Biology subjects

Ikonen, E.

Publications and source records attributed to Ikonen, E..

9 recordsLinked to original sources

bioRxiv2024

Control of Golgi- V-ATPase through Sac1-dependent co-regulation of PI(4)P and cholesterol

Sac1 is a conserved phosphoinositide phosphatase, whose loss-of-function compromises cell and organism viability. Here, we employed acute auxin-inducible Sac1 degradation to identify its immediate downstream effectors in human cells. Most of Sac1 was degraded in [~]1 h, paralleled by increased PI(4)P and decreased cholesterol in the trans-Golgi network (TGN) during the following hour, and superseded by Golgi fragmentation, impaired glycosylation, and selective degradation of TGN proteins by [~]4 h. The TGN disintegration resulted from its acute deacidification caused by disassembly of the Golgi V-ATPase. Mechanistically, Sac1 mediated TGN membrane composition maintained an assembly promoting conformation of the V0a2 subunit. Key phenotypes of acute Sac1 degradation were recapitulated in human differentiated trophoblasts, causing processing defects of chorionic gonadotropin, in line with loss-of-function intolerance of the human SACML1 gene. Collectively, our findings reveal that the assembly of the Golgi V-ATPase is controlled by the TGN membrane via Sac1 fuelled lipid exchange.

cell biology↗

bioRxiv2024

Cholesterol binding to VCAM-1 promotes vascular inflammation

Hypercholesterolemia has long been implicated in endothelial cell (EC) dysfunction, but the mechanisms by which excess cholesterol causes vascular pathology are incompletely understood. Here we used a cholesterol-mimetic probe to map cholesterol-protein interactions in primary human ECs and discovered that cholesterol binds to and stabilizes the adhesion molecule VCAM-1. We show that accessible plasma membrane (PM) cholesterol in ECs is acutely responsive to inflammatory stimuli and that the nonvesicular cholesterol transporter Aster-A regulates VCAM-1 stability in activated ECs by controlling the size of this pool. Deletion of Aster-A in ECs increases VCAM-1 protein, promotes immune cell recruitment to vessels, and impairs pulmonary immune homeostasis. Conversely, depleting cholesterol from the endothelium in vivo dampens VCAM-1 induction in response to inflammatory stimuli. These findings identify cholesterol binding to VCAM-1 as a key step during EC activation and provide a biochemical explanation for the ability of excess membrane cholesterol to promote immune cell recruitment to the endothelium.

physiology↗

bioRxiv2024

Adipogenin Dictates Adipose Tissue Expansion by Facilitating the Assembly of a Dodecameric Seipin Complex

Adipogenin (Adig) is an evolutionarily conserved microprotein and is highly expressed in adipose tissues and testis. Here, we identify Adig as a critical regulator for lipid droplet formation in adipocytes. We determine that Adig interacts directly with seipin, leading to the formation of a rigid complex. We solve the structure of the seipin/Adig complex by Cryo-EM at 2.98[A] overall resolution. Surprisingly, seipin can form two unique oligomers, undecamers and dodecamers. Adig selectively binds to the dodecameric seipin complex. We further find that Adig promotes seipin assembly by stabilizing and bridging adjacent seipin subunits. Functionally, Adig plays a key role in generating lipid droplets in adipocytes. In mice, inducible overexpression of Adig in adipocytes substantially increases fat mass, with enlarged lipid droplets. It also elevates thermogenesis during cold exposure. In contrast, inducible adipocyte-specific Adig knockout mice manifest aberrant lipid droplet formation in brown adipose tissues and impaired cold tolerance.

cell biology↗

bioRxiv2023

One-step generation of auxin-inducible degron cells with high-efficiency homozygous tagging

Auxin-inducible degron (AID) technology is powerful for chemogenetic control of proteolysis. However, generation of human cell lines to deplete endogenous proteins with AID remains challenging. Typically, homozygous degron-tagging efficiency is low and overexpression of an auxin receptor requires additional engineering steps. Here, we establish a one-step genome editing procedure with high-efficiency homozygous tagging and auxin receptor expression. We demonstrate its application in 5 human cell lines, including embryonic stem (ES) cells. The method allowed isolation of AID single-cell clones in 10 days for 11 target proteins with >80% average homozygous degron-tagging efficiency in A431 cells, and >50% efficiency for 5 targets in H9 ES cells. The tagged endogenous proteins were inducibly degraded in all cell lines, including ES cells and ES-cell derived neurons, with robust expected functional readouts. This method facilitates the application of AID for studying endogenous protein functions in human cells, especially in stem cells.

cell biology↗

bioRxiv2022

ATPase activity of DFCP1 controls selective autophagy

Cellular homeostasis is governed by removal of damaged organelles and protein aggregates by selective autophagy mediated by cargo adaptors such as p62/SQSTM1. Autophagosomes can assemble in specialized cup-shaped regions of the endoplasmic reticulum (ER) known as omegasomes, which are characterized by the presence of the ER protein DFCP1/ZFYVE1. The function of DFCP1 is unknown, as are the mechanisms of omegasome formation and constriction. Here, we demonstrate that DFCP1 is an ATPase that dimerizes in an ATP-dependent fashion. Whereas depletion of DFCP1 had a minor effect on bulk autophagic flux, DFCP1 was required to maintain the autophagic flux of p62 under both fed and starved conditions, and this was dependent on its ability to bind and hydrolyse ATP. While DFCP1 mutants defective in ATP binding or hydrolysis localized to forming omegasomes, these omegasomes failed to constrict. Consequently, the release of nascent autophagosomes from omegasomes was markedly delayed. DFCP1 was found to associate with ubiquitinated proteins, and degradation of ubiquitinated cargoes such as protein aggregates, mitochondria and micronuclei was strongly inhibited when DFCP1 was knocked out or mutated. Thus, DFCP1 mediates ATPase-driven constriction of omegasomes to release autophagosomes for selective autophagy.

cell biology↗

bioRxiv2021

Multiparametric assessment of cellular lipid metabolism in hypercholesterolemia

Systematic insight into cellular dysfunctions can improve understanding of disease etiology, risk assessment and patient stratification. We present a multiparametric high-content imaging platform enabling quantification of low-density lipoprotein (LDL) uptake and lipid storage in cytoplasmic droplets of primary leukocyte subpopulations. We validated this platform with samples from 65 individuals with variable blood LDL-cholesterol (LDL-c) levels, including familial hypercholesterolemia (FH) and non-FH subjects. We integrated lipid storage data into a novel readout, lipid mobilization, measuring the efficiency with which cells deplete lipid reservoirs. Lipid mobilization correlated positively with LDL uptake and negatively with hypercholesterolemia and age, improving differentiation of individuals with normal and elevated LDL-c. Moreover, combination of cell-based readouts with a polygenic risk score for LDL-c explained hypercholesterolemia better than the genetic risk score alone. This platform provides functional insights into cellular lipid trafficking from a few mls of blood and is applicable to dissect metabolic disorders, such as hypercholesterolemia. MotivationWe have limited information on how cellular lipid uptake and processing differ between individuals and influence the development of metabolic diseases, such as hypercholesterolemia. Available assays are labor intensive, require skilled personnel and are difficult to scale to higher throughput, making it challenging to obtain systematic functional cell-based data from individuals. To overcome this problem, we established a scalable automated analysis pipeline enabling reliable quantification of multiple cellular readouts, including lipid uptake, storage and mobilization, from different white blood cell populations. This approach provides new personalized insights into the cellular basis of hypercholesterolemia and obesity. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/440471v4_ufig1.gif" ALT="Figure 1"> View larger version (84K): org.highwire.dtl.DTLVardef@9222c1org.highwire.dtl.DTLVardef@27e289org.highwire.dtl.DTLVardef@89ba82org.highwire.dtl.DTLVardef@33c9cd_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2020

Seipin traps triacylglycerols to facilitate their nanoscale clustering in the ER membrane

Seipin is a disk-like oligomeric ER protein important for lipid droplet (LD) biogenesis and triacylglycerol (TAG) delivery to growing LDs. Here we show through biomolecular simulations bridged to experiments that seipin can trap TAGs in the ER bilayer via the luminal hydrophobic helices of the protomers delineating the inner opening of the seipin disk. This promotes the nanoscale sequestration of TAGs at a concentration that by itself is insufficient to induce TAG clustering in a lipid membrane. We identify Ser166 in the 3 helix as a favored TAG occupancy site and show that mutating it compromises the ability of seipin complexes to sequester TAG in silico and to promote TAG transfer to LDs in cells. While seipin-S166D mutant colocalizes poorly with promethin, the association of nascent wild-type seipin complexes with promethin is promoted by TAGs. Together, these results suggest that seipin traps TAGs via its luminal hydrophobic helices, serving as a catalyst for seeding the TAG cluster from dissolved monomers inside the seipin ring, thereby generating a favorable promethin binding interface.

cell biology↗

bioRxiv2018

Mitochondrial biogenesis is transcriptionally repressed in lysosomal lipid storage diseases

Perturbations in mitochondrial function and homeostasis are pervasive in lysosomal storage diseases, but the underlying mechanisms remain unknown. Here, we report a transcriptional program that represses mitochondrial biogenesis and function in lysosomal storage diseases Niemann-Pick type C (NPC) and acid sphingomyelinase deficiency (ASM), in patient cells and mouse tissues. This mechanism is mediated by the transcription factors KLF2 and ETV1, which are both induced in NPC and ASM patient cells. Mitochondrial biogenesis and function defects in these cells are rescued by the silencing of KLF2 or ETV1. Increased ETV1 expression is regulated by KLF2, while the increase of KLF2 protein levels in NPC and ASM stems from impaired signaling downstream sphingosine-1-phosphate receptor 1 (S1PR1), which normally represses KLF2. In patient cells, S1PR1 is undetectable at the plasma membrane and thus unable to repress KLF2. This manuscript provides a mechanistic pathway for the prevalent mitochondrial defects in lysosomal storage diseases.

cell biology↗

bioRxiv2018

Association of tamoxifen resistance and lipid reprogramming in breast cancer.

BackgroundTamoxifen treatment of estrogen receptor (ER)-positive breast cancer reduces mortality by 31%. However, over half of advanced ER-positive breast cancers are intrinsically resistant to tamoxifen and about 40% will acquire the resistance during the treatment.\n\nMethodsIn order to explore mechanisms underlying endocrine therapy resistance in breast cancer and to identify new therapeutic opportunities, we created tamoxifen-resistant breast cancer cell lines that represent the luminal A or the luminal B. Gene expression patterns revealed by RNA-sequencing in seven tamoxifen-resistant variants were compared with their isogenic parental cells. We further examined those transcriptomic alterations in a publicly available patient cohort.\n\nResultsWe show that tamoxifen resistance cannot simply be explained by altered expression of individual genes, common mechanism across all resistant variants, or the appearance of new fusion genes. Instead, the resistant cell lines shared altered gene expression patterns associated with cell cycle, protein modification and metabolism, especially with the cholesterol pathway. In the tamoxifen-resistant T-47D cell variants we observed a striking increase of neutral lipids in lipid droplets as well as an accumulation of free cholesterol in the lysosomes. Tamoxifen-resistant cells were also less prone to lysosomal membrane permeabilization (LMP) and not vulnerable to compounds targeting the lipid metabolism. However, the cells were sensitive to disulfiram, LCS-1, and dasatinib.\n\nConclusionAltogether, our findings highlight a major role of LMP prevention in tamoxifen resistance, and suggest novel drug vulnerabilities associated with this phenotype.

cancer biology↗