressure, membranes reach an instability and undergo a shape transition to a string of connected pearls. First, we use high-speed light-sheet microscopy to show that transient, short-lived pearling events occur spontaneously in the mitochondrial network in every cell type we have examined, including primary fibroblasts, T-cells, neurons, and budding yeast. We present evidence that transient mitochondrial pearling occurs during important biological events, particularly during T cell activation, neuronal firing, and replicative senescence. Using our high-temporal resolution data, we identify two distinct categories of spontaneous pearling, i) internal pressure-driven pearling generated by ionic flux, and ii) external tension-driven pearling generated by the cytoskeleton. By applying live-cell STED and FIB-SEM imaging we document the structural reorganization of inner cristae membranes during mitochondrial pearling and the role of the MICOS complex in regulating the frequency of pearling events. We then establish numerous methods for inducing pearling, including the ability to induce these dynamics with single mitochondrion precision. These methods include ionophores, channel activators, osmotic shock, detergents, laser stimulation, membrane intercalating molecules, chemical fixation, and micro-needle force. These disparate inducers establish three main physical causes of pearling, i) ionic flux producing internal osmotic pressure, ii) membrane packing lowering bending elasticity, and iii) external mechanical force increasing membrane tension. Pearling dynamics thereby reveal a fundamental biophysical facet of mitochondrial biology. We suggest that pearling should take its place beside fission and fusion as a key process of mitochondrial dynamics, with implications for physiology, disease, and aging.

biophysics↗

bioRxiv2024

Pearling Drives Mitochondrial DNA Nucleoid Distribution

The mitochondria of most eukaryotes carry an indispensable second genome (mtDNA), encoding genes engaged in oxidative phosphorylation1. The regular positioning and segregation of mtDNA-containing nucleoids is essential for mitochondrial function and inheritance, as well as cellular health2-5. However, the underlying mechanism driving nucleoid distribution and disaggregation remains unknown6,7. Our data reveal that mitochondria frequently undergo reversible pearling, a biophysical instability that undulates tubules into regularly spaced beads 8, typically triggered by calcium influx. We discovered that physiological pearling imposes a characteristic length scale, simultaneously mediating nucleoid disaggregation and establishing inter-nucleoid distancing with near-maximally achievable precision. We found that lamellar cristae invaginations of the inner mitochondrial membrane play a dual role, determining pearling frequency and duration, and preserving the resulting nucleoid spacing after organelle recovery to a tubular form. Thus, disrupting cristae ultrastructure resulted in more frequent pearling, but also aberrant nucleoid clustering. Our results demonstrate that the distribution of mitochondrial genomes is governed by the interplay between rapid and reversible pearling and cristae ultrastructure, establishing a mechanism for this long-puzzling yet fundamental feature of eukaryotic life, and offering insights into its potential modulation.

cell biology↗

bioRxiv2022

Overactive mitochondrial DNA replisome causes neonatal heart failure via ferroptosis

Increasing mitochondrial DNA (mtDNA) replication and amount have been proposed as therapeutic approaches for mitochondrial dysfunction, but also as a mechanism of premature aging. We addressed this fascinating paradox by enhancing mtDNA replication via two mechanisms: increasing both mtDNA replication licensing and processivity. We crossed mice overexpressing Twinkle helicase (boosting mtDNA replication initiation) with mtDNA mutator mice (exonuclease-deficient mtDNA replicase, increasing mtDNA mutagenesis and replication processivity). The former model is asymptomatic by two years of age, whereas the latter manifests with progeroid symptoms at six months. Surprisingly, the double transgenics demonstrate postnatally halted growth and devastating cardiomyopathy, fatal within weeks. The mice show high mtDNA replication preventing cardiac maturation and the postnatal shift to oxidative metabolism, causing ferroptotic cardiomyocyte death. Our findings emphasize the critical importance of mtDNA replisome regulation for perinatal cardiac maturation. Furthermore, the data implicate ferroptosis as a cell death mechanism for neonatal mitochondrial cardiomyopathies.

molecular biology↗

bioRxiv2018

Quantitative solid-phase assay to measure deoxynucleoside triphosphate pools

Deoxyribonucleoside triphosphates (dNTPs) are the reduced nucleotides used as the building blocks and energy source for DNA replication and maintenance in all living systems. They are present in highly regulated amounts and ratios in the cell, and their balance has been implicated in the most important cell processes, from determining the fidelity of DNA replication to affecting cell fate. Furthermore, many cancer drugs target biosynthetic enzymes in dNTP metabolism, and mutations in genes directly or indirectly affecting these pathways are the cause of devastating diseases. The accurate and systematic measurement of these pools is key to understand the mechanisms behind these diseases and their treatment. We present a new method for measuring dNTP pools from biological samples, utilising the current state-of-the-art polymerase method, modified to a solid-phase setting and optimised for larger scale measurements.

biochemistry↗