Biology subjects

Pereira, R. O.

Publications and source records attributed to Pereira, R. O..

13 recordsLinked to original sources

bioRxiv2026

OPA1-dependent mitochondrial remodeling coordinates TCR signaling and metabolic adaptation during iNKT cell differentiation

Invariant natural killer T (iNKT) cells require mitochondrial metabolism for terminal effector development. We found iNKT cells express elevated levels of proteins regulating mitochondrial membrane dynamics, and identified Opa1, but not Drp1, as selectively required for iNKT cell differentiation. OPA1 deficiency disrupted mitochondrial cristae organization, reduced mitochondrial membrane potential, increased mitochondrial mass, and altered calcium homeostasis in iNKT cells. Bulk and single-cell transcriptomic analyses revealed impaired TCR-responsive gene expression, activation of mitochondrial stress adaptation and integrated stress response, enhanced glycolysis, and retention of immature differentiation features. SCENITH analysis demonstrated increased glycolytic dependence; LDHA became required in Opa1-deficient iNKT cells while dispensable for normal iNKT development, indicating compensatory glycolytic adaptation. EGTA in thymic organ culture partially restored NKT1 marker expression in Opa1-deficient cells. Co-deletion of Drp1 improved mitochondrial morphology, TCR signaling, stress and metabolic adaptation, and partially rescued iNKT cell differentiation, demonstrating that balanced mitochondrial dynamics coordinate mitochondrial function and terminal effector development.

immunology↗

bioRxiv2026

ATF4 Coordinates Transcriptomic and Structural Adaptations in Aging Muscle

Aging is associated with a progressive loss of skeletal muscle function, known as sarcopenia; however, the molecular mechanisms coordinating cellular stress responses and structural adaptations permissive of sarcopenia remain incompletely understood. In our previous studies, we found aging differentially impacted mitochondrial networks by muscle, suggesting unique stress thresholds and response activation. Here, we investigate the role of activating transcription factor 4 (ATF4), a master regulator of the integrated stress response (ISR), in aged quadriceps muscle using complementary patient and aging mouse models. Older adults exhibited a marked decrease in aerobic capacity, muscle strength, and endurance when compared with young participants. These results paralleled findings in aged mice, with significant loss of muscle mass across multiple hindlimb muscles. Ultrastructural analysis revealed substantial age-related changes in mitochondrial morphology, including increased volume, surface area, and branching index, as well as a shift toward larger, more complex mitochondria. Our data indicate that ATF4 binds directly to the promoter region of the gene encoding TFAM, suggesting a transcriptional regulatory relationship to support DNA stability. These structural and transcriptional changes likely impair oxidative capacity and drive a feed-forward cycle of mitochondrial dysfunction and ISR activation. Our findings indicate that ATF4 coordinates transcriptomic and structural adaptations in aging muscle, identifying the ISR pathway as a potential therapeutic target for preserving muscle function in older adults.

cell biology↗

bioRxiv2026

Stress-Encoded Mitochondrial Plasticity: ATF4 Control of Mega-Mitochondria and Nanotunnel Communication

Mitochondrial structural plasticity is a critical adaptive response to cellular stress, yet the transcriptional networks governing the formation of specialized mitochondrial architectures remain poorly defined. Here, we identified and demonstrated that activating transcription factor 4 (ATF4), the master regulator of the integrated stress response, directly regulates mitochondrial morphological remodeling through a novel ATF4-NRF1/Nrf2-MFN2 signaling axis. Using serial block-face scanning electron microscopy and three-dimensional reconstruction in Drosophila flight muscle, primary myotubes, and human skeletal muscle, we show that overexpression of ATF4 promotes significant mitochondrial elongation, increased cristae concentration, enhanced mitochondrial-endoplasmic reticulum contact site (MERC) formation, and the initiation of Mitochondrial Nanotunnels. In contrast, loss of ATF4 results in mitochondrial fragmentation and impaired aerobic capacity. Chromatin immunoprecipitation sequencing reveals direct ATF4 binding at the promoters of the genes encoding NRF1 and Nrf2, which in turn regulate MFN2 expression. Small-molecule inhibition studies further establish that activation of this hierarchical pathway is both necessary and sufficient for stress-induced mitochondrial structural adaptation. Together, these findings position ATF4 as a master regulator of mitochondrial architectural plasticity, providing a direct mechanistic link between cellular stress signaling and organelle remodeling.

cell biology↗

bioRxiv2025

Altered Relaxation and Mitochondria-Endoplasmic Reticulum Contact Sites Precede Major (Mal)adaptations in Aging Skeletal Muscle and are Prevented by Exercise

Sarcopenia, or age-related muscle dysfunction, contributes to morbidity and mortality. Besides decreases in muscle force, sarcopenia is associated with atrophy and fast-to-slow fiber type switching, which is typically secondary to denervation in humans and rodents. However, very little is known about cellular changes preceding these important (mal)adaptations. To this matter, mitochondria and the sarcoplasmic reticulum are critical for tension generation in myofibers. They physically interact at the boundaries of sarcomeres forming subcellular hubs called mitochondria-endo/sarcoplasmic reticulum contacts (MERCs). Yet, whether changes at MERCs ultrastructure and proteome occur early in aging is unknown. Here, studying young adult and older mice we reveal that aging slows muscle relaxation leading to longer excitation-contraction-relaxation (ECR) cycles before maximal force decreases and fast-to-slow fiber switching takes place. We reveal that muscle MERC ultrastructure and mitochondria-associated ER membrane (MAM) protein composition are also affected early in aging and are closely associated with rate of muscle relaxation. Additionally, we demonstrate that regular exercise preserves muscle relaxation rate and MERC ultrastructure in early aging. Finally, we profile a set of muscle MAM proteins involved in energy metabolism, protein quality control, Ca2+ homeostasis, cytoskeleton integrity and redox balance that are inversely regulated early in aging and by exercise. These may represent new targets to preserve muscle function in aging individuals.

physiology↗

bioRxiv2023

ATF4 Expression in Thermogenic Adipocytes is Required for Cold-Induced Thermogenesis in Mice via FGF21-Independent Mechanisms

In brown adipose tissue (BAT), short-term cold exposure induces the activating transcription factor 4 (ATF4), and its downstream target fibroblast growth factor 21 (FGF21). Induction of ATF4 in BAT in response to mitochondrial stress is required for thermoregulation, partially via upregulation of FGF21. In the present study, we tested the hypothesis that Atf4 and Fgf21 induction in BAT are both required for BAT thermogenesis by generating mice selectively lacking either Atf4 (ATF4 BKO) or Fgf21 (FGF21 BKO) in UCP1-expressing adipocytes. After 3 days of cold exposure, core body temperature was significantly reduced in ad-libitum-fed ATF4 BKO mice, which correlated with Fgf21 downregulation in brown and beige adipocytes, and impaired browning of white adipose tissue (WAT). Conversely, despite having reduced browning, FGF21 BKO mice had preserved core body temperature after cold exposure. Mechanistically, ATF4, but not FGF21, regulates amino acid import and metabolism in response to cold, likely contributing to BAT thermogenic capacity under ad libitum-fed conditions. Importantly, under fasting conditions, both ATF4 and FGF21 were required for thermogenesis in cold-exposed mice. Thus, ATF4 regulates BAT thermogenesis by activating amino acid metabolism in BAT in a FGF21-independent manner. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=152 SRC="FIGDIR/small/531964v2_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1d2c3fdorg.highwire.dtl.DTLVardef@414f5forg.highwire.dtl.DTLVardef@1c1fa9eorg.highwire.dtl.DTLVardef@2cc815_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2022

GDF15 Is Required for Cold-Induced Thermogenesis and Contributes to Improve Systemic Metabolic Health Following Loss of OPA1 in Brown Adipocytes

Previously we showed that mice lacking the protein optic atrophy 1 (OPA1 BKO) in brown adipose tissue (BAT) have induction of the activating transcription factor 4 (ATF4), which promotes fibroblast growth factor 21 (FGF21) secretion as a batokine. FGF21 increases metabolic rates at baseline conditions but is dispensable for the resistance to diet-induced obesity (DIO) reported in OPA1 BKO mice [1]. To determine alternative mediators of this phenotype, we performed transcriptome analysis, which revealed increased levels of growth differentiation factor 15 (GDF15) in BAT. To determine if ATF4 induction was mediated by the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), and to evaluate the contribution of GDF15 to the resistance to DIO, we selectively deleted PERK or GDF15 in OPA1 BKO mice. Mice lacking both OPA1 and PERK in BAT had preserved induction of ATF4. Importantly, simultaneous deletion of OPA1 and GDF15 partially reversed the resistance to diet-induced obesity and abrogated the improvements in glucose tolerance. Furthermore, GDF15 mediated cold-induced thermogenesis, likely via sympathetic activation of iWAT. Taken together, our data indicate that PERK is dispensable for ATF4 induction, but GDF15 contributes to the resistance to DIO, and is required for glucose homeostasis and thermoregulation in OPA1 BKO mice.

physiology↗

bioRxiv2022

OPA1 Downregulation in Skeletal Muscle Induces MERC formation in an ATF4-Dependent Manner

Mitochondria and endoplasmic reticulum (ER) contact sites (MERCs) are protein- and lipid-enriched hubs that mediate interorganellar communication by contributing to the dynamic transfer of Ca2+, lipid, and other metabolites between these organelles. Defective MERCs are associated with cellular oxidative stress, neurodegenerative disease, and cardiac and skeletal muscle pathology via mechanisms that are poorly understood. We previously demonstrated that skeletal muscle-specific knockdown (KD) of the mitochondrial fusion mediator optic atrophy 1 (OPA1) induced ER stress and correlated with an induction of Mitofusin-2, a known MERC protein. In the present study, we tested the hypothesis that Opa1 downregulation in skeletal muscle cells alters MERC formation by evaluating multiple myocyte systems, including from mice and Drosophila, and in primary myotubes. Our results revealed that OPA1 deficiency induced tighter and more frequent MERCs in concert with a greater abundance of MERC proteins involved in calcium exchange. Additionally, loss of OPA1 increased the expression of activating transcription factor 4 (ATF4), an integrated stress response (ISR) pathway effector. Reducing Atf4 expression prevented the OPA1-loss-induced tightening of MERC structures. OPA1 reduction was associated with decreased mitochondrial and sarcoplasmic reticulum, a specialized form of ER, calcium, which was reversed following ATF4 repression. These data suggest that mitochondrial stress, induced by OPA1 deficiency, regulates skeletal muscle MERC formation in an ATF4-dependent manner.

biophysics↗

bioRxiv2021

Practices for Measuring 3D Organelle Morphology and Generating Surfaces with Amira

Analysis of 3D structures is of paramount importance in cellular biology. Although light microscopy and transmission electron microscopy (TEM) have remained staples for imaging cellular structures, they lack the ability to image in 3D. However, recent technological advances, such as serial block-face scanning electron microscopy (SBF-SEM) and focused ion beam scanning electron microscopy (FIB-SEM), have allowed researchers to observe cellular ultrastructure in 3D. Here, we propose a standardized protocol using the visualization software Amira to quantify organelle morphologies in 3D; this method allows researchers to produce accurate and reproducible measurements of cellular structure characteristics. We demonstrate this applicability by utilizing SBF-SEM and Amira to quantify mitochondria and endoplasmic reticulum (ER) structures. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/461807v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1c40041org.highwire.dtl.DTLVardef@ecde92org.highwire.dtl.DTLVardef@10870daorg.highwire.dtl.DTLVardef@1290c3b_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

Systematic Transmission Electron Microscopy-Based Identification of Cellular Degradation Machinery

Many interconnected degradation machineries including autophagosomes, lysosomes, and endosomes work in tandem to conduct autophagy, an intracellular degradation system that is crucial for cellular homeostasis. Altered autophagy contributes to the pathophysiology of various diseases, including cancers and metabolic diseases. Although many studies have investigated autophagy to elucidate disease pathogenesis, identification of specific components of the autophagy machinery has been challenging. The goal of this paper is to describe an approach to reproducibly identify and distinguish subcellular structures involved in macro autophagy. We provide methods that help avoid common pitfalls, including a detailed explanation for distinguishing lysosomes and lipid droplets and discuss differences between autophagosomes and inclusion bodies. These methods are based on using transmission electron microscopy (TEM), capable of generating nanometer-scale micrographs of cellular degradation components in a fixed sample. We also utilize serial block face-scanning electron microscopy (SBF-SEM) to offer a protocol for visualizing 3D morphology of degradation machinery. In addition to TEM and 3D reconstruction, we discuss other imaging techniques, such as immunofluorescence and immunogold labeling that can be utilized to reliably and accurately classify cellular organelles. Our results show how these methods may be used to accurately quantify the cellular degradation machinery under various conditions, such as treatment with the endoplasmic reticulum stressor thapsigargin or ablation of the dynamin-related protein 1.

biophysics↗

bioRxiv2021

A comprehensive approach for artifact-free sample preparation and assessment of mitochondrial morphology in tissue and cultured cells

Mitochondrial dynamics (fission, fusion, and the formation of nanotunnels) and morphology are very sensitive to the cellular environment. Mitochondria may be adversely affected by oxidative stress, changes in calcium levels, and hypoxia. Investigating the precise relationship between organelle structure and function requires methods that can adequately preserve mitochondria while providing accurate, quantitative measurements of morphological attributes. Here, we demonstrate a practical approach for preserving and measuring fine structural changes using two-dimensional, high-resolution electron micrographs. This approach is further applicable for three-dimensional volume renderings, obtained using serial block-face and focused ion beam-scanning electron microscopy, highlighting the specific advantages of these techniques. Additionally, this study defines a set of quantifiable metrics that can be applied to measure mitochondrial architecture and other organellar structures. Finally, we validated specimen preparation methods that avoid the introduction of morphological artifacts that may interfere with mitochondrial appearance and do not require whole-animal perfusion.

cell biology↗

bioRxiv2021

A Universal Approach to Analyzing Transmission Electron Microscopy with ImageJ

2Transmission electron microscopy (TEM) is a scientific research standard for producing nanometer-resolution ultrastructural images of subcellular components within cells and tissues. Mitochondria, endoplasmic reticulum (ER), lysosomes, and autophagosomes are organelles of particular interest to those investigating metabolic disorders. However, there is no clear consensus amongst regarding the best methods for quantifying the features of organelles in TEM images. In this protocol, we propose a standardized approach to accurately measure the morphology of these important subcellular structures using the free program ImageJ, developed by the National Institutes of Health (NIH). Specifically, we detail procedures for obtaining mitochondrial length, width, area, and circularity, in addition to assessing cristae morphology. We further provide methods for measuring interactions between the mitochondria and ER and measuring the length and width of lysosomes and autophagosomes. This standardized method can be used to quantify key features of organelle morphology, allowing investigators to produce accurate and reproducible measurements of organelle structures in their experimental samples. 1 SUMMARYWe discuss a standardized method for measuring and quantifying organelle features using transmission electron microscopy and accessing for interactions between subcellular structures; organelles of focus include mitochondria, endoplasmic reticulum, lysosomes, and autophagosomes.

biophysics↗

bioRxiv2021

Cardiac Specific Overexpression of Transcription Factor EB (TFEB) in Normal Hearts Induces Pathologic Cardiac Hypertrophy and Lethal Cardiomyopathy

TFEB promotes lysosomal biogenesis, autophagy, and lysosomal exocytosis. The present study characterized the consequence of inducible TFEB overexpression in cardiomyocytes in vivo. We generated cardiomyocyte-specific doxycycline inducible (Tet off) mice to achieve spatial and temporal control of TFEB overexpression, by crossing TFEB transgenic mice with mice harboring the tTA transgene (TFEB/tTA). Two weeks after doxycycline removal, an 8-fold increase in TFEB protein expression was observed in transgenic hearts. Heart weight normalized to tibia length was increased by 2.5-fold following TFEB overexpression (TFEB/tTA), characterized by induction of markers of pathological hypertrophy, such as Nppa, Nppb and Acta1, progressive contractile dysfunction and cardiac dilatation. Overexpression of TFEB resulted in premature death, associated with high degree AV block. Reversal of TFEB overexpression normalized cardiac structure and function. Mitochondrial respiration and ATP levels were preserved after 2-weeks of TFEB induction, despite reduced mitochondrial (OXPHOS) protein expression, mtDNA content, and altered mitochondrial morphology. Signaling through mTOR was induced in TFEB/tTA mice, and when inhibited by rapamycin treatment for 4 weeks, partially offset left ventricular dysfunction. Transcriptome analysis revealed early suppression of mitochondrial metabolic pathways, induction of fibrosis and altered calcium signaling. MCOLN1, a lysosomal calcium release channel, the calcineurin target RCAN1.4, and the mitochondrial calcium uniporter (MCU) were strikingly induced in TFEB/tTA mice. In summary, persistent overexpression of TFEB at high levels (8-fold protein upregulation) in cardiomyocytes promotes pathologic cardiac hypertrophy via suppression of mitochondrial bioenergetic pathways and activation of pro-fibrotic and calcium regulatory pathways.

cell biology↗

bioRxiv2021

OPA1 Deletion in Brown Adipose Tissue Improves Thermoregulation and Systemic Metabolism via FGF21

Adrenergic stimulation of brown adipocytes alters mitochondrial dynamics, including proteolytic processing of the mitochondrial fusion protein optic atrophy 1 (OPA1). However, direct mechanisms linking OPA1 to brown adipose tissue (BAT) physiology are incompletely understood. By deleting OPA1 selectively in BAT (OPA1 BAT KO), we demonstrate that OPA1 is required for cold-induced thermogenesis. Unexpectedly, OPA1 deficiency induced fibroblast growth factor 21 (FGF21) as a BATokine in an activating transcription factor 4 (ATF4)- dependent manner. BAT-derived FGF21 mediates an adaptive response in OPA1 BAT KO mice, by inducing browning of white adipose tissue (WAT), increasing resting metabolic rates, and improving thermoregulation. However, FGF21 does not mediate the resistance to diet-induced obesity observed in these animals. These findings reveal a requirement for OPA1 in BAT thermogenesis, and uncovers a homeostatic mechanism of BAT-mediated metabolic protection governed by an ATF4-FGF21 axis, that is activated independently of BAT thermogenic function.

physiology↗