Biology subjects

Miaczynska, M.

Publications and source records attributed to Miaczynska, M..

8 recordsLinked to original sources

bioRxiv2025

p70S6 kinase-dependent phosphorylation of the μ2 subunit of the AP2 adaptor complex is needed for clathrin-mediated endocytosis.

Clathrin-mediated endocytosis (CME) internalizes cell-surface receptors via clathrin-coated invaginations of the plasma membrane. Both clathrin and endocytic cargo are recruited to these sites by the adaptor protein complex AP2. AP2 cycles between a closed cytoplasmic conformation and an open membrane-bound state, and efficient CME requires both conformations and their dynamic interconversion. The mechanisms regulating these conformational changes, which include post-translational modifications of the AP2, remain incompletely understood. Here, we report that p70S6 kinase phosphorylates the {micro}2 subunit of the AP2 and that the phosphorylation of serine 45 (S45) depends on p70S6K activity. Loss of S45-{micro}2 phosphorylation results in decreased internalization of canonical CME cargo such as transferrin and PDGF receptors. In Caenorhabditis elegans, lack of S45-{micro}2 phosphorylation produces directionally similar but markedly weaker phenotypes than AP2 loss of function. Live imaging and in silico dynamic modelling suggest that S45-2 phosphorylation has impact on the conformational changes of the AP2 complex. These findings identify a p70S6K-dependent mechanism that modulates AP2 function and further strengthen the importance of post-translational regulation in controlling CME. Summary statementClathrin-mediated endocytosis is essential for receptor internalization and cell signaling. This study identifies a novel regulatory mechanism in which p70S6K-mediated phosphorylation of the AP2 subunit 2 at Ser45 modulates AP2 conformational dynamics and facilitates receptor endocytosis.

cell biology↗

bioRxiv2024

NF-kappaB and JNK pathways mediate metabolic adaptation upon ESCRT-I deficiency

Endosomal Sorting Complexes Required for Transport (ESCRTs) are crucial for delivering membrane receptors or intracellular organelles for lysosomal degradation. Yet, how ESCRT dysfunction affects cell metabolism remained elusive. To address this, we analyzed transcriptomes of cells lacking TSG101 or VPS28 proteins, components of ESCRT-I subcomplex. ESCRT-I deficiency reduced the expression of genes encoding enzymes involved in oxidation of fatty acids and amino acids, and increased the expression of genes encoding glycolytic enzymes. Although depletion of ESCRT-I components did not impair mitochondrial biogenesis and ATP-linked respiration it caused intracellular accumulation of lipids and increased lactate production, hallmarks of aerobic glycolysis. Mechanistically, the observed transcriptional reprogramming towards glycolysis in the absence of ESCRT-I occurred due to activation of the canonical NF{kappa}B and JNK signaling pathways. Moreover, inhibiting lysosomal activity phenocopied the altered expression of metabolic genes and lipid homeostasis observed for ESCRT-I deficiency, indicating that ESCRT-I restricts glycolysis by mediating lysosomal degradation.

cell biology↗

bioRxiv2023

Ammonia inhibits antitumor activity of NK cells by decreasing mature perforin

Immunotherapy revolutionized cancer treatment in the last decade. Natural killer (NK) cells are one of the key host immunity components against malignant cells. Thus, they are currently extensively investigated in the field of immunotherapy of cancer. Different approaches have been developed to improve the antitumor activity of NK cells. Nonetheless, tumor microenvironment remains an obstacle to effective NK cell-based therapies. Here, we demonstrated that a cancer-conditioned medium suppresses the anti-tumor activity of NK cells. Further, we found that ammonia, a by-product of cancer cell metabolism, accumulates in the cancer-conditioned medium and tumor microenvironment. We identified that ammonia impairs the cytotoxicity of NK cells as well as the effectiveness of antibody-based and chimeric antigen receptor (CAR)-NK-based therapies in vitro. Inhibited activity of NK cells was caused by decreased levels of perforin. This effect was dependent on the lysosomotropic features of ammonia and its ability to increase pH in acidic compartments. In consequence, upon contact with ammonia the mature form of perforin was decreased in NK cells leading to their dysfunction. Our findings demonstrate that in addition to its previously described role of promoting tumor growth as a nitrogen source for tumor biomass ammonia could promote tumor escape as an NK cells immune checkpoint. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=66 SRC="FIGDIR/small/567708v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@19db6a5org.highwire.dtl.DTLVardef@1424903org.highwire.dtl.DTLVardef@c795d7org.highwire.dtl.DTLVardef@13e7a8e_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LICancer-conditioned medium suppresses the antitumor activity of NK cells C_LIO_LIAmmonia accumulates in conditioned medium and in the tumor microenvironment C_LIO_LIImpaired cytotoxicity of NK cells is caused by ammonia that decreases perforin levels C_LIO_LIAmmonia causes NK cell dysfunction C_LI

cancer biology↗

bioRxiv2023

Liver sinusoidal endothelial cells constitute a major route for hemoglobin clearance

Mild rupture of aged erythrocytes occurs physiologically in the spleen, leading to the release of hemoglobin (Hb), while pathological hemolysis characterizes several diseases. The detoxification of Hb has traditionally been attributed to the sequestration of Hb-haptoglobin complexes by macrophages. However, this process remains incompletely studied in animal models or primary cells, leaving the precise mechanisms of Hb clearance elusive. Using mice and primary liver cell cultures (murine and human), we uncovered that Hb uptake is chiefly performed by liver sinusoidal endothelial cells (LSECs) and involves macropinocytosis. Consistently, mouse LSECs displayed proteomic signatures indicative of active heme catabolism, ferritin iron storage, antioxidant defense, and macropinocytic capacity. LSECs also exhibited high iron content and the expression of hepcidin-regulated iron exporter ferroportin. Using erythrocyte/Hb transfusion assays in mice, we demonstrated that while splenic macrophages excel in phagocytosis of erythrocytes, LSECs primarily scavenge Hb and Kupffer cells clear erythrocyte membranes, the spleen-borne hemolysis products delivered to the liver via the portal circulation. High-dose Hb injections resulted in transient hepatic iron retention, early LSEC-specific induction of heme-catabolizing Hmox1 and iron-sensing Bmp6, culminating in hepcidin-mediated temporary hypoferremia. Transcriptional induction of Bmp6 in mice was phenocopied by erythrocyte lysis upon phenylhydrazine or iron citrate injection, although the latter elicited a distinct LSEC transcriptional signature compared to Hb. In conclusion, we identify LSECs as key Hb scavengers, a function that establishes the spleen-to-liver axis for iron recycling and contributes to heme detoxification during hemolysis, coupled with the induction of the BMP6-hepcidin axis to restore iron homeostasis.

cell biology↗

bioRxiv2021

ESCRT-I controls lysosomal membrane protein homeostasis and restricts MCOLN1-dependent TFEB/TFE3 signaling.

Within the endolysosomal pathway in mammalian cells, ESCRT complexes facilitate degradation of proteins residing in endosomal membranes. Recent studies revealed that yeast ESCRT machinery also sorts ubiquitinated proteins from the vacuolar membrane for degradation in the vacuole lumen. However, whether mammalian ESCRTs perform a similar function at lysosomes remained unknown. Here, we show that ESCRT-I restricts the size of lysosomes and promotes degradation of proteins from lysosomal membranes, including MCOLN1, a Ca2+ channel protein. Upon ESCRT-I depletion, the lysosomal accumulation of non-degraded proteins coincided with elevated expression of genes annotated to cholesterol biosynthesis and biogenesis of lysosomes, indicative of response to lysosomal stress. Accordingly, the lack of ESCRT-I promoted abnormal cholesterol accumulation in lysosomes and activated TFEB/TFE3 transcription factors. Finally, we discovered that in contrast to basal TFEB/TFE3 signaling that depended on the availability of exogenous lipids, the stress-induced activation of this pathway was Ca2+-MCOLN1-dependent. Hence, we provide evidence that ESCRT-I is crucial for maintaining lysosomal homeostasis and we elucidate mechanisms distinguishing basal from lysosomal stress-induced TFEB/TFE3 signaling.

cell biology↗

bioRxiv2020

Concurrent depletion of Vps37 proteins evokes ESCRT-I destabilization and profound cellular stress responses

ABSTRACTMolecular details of how endocytosis contributes to oncogenesis remain elusive. Our in silico analysis of colorectal cancer (CRC) patients revealed stage-dependent alterations in the expression of 113 endocytosis-related genes. Among them transcription of the Endosomal Sorting Complex Required for Transport (ESCRT)-I component VPS37B was decreased in the advanced stages of CRC. Expression of other ESCRT-I core subunits remained unchanged in the investigated dataset. We analyzed an independent cohort of CRC patients showing also reduced VPS37A mRNA and protein abundance. Transcriptomic profiling of CRC cells revealed non-redundant functions of Vps37 proteins. Knockdown of VPS37A and VPS37B triggered p21-mediated inhibition of cell proliferation and sterile inflammatory response driven by the Nuclear Factor (NF)-κB transcription factor and associated with mitogen-activated protein kinase signaling. Co-silencing of VPS37C further potentiated activation of these independently induced processes. The type and magnitude of transcriptional alterations correlated with the differential ESCRT-I stability upon individual and concurrent Vps37 depletion. Our study provides novel insights into cancer cell biology by describing cellular stress responses that are associated with ESCRT-I destabilization, which might occur in CRC patients.SUMMARY STATEMENT Endosomal Sorting Complex Required for Transport (ESCRT)-I destabilization upon concurrent depletion of Vps37 proteins is linked to the activation of sterile inflammatory response and cell growth inhibition.View Full Text

cell biology↗

bioRxiv2020

Splicing variation of BMP2K balances endocytosis, COPII trafficking and autophagy in erythroid cells

Intracellular transport undergoes remodeling upon cell differentiation, which involves cell type-specific regulators. Bone morphogenetic protein 2-inducible kinase (BMP2K) has been potentially implicated in endocytosis and cell differentiation but its molecular functions remained unknown. We discovered that its longer (L) and shorter (S) splicing variants regulate erythroid differentiation in a manner unexplainable by their involvement in AP-2 adaptor phosphorylation and endocytosis. However, both variants interacted with SEC16A whose silencing in K562 erythroid leukemia cells affected generation of COPII assemblies and induced autophagic degradation. Variant-specific depletion approach showed that BMP2K isoforms constitute a BMP2K-L/S regulatory system. Therein, L promotes while S restricts recruitment of SEC31A to SEC24B-containing COPII structures forming at SEC16A-positive ER exit sites. Finally, we found L to promote and S to restrict autophagic degradation. Hence, we propose that BMP2K-L favors SEC16A-dependent intracellular processes important for erythroid maturation, such as COPII trafficking and autophagy, in a manner inhibited by BMP2K-S.

cell biology↗

bioRxiv2020

GAS6-AXL signaling triggers actin remodeling and macropinocytosis that drive cancer cell invasion

AXL, a member of the TAM (TYRO3, AXL, MER) receptor tyrosine kinase family, and its ligand GAS6 are implicated in oncogenesis and metastasis of many cancer types. However, the exact cellular processes activated by GAS6-AXL remain largely unexplored. Here, we identified an interactome of AXL and revealed its associations with proteins regulating actin dynamics. Consistently, GAS6-mediated AXL activation triggered actin remodeling manifested by peripheral membrane ruffling and circular dorsal ruffles (CDRs). This further promoted macropinocytosis that mediated the internalization of GAS6-AXL complexes and sustained survival of glioblastoma cells grown under glutamine-deprived conditions. GAS6-induced CDRs contributed to focal adhesion (FA) turnover, cell spreading and elongation. Consequently, AXL activation by GAS6 drove invasion of cancer cells in a spheroid model. All these processes required the kinase activity of AXL but not TYRO3, and downstream activation of PI3K. We propose that GAS6-AXL signaling induces multiple actin-driven cytoskeletal rearrangements and macropinocytosis that jointly contribute to cancer cell invasion.

cell biology↗