Biology subjects

Sancak, Y.

Publications and source records attributed to Sancak, Y..

12 recordsLinked to original sources

bioRxiv2026

A Modular Platform for Purification of Organelle-associated Mitochondria Reveals Functional Specialization at Organelle Contact Sites

Mitochondria perform diverse metabolic and signaling functions, yet how these activities are spatially organized within the mitochondrial network of cells remains poorly understood. Organelle contact sites are spatially restricted hubs that regulate mitochondrial metabolism, signaling, and dynamics, and are therefore well positioned to influence mitochondrial specialization. Investigation of contact site-associated mitochondrial populations has been hindered by a lack of methods to isolate these subpopulations. Here, we develop Organelle Contact-dependent Affinity Purification (ORCA), a workflow for the isolation and analysis of subpopulations of intact mitochondria and associated proteins defined by their organelle contacts. ORCA revealed distinct proteomes for mitochondria associated with the endoplasmic reticulum, lysosomes, peroxisomes, and the Golgi apparatus, demonstrating that organelle contacts define biochemically specialized mitochondrial populations. Focused analysis of Golgi-associated mitochondria showed enrichment of mitochondrial ribosomes and increased mitochondrial translation, revealing an unexpected role for Golgi-mitochondria contacts in regulating mitochondrial protein homeostasis. ORCA also identified the previously uncharacterized Golgi protein KIAA0930/GMO1 as an evolutionarily conserved regulator of oxidative phosphorylation at Golgi-mitochondria contacts. Together, our findings establish ORCA as a broadly applicable approach for investigating the spatial organization of intracellular organelles and reveal organelle contacts as key determinants of mitochondrial specialization.

cell biology↗

bioRxiv2025

Radiolabeling Isolated Mitochondria with Tc-99m: A First-in-Field Protocol and Early Feasibility Findings

Mitochondrial transplantation is a promising but still experimental strategy for treating ischemic and metabolic disorders. A key barrier to its advancement is the lack of scalable, non-invasive methods for tracking transplanted extracellular mitochondria in vivo. Technetium-99m (Tc-99m) radiopharmaceuticals, widely used in SPECT imaging, may offer a clinically compatible solution. Cryopreserved mitochondria derived from HEK-293 cells were incubated with Tc-99m sestamibi, tetrofosmin, pertechnetate, or control solutions. After brief incubation and washing, mitochondrial pellets were analyzed for retained radioactivity. ATP content was measured to assess metabolic function, and electron microscopy was used to evaluate ultrastructural integrity. Tc-99m sestamibi and tetrofosmin showed labeling efficiencies of 2.74% and 2.68%, respectively. Pertechnetate demonstrated minimal uptake (0.34%). Radiolabeled mitochondria retained ATP production comparable to controls. Electron microscopy showed preserved double membranes and cristae. Controls confirmed assay specificity and viability. To our knowledge, this is the first report of radiolabeling isolated mitochondria with clinically approved Tc-99m agents. This platform supports the development of SPECT-compatible protocols for visualizing viable transplanted mitochondria in recipient tissues.

cell biology↗

bioRxiv2025

ER-Localized Ceramide Accumulation Due to Disrupted CERT Transport Contributes to Replicative Senescence

Ceramides regulate diverse cellular processes through compartment-specific accumulation. While mitochondrial ceramide accumulation promotes apoptosis, its regulation and function during senescence remain incompletely understood. Here, we integrate lipidomics, transcriptomics, Raman spectroscopy and biochemical characterizations to define sphingolipid remodeling in replicative senescence. Senescent cells exhibit elevated ceramide levels and depletion of very-long-chain sphingomyelins, despite unaltered sphingomyelin synthase 1 expression, implicating impaired ceramide-sphingomyelin turnover. Pharmacological inhibition of ceramide transfer protein (CERT), the ER-to-Golgi ceramide transporter, phenocopies this sphingolipid remodeling and enhances senescence markers, suggesting disrupted ceramide trafficking as a driver of senescence. Raman spectroscopy suggests ceramide accumulation localized to the ER, with no enrichment at mitochondria, consistent with a non-apoptotic phenotype. Analysis of ER-enriched fractions confirm increased ceramide levels in ER fractions of senescent cells. Mechanistically, ceramide accumulation at the ER can contribute to ER stress, a key component of the senescence program. These findings identify altered ceramide trafficking as a contributor to ER stress and highlight ER-localized ceramide as a critical component of senescence-associated sphingolipid remodeling. SignificanceHow ceramides function during replicative senescence has remained poorly understood. In this study, we demonstrate that senescent cells accumulate ceramides at the endoplasmic reticulum (ER), revealing a fundamentally different mode of ceramide regulation and function in senescence. By integrating lipidomics, enzymatic assays, Raman-BCA imaging, and biochemical assays, we show that impaired ER-to-Golgi ceramide transport leads to ER-localized ceramide retention. Disrupting ceramide trafficking through inhibition of the ceramide transfer protein CERT phenocopies senescence-associated lipid remodeling and enhances senescence markers, establishing altered ceramide transport as a mechanistic contributor to this process. These findings link impaired ceramide trafficking to replicative senescence and suggest that ceramide function in senescence is linked to its retention at the ER.

cell biology↗

bioRxiv2024

Mechanism of MCUB-dependent inhibition of mitochondrial calcium uptake

Mitochondrial Ca2+ levels are regulated to balance stimulating respiration against the harm of Ca2+ overload. Contributing to this balance, the main channel transporting Ca2+ into the matrix, the mitochondrial Ca2+ uniporter, can incorporate a dominant-negative subunit (MCUB). MCUB is homologous to the pore-forming subunit MCU, but when present in the pore-lining tetramer, inhibits Ca2+ transport. Here, using cell lines deleted of both MCU and MCUB, we identify three factors that contribute to MCUB-dependent inhibition. First, MCUB protein requires MCU to express. The effect is mediated via the N-terminal domain (NTD) of MCUB. Replacement of the MCUB NTD with the MCU NTD recovers autonomous expression but fails to rescue Ca2+ uptake. Surprisingly, mutations to MCUB that affect interactions with accessory subunits or the conduction pore all failed to rescue Ca2+ uptake, suggesting the mechanism of inhibition may involve global rearrangements. Second, using concatemeric tetramers with varying MCU:MCUB ratios, we find that MCUB incorporation does not abolish conduction, but rather inhibits Ca2+ influx proportional to the amount of MCUB present in the channel. Reducing rather than abolishing Ca2+ transport is consistent with MCUB retaining the highly-conserved selectivity filter DIME sequence. Finally, we apply live-cell Forster resonance energy transfer to establish that the endogenous stoichiometry is 2:2 MCU:MCUB. Taken together, our results suggest MCUB preferentially incorporates into nascent uniporters, and the amount of MCUB protein present linearly correlates with the degree of inhibition of Ca2+ transport, creating a precise, tunable mechanism for cells to regulate mitochondrial Ca2+ uptake.

cell biology↗

bioRxiv2024

Mitochondrial Calcium Signaling Regulates Branched-Chain Amino Acid Catabolism in Fibrolamellar Carcinoma

Metabolic adaptations in response to changes in energy supply and demand are essential for survival. The mitochondrial calcium uniporter plays a key role in coordinating metabolic homeostasis by regulating TCA cycle activation, mitochondrial fatty acid oxidation, and cellular calcium signaling. However, a comprehensive analysis of uniporter-regulated mitochondrial pathways has remained unexplored. Here, we investigate metabolic consequences of uniporter loss- and gain-of-function using uniporter knockout cells and the liver cancer fibrolamellar carcinoma (FLC), which we demonstrate to have elevated mitochondrial calcium levels. Our results reveal that branched-chain amino acid (BCAA) catabolism and the urea cycle are uniporter-regulated metabolic pathways. Reduced uniporter function boosts expression of BCAA catabolism genes, and the urea cycle enzyme ornithine transcarbamylase (OTC). In contrast, high uniporter activity in FLC suppresses their expression. This suppression is mediated by reduced expression of the transcription factor KLF15, a master regulator of liver metabolism. Thus, uniporter responsive calcium signaling plays a central role in FLC-associated metabolic changes, including hyperammonemia. Our study identifies an important role for mitochondrial calcium signaling in metabolic adaptation through transcriptional regulation of metabolism and elucidates its importance for BCAA and ammonia metabolism in FLC.

cell biology↗

bioRxiv2024

High-Throughput Identification of Calcium Regulated Proteins Across Diverse Proteomes

Calcium ions play important roles in nearly every biological process, yet whole-proteome analysis of calcium effectors has been hindered by lack of high-throughput, unbiased, and quantitative methods to identify proteins-calcium engagement. To address this, we adapted protein thermostability assays in the budding yeast, human cells, and mouse mitochondria. Based on calcium-dependent thermostability, we identified 2884 putative calcium-regulated proteins across human, mouse, and yeast proteomes. These data revealed calcium engagement of novel signaling hubs and cellular processes, including metabolic enzymes and the spliceosome. Cross-species comparison of calcium-protein engagement and mutagenesis experiments identified residue-specific cation engagement, even within well-known EF-hand domains. Additionally, we found that the dienoyl-CoA reductase DECR1 binds calcium at physiologically-relevant concentrations with substrate-specific affinity, suggesting direct calcium regulation of mitochondrial fatty acid oxidation. These unbiased, proteomic analyses of calcium effectors establish a key resource to dissect cation engagement and its mechanistic effects across multiple species and diverse biological processes.

cell biology↗

bioRxiv2023

Synchronized long-read genome, methylome, epigenome, and transcriptome for resolving a Mendelian condition

Resolving the molecular basis of a Mendelian condition (MC) remains challenging owing to the diverse mechanisms by which genetic variants cause disease. To address this, we developed a synchronized long-read genome, methylome, epigenome, and transcriptome sequencing approach, which enables accurate single-nucleotide, insertion-deletion, and structural variant calling and diploid de novo genome assembly, and permits the simultaneous elucidation of haplotype-resolved CpG methylation, chromatin accessibility, and full-length transcript information in a single long-read sequencing run. Application of this approach to an Undiagnosed Diseases Network (UDN) participant with a chromosome X;13 balanced translocation of uncertain significance revealed that this translocation disrupted the functioning of four separate genes (NBEA, PDK3, MAB21L1, and RB1) previously associated with single-gene MCs. Notably, the function of each gene was disrupted via a distinct mechanism that required integration of the four omes to resolve. These included nonsense-mediated decay, fusion transcript formation, enhancer adoption, transcriptional readthrough silencing, and inappropriate X chromosome inactivation of autosomal genes. Overall, this highlights the utility of synchronized long-read multi-omic profiling for mechanistically resolving complex phenotypes.

genetics↗

bioRxiv2023

Mitosis exit followed by death in interphase prevents the development of polyploid giant cancer cells.

Microtubule targeting agents (MTAs) are commonly prescribed to treat cancers and predominantly kill cancer cells in mitosis. Significantly, some MTA-treated cancer cells can escape death in mitosis and exit mitosis, and become malignant polyploid giant cancer cells (PGCC). Considering the low number of malignant cells undergoing mitosis in tumor tissue, killing these cells in interphase may represent a favored antitumor approach. We discovered that ST-401, a mild inhibitor of microtubule assembly, preferentially kills cancer cells in interphase as opposed to mitosis, and avoids the development of PGCC. Single cell RNA sequencing identified mRNA transcripts regulated by ST-401, including mRNAs involved in ribosome and mitochondrial functions. Accordingly, ST-401 induces an integrated stress response and promotes mitochondria fission accompanied by a reduction in energy metabolism. This cell response may underly death in interphase and avoid the development of PGCC.

cell biology↗

bioRxiv2023

Acylation of MLKL impacts its function in necroptosis

Mixed lineage kinase domain-like (MLKL) is a key signaling protein of necroptosis. Upon activation by phosphorylation, MLKL translocates to the plasma membrane and induces membrane permeabilization which contributes to the necroptosis-associated inflammation. Membrane binding of MLKL is initially initiated by the electrostatic interactions between the protein and membrane phospholipids. We previously showed that MLKL and its phosphorylated form (pMLKL) are S-acylated during necroptosis. Here, we characterize acylation sites of MLKL and identify multiple cysteines that can undergo acylation with an interesting promiscuity at play. Our results show that MLKL and pMLKL undergo acylation at a single cysteine, C184, C269 and C286 are the possible acylation sites. Using all atom molecular dynamic simulations, we identify differences that the acylation of MLKL causes at the protein and membrane level. Through systematic investigations of the S-palmitoyltransferases that might acylate MLKL in necroptosis, we showed that zDHHC21 activity has the strongest effect on pMLKL acylation, inactivation of which profoundly reduced the pMLKL levels in cells and improved membrane integrity. These results suggest that blocking the acylation of pMLKL destabilizes the protein at the membrane interface and causes its degradation, ameliorating necroptotic activity. At a broader level, our findings shed light on the effect of S-acylation on MLKL functioning in necroptosis and MLKL-membrane interactions mediated by its acylation.

biochemistry↗

bioRxiv2022

Mathematical modeling and biochemical analysis support partially ordered CaM-MLCK binding

Activation of myosin light-chain kinase (MLCK) by calcium ions (Ca2+) and calmodulin (CaM) plays an important role in numerous cellular functions including vascular smooth muscle contraction and cellular motility. Despite extensive biochemical analysis of this system, aspects of the mechanism of activation remain controversial, and competing theoretical models have been proposed for the binding of Ca2+ and CaM to MLCK. The models are analytically solvable for an equilibrium steady state and give rise to distinct predictions that hold regardless of the numerical values assigned to parameters. These predictions form the basis of a recently proposed, multi-part experimental strategy for model discrimination. Here we implement this strategy by measuring CaM-MLCK binding using an in vitro FRET system. This system uses the CaM-binding region of smooth muscle MLCK protein to link two fluorophores to form an MLCK FRET Reporter (FR). Biochemical and biophysical experiments have established that FR can be reliably used to analyze MLCK-CaM binding. We assessed the binding of either wild-type CaM, or mutant CaM with one or more defective EF-hand domains, to FR. Interpretation of binding data in light of the mathematical models suggests a partially ordered mechanism for binding of CaM to MLCK. Complementary data collected using orthogonal approaches that directly quantify CaM-MLCK binding further supports our conclusions.

biochemistry↗

bioRxiv2019

Mitochondrial clearance of Ca2+ controls insulin secretion

Transport of Ca2+ from the cytosol to the mitochondrial matrix of insulin-secreting pancreatic {beta}-cells facilitates nutrient-mediated insulin secretion. However, the underlying mechanism is unclear. The establishment of the molecular identity of the mitochondrial Ca2+ uniporter (MCU) and associated proteins has allowed mitochondrial Ca2+ transport to be modified in intact cells. We examined the consequences of deficiency of the accessory protein, MICU2, in rat and human insulin-secreting cell lines as well as in mouse islets. Glucose-induced mitochondrial Ca2+ elevation and inner membrane hyperpolarization were reduced, together with cytosolic ATP/ADP-ratios and insulin secretion. Insulin secretion in Micu2 knock out mice was attenuated in vitro as well as in vivo. While KCl-evoked sub-plasmalemmal Ca2+ increases were more pronounced, the global cytosolic Ca2+ response was, surprisingly, diminished in MICU2-deficient cells. These findings were supported by selective inhibition of mitochondrial Ca2+ uptake by mitochondrial depolarization. It is concluded that mitochondrial Ca2+ transport plays an additional and hitherto unrecognized role in stimulated {beta}-cells by regulating net Ca2+ entry across the plasma membrane. This is likely accounted for by clearing of sub-plasmalemmal Ca2+ levels by mitochondria located near the plasma membrane.

molecular biology↗

bioRxiv2019

Molecular basis of EMRE-dependence of the human mitochondrial calcium uniporter

The mitochondrial uniporter is calcium-activated calcium channel complex critical for cellular signaling and bioenergetics. MCU, the pore-forming subunit of the uniporter, contains two transmembrane domains and is found in all major eukaryotic taxa. In amoeba and fungi, MCU homologs are sufficient to form a functional calcium channel, whereas human MCU exhibits a strict requirement for the metazoan-specific, single-pass transmembrane protein EMRE for conductance. Here, we exploit this evolutionary divergence to decipher the molecular basis of the human MCUs dependence on EMRE. By systematically generating chimeric proteins that consist of EMRE-independent D. discoideum MCU (DdMCU) and H. sapiens MCU (HsMCU), we converged on a stretch of 10 amino acids in DdMCU that can be transplanted to HsMCU to render it EMRE-dependent. We call this region in human MCU the EMRE-dependence domain (EDD). Crosslinking experiments show that HsEMRE directly interacts with MCU at both of its transmembrane domains as well as the EDD. Based on previously published structures of fungal MCU homologs, the EDD segment is located distal to the calcium pores selectivity filter and appears flexible. We propose that EMRE stabilizes EDD of MCU, permitting both channel opening and calcium conductance

biochemistry↗