Search bioRxiv⌕ Search

Biology subjects

Zaganjor, E.

Publications and source records attributed to Zaganjor, E..

9 recordsLinked to original sources

QuantEM: An optimized platform of vision transformer-based models for segmentation and analysis of electron microscopy data

Electron microscopy (EM) is essential for resolving cellular ultrastructure, yet quantitative analysis remains limited by labor-intensive segmentation and the scarcity of generalizable models. Here we present QuantEM, an open-source platform for segmentation and analysis of EM data across imaging modalities, tissues, and species. We assembled the largest curated collection of intracellular EM datasets to date, comprising over 15,000 two-dimensional images and 1,700 three-dimensional acquisitions from more than 600 datasets, including nearly 4,000 newly released acquisitions. Using this resource, we trained an EM-specific vision transformer foundation model and systematically optimized adaptation strategies for organelle segmentation. QuantEM provides pretrained models for mitochondria, endoplasmic reticulum, nuclei, and lipid droplets, integrated with interactive proofreading and downstream quantitative analyses through standalone and napari interfaces. Across diverse naive datasets, QuantEM consistently matches or exceeds existing models on zero-shot segmentation while requiring less data for finetuning. We further demonstrate its utility by revealing previously unrecognized subcellular compartmentation of hepatic glucokinase using immuno-electron microscopy.

cell biology↗

Glutamine Metabolism Supports α cell Mass and Glucagon Secretion

The liver- cell axis is a finely tuned biological rheostat that regulates whole body amino acid availability. Pancreatic cells secrete glucagon that regulates amino acid catabolism through gluconeogenesis and ureagenesis, yet the mechanisms linking amino acid levels to cell growth and function are not fully understood. Here, we identify glutaminase, the enzyme that catalyzes glutamine catabolism, as a critical cell regulator. Glutaminase is highly enriched in cells across species. cell expression of glutaminase is required for nutrient-dependent mTORC1 activation, suppression of AMPK signaling, and sustained expression of the glutamine transporter SLC38A5. This establishes a feed-forward loop linking glutamine metabolism to amino acid sensing and growth. Reduced glutaminase activity impairs dynamic glucagon secretion in response to low glucose and amino acids. Together, these findings highlight the importance of glutamine metabolism in cell growth and hormone secretion and suggest it may play a role in cell adaptation to hyperaminoacidemia.

physiology↗

Systemic inhibition of de novo purine biosynthesis prevents weight gain and improves metabolic health by increasing thermogenesis and decreasing food intake

ObjectiveObesity is a major health concern, largely because it contributes to type 2 diabetes mellitus (T2DM), cardiovascular disease, and various malignancies. Increase in circulating amino acids and lipids, in part due to adipose dysfunction, have been shown to drive obesity-mediated diseases. Similarly, elevated purines and uric acid, a degradation product of purine metabolism, are found in the bloodstream and in adipose tissue. These metabolic changes are correlated with metabolic syndrome, but little is known about the physiological effects of targeting purine biosynthesis. MethodsTo determine the effects of purine biosynthesis on organismal health we treated mice with mizoribine, an inhibitor of inosine monophosphate dehydrogenase 1 and 2 (IMPDH1/2), key enzymes in this pathway. Mice were fed either a low-fat (LFD; 13.5% kcal from fat) or a high-fat (HFD; 60% kcal from fat) diet for 30 days during drug or vehicle treatment. We ascertained the effects of mizoribine on weight gain, body composition, food intake and absorption, energy expenditure, and overall metabolic health. ResultsMizoribine treatment prevented mice on a HFD from gaining weight, but had no effect on mice on a LFD. Body composition analysis demonstrated that mizoribine significantly reduced fat mass but did not affect lean mass. Although mizoribine had no effect on lipid absorption, food intake was reduced. Furthermore, mizoribine treatment induced adaptive thermogenesis in skeletal muscle by upregulating sarcolipin, a regulator of muscle thermogenesis. While mizoribine-treated mice exhibited less adipose tissue than controls, we did not observe lipotoxicity. Rather, mizoribine-treated mice displayed improved glucose tolerance and reduced ectopic lipid accumulation. ConclusionsInhibiting purine biosynthesis prevents mice on a HFD from gaining weight, and improves their metabolic health, to a significant degree. We also demonstrated that the purine biosynthesis pathway plays a previously unknown role in skeletal muscle thermogenesis. A deeper mechanistic understanding of how purine biosynthesis promotes thermogenesis and decreases food intake may pave the way to new anti-obesity therapies. Crucially, given that many purine inhibitors have been FDA-approved for use in treating various conditions, our results indicate that they may benefit overweight or obese patients. HighlightsO_LIA purine biosynthesis inhibitor, mizoribine, protects against diet-induced weight gain C_LIO_LIMizoribine prevents fat mass gain in high-fat diet-fed male mice C_LIO_LIMizoribine reduces food intake and increases thermogenesis C_LIO_LIMizoribine induces expression of sarcolipin, a regulator of thermogenesis C_LIO_LIMizoribine treatment reduces ectopic lipids and increases glucose tolerance C_LI

physiology↗

MICOS Complex Loss Governs Age-Associated Murine Mitochondrial Architecture and Metabolism in the Liver, While Sam50 Dictates Diet Changes

Background & AimsAging is associated with a significant decline in mitochondrial function in the liver, leading to an increased risk of liver disease. This study examines age-related changes in the mitochondrial structure of human and murine livers using a combination of Serial Block-Face Scanning Electron Microscopy (SBF-SEM) and mass spectrometry approaches. MethodsThis study integrates mitochondrial structure analysis in a murine model with an analysis of liver architecture, lipogenesis, and genetically regulated gene expression in human cohorts. We explored the Mitochondrial Contact Site and Cristae Organizing System (MICOS) complex using SBF-SEM, three-dimensional reconstruction with Amira software, and mass spectrometry techniques. ResultsAging leads to a reduction in mitochondrial size and complexity, resulting in changes in the metabolomic and lipidomic profiles of murine liver cells that are comparable to those observed in aged human samples. We find that genetically modeled expression of MICOS complex genes OPA1 and CHCHD3 is associated with chronic liver disease phenotypes within a large biobank population. Furthermore, we observed dysregulated mitochondrial calcium handling and increased oxidative stress due to the disruption of the MICOS complex. ConclusionOur study highlights the age-associated decline in mitochondrial complexity and metabolic regulation within the aging murine liver and the human population. We have identified that these changes are partially attributable to the age-related loss of the MICOS complex. Impact and implicationsThis study offers new insights into the changes to mitochondrial ultrastructure that occur during aging. Using SBF-SEM, the quantification of young and aged murine mitochondrial structure was performed, which had previously been an underexplored avenue for measuring mitochondrial changes. The discovery of mitochondrial ultrastructural changes, in conjunction with measurements of age-associated metabolic alterations and gene association data, provides a model for how changes in MICOS expression may modulate age-related impairment of hepatic mitochondria. These results provide a new model by which changes in MICOS protein expression may both cause and be a potential therapeutic target for age-related impairment in hepatic function. HighlightsDecreased modeled expression of CHCHD3 in individuals of European genetic ancestry is linked to liver transplant and cirrhosis, while decreased modeled expression of OPA1 in individuals of African genetic ancestry is associated with chronic liver disease and cirrhosis. Aging alters liver lipid accumulation, MICOS mRNA levels, and disease markers. Aging reduces the volume and complexity of murine liver ultrastructure. Aging and diet significantly alter the MICOS complex in mice. Knockdown of Mic60 and Chchd6 lowers Ca2+ uptake, retention, and induces oxidative stress in HepG2 cells. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/599846v3_ufig1.gif" ALT="Figure 1000"> View larger version (47K): org.highwire.dtl.DTLVardef@1cdd61corg.highwire.dtl.DTLVardef@a3fb74org.highwire.dtl.DTLVardef@1d1ad36org.highwire.dtl.DTLVardef@c2e55f_HPS_FORMAT_FIGEXP M_FIG C_FIG Liver aging causes metabolic, lipidomic, and mitochondrial structural alterations, reflecting age-dependent losses in the MICOS complex. Key components of the MICOS complex (MIC60, CHCHD3 and CHCHD6) are illustrated.

physiology↗

The MICOS Complex Regulates Mitochondrial Structure and Oxidative Stress During Age-Dependent Structural Deficits in the Kidney

Due to aging, the efficiency of kidney function begins to decrease. Dysfunction in mitochondria and their cristae is a hallmark of aging. Therefore, age-related decline in kidney function could be attributed to changes in mitochondrial ultrastructure, increased reactive oxygen species, and alterations in metabolism and lipid composition. We sought to understand how mitochondrial ultrastructure is altered over time in tubular kidney cells. A serial block facing-scanning electron microscope and manual segmentation using the Amira software were employed to visualize murine kidney samples during the aging process at 3 months (young) and 2 years (old). We found that 2-year mitochondria are more fragmented with many uniquely shaped mitochondria observed across aging, concomitant with shifts in ROS, metabolomics, and lipid homeostasis. Furthermore, we demonstrate that the mitochondrial contact site and cristae organizing system (MICOS) complex is impaired in the kidney during aging. Disruption of the MICOS complex resulted in altered mitochondrial metabolic function and increased ROS levels. We found significant, detrimental structural changes in the mitochondria of aged kidney tubules, suggesting a potential mechanism underlying the increased frequency of kidney disease with aging. We hypothesize that disruption of the MICOS complex exacerbates mitochondrial dysfunction, creating a vicious cycle of mitochondrial degradation and oxidative stress, which impacts kidney health. Impact and ImplicationsDue to aging, the efficiency of kidney function begins to decrease, and the risk of kidney diseases may increase; however, the specific regulators of mitochondrial age-related changes are poorly understood. This study demonstrates that the MICOS complex may be a target for mitigating age-related mitochondrial changes. The MICOS complex is associated with oxidative stress and calcium dysregulation, which also arise in many kidney pathologies. HighlightsO_LIAging alters the MICOS mRNA levels and disease markers. C_LIO_LIAging reduces cristae architecture, mitochondrial volume and complexity in murine kidney ultrastructure C_LIO_LIReducing MIC60 and CHCHD6 lowers Ca2+ uptake and retention and induces oxidative stress in HEK cells. C_LIO_LIMetabolomic Profiling revealed that NAD+ and amino acid metabolism were altered in aged kidneys. C_LIO_LIMICOS deficiency alters the reduced basal, ATP-linked, maximal capacity and spare capacity. C_LIO_LIDecreased modeled expression of CHCHD6 in individuals of European genetic ancestry is linked to chronic kidney disease, whereas decreased modeled expression of OPA1 in individuals of African genetic ancestry is associated with chronic kidney disease. C_LI Graphical AbstractKidney aging causes a decline in the MICOS complex, concomitant with metabolic, lipidomic, and mitochondrial structural alterations. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/598108v3_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@1a868f0org.highwire.dtl.DTLVardef@1817bfborg.highwire.dtl.DTLVardef@1f2a1a3org.highwire.dtl.DTLVardef@520692_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

Quantitative Assessment of Morphological Changes in Lipid Droplets and Lipid-Mito Interactions with Aging in Brown Adipose

The physical characteristics of brown adipose tissue (BAT) are defined by the presence of multilocular lipid droplets (LD) within the brown adipocytes and a high abundance of iron-containing mitochondria, which give it its characteristic color. Normal mitochondrial function is, in part, regulated by organelle-to-organelle contacts. Particularly, the contact sites that mediate mitochondria-LD interactions are thought to have various physiological roles, such as the synthesis and metabolism of lipids. Aging is associated with mitochondrial dysfunction, and previous studies show that there are changes in mitochondrial structure and proteins that modulate organelle contact sites. However, how mitochondria-LD interactions change with aging has yet to be fully clarified. Therefore, we sought to define age-related changes in LD morphology and mitochondria-lipid interactions in BAT. We examined the three-dimensional morphology of mitochondria and LDs in young (3-month) and aged (2-year) murine BAT using serial block face-scanning electron microscopy and the Amira program for segmentation, analysis, and quantification. Analysis showed reductions in LD volume, area, and perimeter in aged samples compared to young samples. Additionally, we observed changes in LD appearance and type in aged samples compared to young samples. Notably, we found differences in mitochondrial interactions with LDs, which could implicate that these contacts may be important for energetics in aging. Upon further investigation, we also found changes in mitochondrial and cristae structure for mitochondria interacting with LD lipids. Overall, these data define the nature of LD morphology and organelle-organelle contacts during aging and provide insight into LD contact site changes that interconnect biogerontology and mitochondrial functionality, metabolism, and bioactivity in aged BAT. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/559135v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@e5b7org.highwire.dtl.DTLVardef@e965e5org.highwire.dtl.DTLVardef@120c7e9org.highwire.dtl.DTLVardef@124bfb5_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract Workflow highlighting the process of murine interscapular BAT extraction, processing and imaging serial block facing-scanning electron microscopy and using Amira software for 3D reconstruction of LDs and mitochondrial lipid contact sites to elucidate their structure across aging. C_FIG

biophysics↗

Defining Mitochondrial Cristae Morphology Changes Induced by Aging in Brown Adipose Tissue

Mitochondria are required for energy production and even give brown adipose tissue (BAT) its characteristic color due to their high iron content and abundance. The physiological function and bioenergetic capacity of mitochondria are connected to the structure, folding, and organization of its inner-membrane cristae. During the aging process, mitochondrial dysfunction is observed, and the regulatory balance of mitochondrial dynamics is often disrupted, leading to increased mitochondrial fragmentation in aging cells. Therefore, we hypothesized that significant morphological changes in BAT mitochondria and cristae would be present with aging. We developed a quantitative three-dimensional (3D) electron microscopy approach to map cristae network organization in mouse BAT to test this hypothesis. Using this methodology, we investigated the 3D morphology of mitochondrial cristae in adult (3-month) and aged (2-year) murine BAT tissue via serial block face-scanning electron microscopy (SBF-SEM) and 3D reconstruction software for manual segmentation, analysis, and quantification. Upon investigation, we found increases in mitochondrial volume, surface area, and complexity and decreased sphericity in aged BAT, alongside significant decreases in cristae volume, area, perimeter, and score. Overall, these data define the nature of the mitochondrial structure in murine BAT across aging. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/540609v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@96099borg.highwire.dtl.DTLVardef@50b4f4org.highwire.dtl.DTLVardef@983092org.highwire.dtl.DTLVardef@19ceb9b_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO Overview of serial block facing-scanning electron microscopy (SBF-SEM) workflow, data segmentation, and 3D analysis of mitochondria using Amira software for murine interscapular BAT. C_FIG

biophysics↗

Cycling cancer persister cells arise from lineages with distinct transcriptional and metabolic programs

Non-genetic mechanisms have recently emerged as important drivers of therapy failure in cancer (Salgia and Kulkarni, 2018), where some cancer cells can enter a reversible drug-tolerant persister state in response to treatment (Vallette et al., 2019). While most cancer persisters, like their bacterial counterparts, remain arrested in the presence of drug, a rare subset of cancer persisters can re-enter the cell cycle under constitutive drug treatment (Sharma et al., 2010). Little is known about the non-genetic mechanisms that enable cancer persisters to maintain proliferative capacity in the presence of drug. Here, using time-lapse imaging, we found that cycling persisters emerge early in the course of treatment of EGFR-mutant lung cancer cells with the EGFR inhibitor osimertinib. To study this rare, transiently-resistant, proliferative persister population we developed Watermelon, a new high-complexity expressed barcode lentiviral library for simultaneous tracing each cells clonal origin, proliferative state, and transcriptional state. Analysis of Watermelon-transduced PC9 cells demonstrated that cycling and non-cycling persisters arise from different pre-existing cell lineages with distinct transcriptional and metabolic programs. The proliferative capacity of persisters is associated with an upregulation of antioxidant gene programs and a metabolic shift to fatty acid oxidation in specific subpopulations of tumor cells. Mitigating oxidative stress or blocking metabolic reprograming significantly alters the fraction of cycling persister cells. In human tumors, programs associated with cycling persisters were induced in malignant cells in response to multiple tyrosine kinase inhibitors. The Watermelon system enabled the identification of rare persister lineages, that are preferentially poised through specific gene programs to proliferate under drug pressure, thus exposing new vulnerabilities that can be targeted to delay or even prevent disease recurrence.

cancer biology↗