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Nature Communications Apr 2022Macropinocytosis, an evolutionarily conserved mechanism mediating nonspecific bulk uptake of extracellular fluid, has been ascribed diverse functions. How nascent...
Macropinocytosis, an evolutionarily conserved mechanism mediating nonspecific bulk uptake of extracellular fluid, has been ascribed diverse functions. How nascent macropinosomes mature after internalization remains largely unknown. By searching for proteins that localize on macropinosomes during the Rab5-to-Rab7 transition stage in Dictyostelium, we uncover a complex composed of two proteins, which we name PripA and TbcrA. We show that the Rab5-to-Rab7 conversion involves fusion of Rab5-marked early macropinosomes with Rab7-marked late macropinosomes. PripA links the two membrane compartments by interacting with PI(3,4)P and Rab7. In addition, PripA recruits TbcrA, which acts as a GAP, to turn off Rab5. Thus, the conversion to Rab7 is linked to inactivation of the upstream Rab5. Consistently, disruption of either pripA or tbcrA impairs Rab5 inactivation and macropinocytic cargo processing. Therefore, the PripA-TbcrA complex is the central component of a Rab GAP cascade that facilitates programmed Rab switch and efficient cargo trafficking during macropinosome maturation.
Topics: Biological Transport; Dictyostelium; Endosomes; Pinocytosis; rab GTP-Binding Proteins
PubMed: 35379834
DOI: 10.1038/s41467-022-29503-1 -
Sub-cellular Biochemistry 2022The distinct movements of macropinosome formation and maturation have corresponding biochemical activities which occur in a defined sequence of stages and transitions...
The distinct movements of macropinosome formation and maturation have corresponding biochemical activities which occur in a defined sequence of stages and transitions between those stages. Each stage in the process is regulated by variously phosphorylated derivatives of phosphatidylinositol (PtdIns) which reside in the cytoplasmic face of the membrane lipid bilayer. PtdIns derivatives phosphorylated at the 3' position of the inositol moiety, called 3' phosphoinositides (3'PIs), regulate different stages of the sequence. 3'PIs are synthesized by numerous phosphoinositide 3'-kinases (PI3K) and other lipid kinases and phosphatases, which are themselves regulated by small GTPases of the Ras superfamily. The combined actions of these enzymes localize four principal species of 3'PI to distinct domains of the plasma membrane or to discrete organelles, with distinct biochemical activities confined to those domains. Phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P) and phosphatidylinositol (3,4)-bisphosphate (PtdIns(3,4)P) regulate the early stages of macropinosome formation, which include cell surface ruffling and constrictions of circular ruffles which close into macropinosomes. Phosphatidylinositol 3-phosphate (PtdIns3P) regulates macropinosome fusion with other macropinosomes and early endocytic organelles. Phosphatidylinositol (3,5)-bisphosphate (PtdIns(3,5)P) mediates macropinosome maturation and shrinkage, through loss of ions and water, and subsequent traffic to lysosomes. The different characteristic rates of macropinocytosis in different cell types indicate levels of regulation which may be governed by the cell's capacity to generate 3'PIs.
Topics: Cell Membrane; Endosomes; Phosphatidylinositols; Phosphoric Monoester Hydrolases; Pinocytosis
PubMed: 35378706
DOI: 10.1007/978-3-030-94004-1_7 -
Journal of Experimental & Clinical... Mar 2022Macropinocytosis, an important nutrient-scavenging pathway in certain cancer cells, allows cells to compensate for intracellular amino acid deficiency under...
BACKGROUND
Macropinocytosis, an important nutrient-scavenging pathway in certain cancer cells, allows cells to compensate for intracellular amino acid deficiency under nutrient-poor conditions. Ferroptosis caused by cysteine depletion plays a pivotal role in sorafenib responses during hepatocellular carcinoma (HCC) therapy. However, it is not known whether macropinocytosis functions as an alternative pathway to acquire cysteine in sorafenib-treated HCC, and whether it subsequently mitigates sorafenib-induced ferroptosis. This study aimed to investigate whether sorafenib drives macropinocytosis induction, and how macropinocytosis confers ferroptosis resistance on HCC cells.
METHODS
Macropinocytosis, both in HCC cells and HCC tissues, was evaluated by measuring TMR-dextran uptake or lysosomal degradation of DQ-BSA, and ferroptosis was evaluated via C11-BODIPY fluorescence and 4-HNE staining. Sorafenib-induced ferroptosis and macropinocytosis were validated in tumor tissues taken from HCC patients who underwent ultrasound-guided needle biopsy.
RESULTS
Sorafenib increased macropinocytosis in human HCC specimens and xenografted HCC tissues. Sorafenib-induced mitochondrial dysfunction was responsible for activation of PI3K-RAC1-PAK1 signaling, and amplified macropinocytosis in HCC. Importantly, macropinocytosis prevented sorafenib-induced ferroptosis by replenishing intracellular cysteine that was depleted by sorafenib treatment; this rendered HCC cells resistant to sorafenib. Finally, inhibition of macropinocytosis by amiloride markedly enhanced the anti-tumor effect of sorafenib, and sensitized resistant tumors to sorafenib.
CONCLUSION
In summary, sorafenib induced macropinocytosis, which conferred drug resistance by mitigating sorafenib-induced ferroptosis. Thus, targeting macropinocytosis is a promising therapeutic strategy to facilitate ferroptosis-based therapy for HCC.
Topics: Animals; Carcinoma, Hepatocellular; Cysteine; Female; Ferroptosis; Humans; Liver Neoplasms; Male; Mice; Pinocytosis; Protein Kinase Inhibitors; Sorafenib
PubMed: 35287706
DOI: 10.1186/s13046-022-02296-3 -
Nature Communications Feb 2022Hepatocellular carcinoma (HCC) invariably exhibits inadequate O (hypoxia) and nutrient supply. Hypoxia-inducible factor (HIF) mediates cascades of molecular events that...
Hepatocellular carcinoma (HCC) invariably exhibits inadequate O (hypoxia) and nutrient supply. Hypoxia-inducible factor (HIF) mediates cascades of molecular events that enable cancer cells to adapt and propagate. Macropinocytosis is an endocytic process initiated by membrane ruffling, causing the engulfment of extracellular fluids (proteins), protein digestion and subsequent incorporation into the biomass. We show that macropinocytosis occurs universally in HCC under hypoxia. HIF-1 activates the transcription of a membrane ruffling protein, EH domain-containing protein 2 (EHD2), to initiate macropinocytosis. Knockout of HIF-1 or EHD2 represses hypoxia-induced macropinocytosis and prevents hypoxic HCC cells from scavenging protein that support cell growth. Germline or somatic deletion of Ehd2 suppresses macropinocytosis and HCC development in mice. Intriguingly, EHD2 is overexpressed in HCC. Consistently, HIF-1 or macropinocytosis inhibitor suppresses macropinocytosis and HCC development. Thus, we show that hypoxia induces macropinocytosis through the HIF/EHD2 pathway in HCC cells, harnessing extracellular protein as a nutrient to survive.
Topics: Animals; Carcinoma, Hepatocellular; Carrier Proteins; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Liver Neoplasms; Mice; Mice, Knockout; Pinocytosis; Proof of Concept Study; Tumor Hypoxia; Xenograft Model Antitumor Assays
PubMed: 35177645
DOI: 10.1038/s41467-022-28618-9 -
Cells Jan 2022To fight neurodegenerative diseases, several therapeutic strategies have been proposed that, to date, are either ineffective or at the early preclinical stages....
To fight neurodegenerative diseases, several therapeutic strategies have been proposed that, to date, are either ineffective or at the early preclinical stages. Intracellular protein aggregates represent the cause of about 70% of neurodegenerative disorders, such as Alzheimer's disease. Thus, autophagy, i.e., lysosomal degradation of macromolecules, could be employed in this context as a therapeutic strategy. Searching for a compound that stimulates this process led us to the identification of a 37/67kDa laminin receptor inhibitor, NSC48478. We have analysed the effects of this small molecule on the autophagic process in mouse neuronal cells and found that NSC48478 induces the conversion of microtubule-associated protein 1A/1B-light chain 3 (LC3-I) into the LC3-phosphatidylethanolamine conjugate (LC3-II). Interestingly, upon NSC48478 treatment, the contribution of membranes to the autophagic process derived mainly from the non-canonical m-TOR-independent endocytic pathway, involving the Rab proteins that control endocytosis and vesicle recycling. Finally, qRT-PCR analysis suggests that, while the expression of key genes linked to canonical autophagy was unchanged, the main genes related to the positive regulation of endocytosis (pinocytosis and receptor mediated), along with genes regulating vesicle fusion and autolysosomal maturation, were upregulated under NSC48478 conditions. These results strongly suggest that 37/67 kDa inhibitor could be a useful tool for future studies in pathological conditions.
Topics: Animals; Autophagy; Laminin; Mice; Microtubule-Associated Proteins; Naphthols; Receptors, Laminin
PubMed: 35159276
DOI: 10.3390/cells11030466 -
Theranostics 2022KRAS mutations are one of the most common gene mutations linked to cancer, presenting in approximately 25% of all tumors, especially pancreatic, lung, and colorectal... (Review)
Review
KRAS mutations are one of the most common gene mutations linked to cancer, presenting in approximately 25% of all tumors, especially pancreatic, lung, and colorectal cancers. Mutant KRAS has long been considered an undruggable target, stalling progress in direct KRAS targeting for many years, while targeted drug delivery into KRAS mutant cells utilizing their transformed metabolic behavior might present an alternative opportunity. Macropinocytosis, a nonselective, fluid-phase, endocytic route, was found to be upregulated as a metabolic feature in KRAS-driven tumors and plays a critical role in nutrient acquisition from extracellular fluids. With the observation that a variety of drug delivery systems could be internalized by KRAS mutant cancer cells through macropinocytosis, exploiting macropinocytosis for intracellular delivery of therapeutics into KRAS mutant tumor cells is emerging as a new drug delivery expedition. In this article, we summarized cancer biology studies that examined KRAS mutation-induced macropinocytosis, reviewed recent studies exploiting macropinocytosis enhancement for KRAS mutant cancer cell-selective drug delivery, and discussed the potential opportunities, challenges and pitfalls of this strategy.
Topics: Cell Line, Tumor; Drug Delivery Systems; Humans; Mutation; Neoplasms; Pinocytosis; Proto-Oncogene Proteins p21(ras)
PubMed: 35154489
DOI: 10.7150/thno.67889 -
Journal of Cell Science Feb 2022The internalization of solutes by macropinocytosis provides an essential route for nutrient uptake in many cells. Macrophages increase macropinocytosis in response to...
The internalization of solutes by macropinocytosis provides an essential route for nutrient uptake in many cells. Macrophages increase macropinocytosis in response to growth factors and other stimuli. To test the hypothesis that nutrient environments modulate solute uptake by macropinocytosis, this study analyzed the effects of extracellular amino acids on the accumulation of fluorescent fluid-phase probes in murine macrophages. Nine amino acids, added individually or together, were capable of suppressing macropinocytosis in murine bone marrow-derived macrophages stimulated with the growth factors colony stimulating factor 1 (CSF1) or interleukin 34, both ligands of the CSF1 receptor (CSF1R). The suppressive amino acids did not inhibit macropinocytosis in response to lipopolysaccharide, the chemokine CXCL12, or the tumor promoter phorbol myristate acetate. Suppressive amino acids promoted release of CSF1R from cells and resulted in the formation of smaller macropinosomes in response to CSF1. This suppression of growth factor-stimulated macropinocytosis indicates that different nutrient environments modulate CSF1R levels and bulk ingestion by macropinocytosis, with likely consequences for macrophage growth and function.
Topics: Amino Acids; Animals; Endosomes; Macrophage Colony-Stimulating Factor; Macrophages; Mice; Pinocytosis; Receptor Protein-Tyrosine Kinases
PubMed: 35107133
DOI: 10.1242/jcs.259284 -
Placenta Jul 2022Prolonged pregnancy describes a pregnancy that progresses beyond 42 weeks' gestation (294 days). In humans, prolonged pregnancy is associated with increasing perinatal... (Review)
Review
Prolonged pregnancy describes a pregnancy that progresses beyond 42 weeks' gestation (294 days). In humans, prolonged pregnancy is associated with increasing perinatal mortality, neonatal compromise and birth by Caesarean section. The underpinning reasons behind these increased risks are unknown; one potential explanation is reduced placental function due to ageing processes. This review describes the structural and functional changes seen in prolonged pregnancy in humans and in animal models. Prolonged pregnancies are associated with reduced placental growth, leading to an increase in fetal to placental weight ratio. Microscopic changes include aggregation of syncytiotrophoblast nuclei, reduced villous vascularity with a concomitant impairment of trophoblast transport processes (reduced pinocytosis); this is associated with increased evidence of oxidative stress, with downstream consequences including cellular senescence, autophagy and apoptosis; importantly many of these changes are similar to fetal growth restriction and pre-eclampsia. Thus, we argue that these observations provide evidence of ageing within the placenta, which may initially be adaptive but can become pathological leading to a reduction in placental function. This provides a biological basis for the increased risk of adverse outcomes observed in prolonged pregnancies. Greater insight into the effects and risks of placental ageing may be useful to guide clinicians on the management of prolonged pregnancies.
Topics: Animals; Cesarean Section; Female; Fetal Growth Retardation; Humans; Placenta; Pre-Eclampsia; Pregnancy; Pregnancy, Prolonged
PubMed: 35058067
DOI: 10.1016/j.placenta.2022.01.009 -
Current Opinion in Virology Feb 2022Endocytosis is used by eukaryotic cells for ingesting external objects. Importantly, endocytosis is a major process that determines phage pharmacokinetics in vivo.... (Review)
Review
Endocytosis is used by eukaryotic cells for ingesting external objects. Importantly, endocytosis is a major process that determines phage pharmacokinetics in vivo. Either dissemination of phages throughout the system or phage clearance engages cellular uptake of phage particles. Here we discuss phage uptake by mammalian cells, focusing on mechanisms and pathways involved. Of note, cellular uptake of phage virions was first observed in professional phagocytes, such as macrophages or granulocytes. For this reason, it was historically referred to as 'phagocytosis'. The modern definition of phagocytosis, however, identifies this process as a type of endocytosis within a larger repertoire of endocytic pathways, such as macropinocytosis, clathrin-mediated endocytosis, and caveolar endocytosis, which have all been included in the scope of this review.
Topics: Animals; Bacteriophages; Caveolae; Endocytosis; Mammals; Phagocytosis; Pinocytosis
PubMed: 34968792
DOI: 10.1016/j.coviro.2021.12.009 -
Anatomy & Cell Biology Jun 2022The anti-aging effects of are extensively investigated. Nisin is an antimicrobial peptide produced by subsp. . We previously reported that 24-hour nisin treatment...
The anti-aging effects of are extensively investigated. Nisin is an antimicrobial peptide produced by subsp. . We previously reported that 24-hour nisin treatment disturbs the intermediate filament distribution in human keratinocytes. Additionally, we showed that the ring-like distribution of the intermediate filament proteins, cytokeratin (CK) 5 and CK17 is a marker of nisin action. However, two questions remained unanswered: 1) What do the CK5 and CK17 ring-like distributions indicate? 2) Is nisin ineffective under the experimental conditions wherein CK5 and CK17 do not exhibit a ring-like distribution? Super resolution microscopy revealed that nisin treatment altered CK5 and CK17 distribution, making them spherical rather than ring-like, along with actin incorporation. This spherical distribution was not induced by the suppression of endocytosis. The possibility of a macropinocytosis-like phenomenon was indicated, because the spherical distribution was >1 µm in diameter and the spherical distribution was suppressed by macropinocytosis inhibiting conditions, such as the inclusion of an actin polymerization inhibitor and cell migration. Even when the spherical distribution of CK5 and CK17 was not induced, nisin induced derangement of the cell membrane. Nisin treatment for 30 minutes deranged the regular arrangement of the lipid layer (flip-flop); the transmembrane structure of the CK5-desmosome or CK17-desmosome protein complex was disturbed. To the best of our knowledge, this is the first study to report that CK5 and CK17 in a spherical distribution could be involved in a macropinosome-like structure, under certain conditions of nisin action in keratinocytes.
PubMed: 34903675
DOI: 10.5115/acb.21.168