Type Of Endocytosis Research Articles

Abstract 4452: The macropinosome: Uncovering the molecular anatomy of an oncogene-driven organelle

Abstract As an integral aspect of the metabolic reprogramming that occurs in cancer, oncogenic KRAS mutations drive the stimulation of macropinocytosis, a type of endocytosis that mediates nonselective fluid-phase uptake. Using KRAS-driven models of pancreatic ductal adenocarcinoma (PDAC), we were the first to demonstrate that macropinocytosis functions in tumor cells as a nutrient acquisition pathway. Macropinocytosis triggers the internalization of extracellular proteins via discrete endocytic vesicles called macropinosomes. The incoming protein cargo is targeted for lysosome-dependent degradation, causing the intracellular release of amino acids. These protein-derived amino acids support metabolic fitness by contributing to the intracellular amino acid pools, as well as to the biosynthesis of central carbon metabolites. In this way, macropinocytosis represents a novel amino acid supply route that tumor cells use to survive the nutrient-poor conditions of the tumor microenvironment. While the tenets of the signal transduction events that drive macropinocytosis in cancer have emerged, a detailed picture of the macropinosome itself is not yet available. With the aid of nanotechnology, we have developed a methodology to isolate a pure fraction of macropinosomes from PDAC cells. Using a large-scale proteomic approach, we have been able to generate a list of proteins that reside in macropinosomes. The specificity of this approach was confirmed through the identification and validation of proteins known to specifically localize to these organelles in different contexts, such as Rab5 or SDC1. Thanks to this innovative method, we have demonstrated the involvement of two calcium channels, ATP2B1 and ATP2B4, in the formation of macropinosomes. By elucidating the molecular anatomy of KRAS-driven macropinosomes, we will gain further insight into the regulation and function of macropinocytosis in cancer. Moreover, having a clearer picture of the macropinosome will make us better positioned to exploit this pathway therapeutically and deliver breakthroughs to patients. Citation Format: Ambroise Manceau, Koen M. Galenkamp, Cheska M. Galapate, Cosimo Commisso. The macropinosome: Uncovering the molecular anatomy of an oncogene-driven organelle [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4452.

Abstract B088: The macropinosome: Uncovering the molecular anatomy of an oncogene-driven organelle

Abstract As an integral aspect of the metabolic reprogramming that occurs in cancer, oncogenic KRAS mutations drive the stimulation of macropinocytosis, a type of endocytosis that mediates nonselective fluid-phase uptake. Using KRAS-driven models of pancreatic ductal adenocarcinoma (PDAC), we were the first to demonstrate that macropinocytosis functions in tumor cells as a nutrient acquisition pathway. Macropinocytosis triggers the internalization of extracellular proteins via discrete endocytic vesicles called macropinosomes. The incoming protein cargo is targeted for lysosome-dependent degradation, causing the intracellular release of amino acids. These protein-derived amino acids support metabolic fitness by contributing to the intracellular amino acid pools, as well as to the biosynthesis of central carbon metabolites. In this way, macropinocytosis represents a novel amino acid supply route that tumor cells use to survive the nutrient-poor conditions of the tumor microenvironment. While the tenets of the signal transduction events that drive macropinocytosis in cancer have emerged, a detailed picture of the macropinosome itself is not yet available. With the aid of nanotechnology, we have developed a methodology to isolate a pure fraction of macropinosomes from PDAC cells. Using a large-scale proteomic approach, we have been able to generate a list of proteins that reside in macropinosomes. The specificity of this approach was confirmed through the identification and validation of proteins known to specifically localize to these organelles in different contexts, such as Rab5 or SDC1. Thanks to this innovative method, we have demonstrated the involvement of two calcium channels, ATP2B1 and ATP2B4, in the formation of macropinosomes. By elucidating the molecular anatomy of KRAS-driven macropinosomes, we will gain further insight into the regulation and function of macropinocytosis in cancer. Moreover, having a clearer picture of the macropinosome will make us better positioned to exploit this pathway therapeutically and deliver breakthroughs to patients. Citation Format: Ambroise Manceau, Koen M.O. Galenkamp, Cheska M. Galapate, Cosimo Commisso. The macropinosome: Uncovering the molecular anatomy of an oncogene-driven organelle [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B088.

A macropinocytosis-related gene signature predicts the prognosis and immune microenvironment in hepatocellular carcinoma.

Available treatments for hepatocellular carcinoma (HCC), a common human malignancy with a low survival rate, remain unsatisfactory. Macropinocytosis (MPC), a type of endocytosis that involves the non-specific uptake of dissolved molecules, has been shown to contribute to HCC pathology; however, its biological mechanism remains unknown. The current study identified 27 macropinocytosis-related genes (MRGs) from 71 candidate genes using bioinformatics. The R software was used to create a prognostic signature model by filtering standardized mRNA expression data from HCC patients and using various methods to verify the reliability of the model and indicate immune activity. The prognostic signature was constructed using seven MPC-related differentially expressed genes, GSK3B, AXIN1, RAC1, KEAP1, EHD1, GRB2, and SNX5, through LASSO Cox regression. The risk score was acquired from the expression of these genes and their corresponding coefficients. HCC patients in the discovery and validation cohorts were stratified, and the survival of low-risk score patients was improved in both cohorts. Time-dependent ROC analysis indicated that the model's prediction reliability was the highest in the short term. Subsequent immunologic analysis, including KEGG, located the immune action pathway of the differentially expressed genes in the direction of the cancer pathway, etc. Immune infiltration and immune checkpoint tests provided valuable guidance for future follow-up experiments. A risk model with MRGs was constructed to effectively predict HCC patient prognoses and suggest changes in the immune microenvironment during the disease process. The findings should benefit the development of a prognostic stratification and treatment strategy for HCC.

Abstract 888: Elucidating the mechanisms of eATP-induced changes in cell growth, energy metabolism, and cancer stem cells in human non-small lung cancer A549 cells

Abstract The tumor microenvironment (TME) has concentrations of extracellular ATP (eATP) 103-104 times higher than normal healthy tissues, implying that eATP may have important roles in tumor development and progression. Our previous study, cited by a Nature Reviews Cancer paper, showed that the extracellular ATP (eATP) is internalized by tumors via macropinocytosis, a type of endocytosis, to increase intracellular ATP (iATP) levels and support tumor cells growth, drug resistance, and cancer cell movement by initiating epithelial to mesenchymal transition (EMT). Recently, our lab found that eATP functions in promoting cancer cell growth, metastasis are similar to those of TGF-β, a well-known EMT and cancer stem cells (CSCs) inducer. Increasing reports state that the EMT and CSCs share some molecular links, but the detailed mechanisms and inductor of them are still far from clear. In this report, eATP’s function in CSCs induction was investigated first. Result shows that eATP treatment significantly induces the protein expression level of CSC related makers, increases cell colony numbers, and cell populations with CSC markers expressed in human lung cancer A549 cells. In addition, RNA-sequencing analysis reveals the gene Stanniocalcin 1 (STC1) is significantly upregulated in both eATP and TGF-β treatments. STC1 gene has been reported to be a competitive biomarker in the tracking of cancers’ progressing. Besides, STC1 exhibits concentration-dependent stimulation effects on electron transportation chain (ETC) and respiration. However, the detail function of STC1 gene in eATP induced cell growth is unknown. To find out the mechanism, CRISPR-Cas9 technique was used to generate STC1 gene targeted knockout (STC1 KO) A549 cells. STC1 KO cells show slower cell growth rate, lower intracellular ATP level and lower oxygen consumption rate (OCR) when compared with normal A549 cells in vitro. These results strongly indicate STC1 gene involved in eATP induced cell growth by alteration iATP level and metabolic profile. Overall, this study not only unravels new role of eATP in CSC induction, but also provides new mechanism of eATP induced cell growth by altering the protein expression level of STC1. Citation Format: Jingwen Song, Xiaozhuo Chen. Elucidating the mechanisms of eATP-induced changes in cell growth, energy metabolism, and cancer stem cells in human non-small lung cancer A549 cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 888.

The Study of Cyclosporin A Nanocrystals Uptake and Transport across an Intestinal Epithelial Cell Model.

Cyclosporin A nanocrystals (CsA-NCs) interaction with Caco-2 cells were investigated in this study, including cellular uptake and transport across Caco-2 cell monolayers. CsA-NCs of 165 nm, 240 nm and 450 nm were formulated. The dissolution of CsA-NCs was investigated by paddle method. The effect of size, concentration and incubation time on cellular uptake and dissolution kinetics of CsA-NCs in cells were studied. Uptake mechanisms were also evaluated using endocytotic inhibitors and low temperature (4 °C). The cell monolayers were incubated with each diameter CsA-NCs to evaluate the effect of size on the permeation characteristics of CsA across the intestinal mucosa. The results of dissolution study showed that 165 nm CsA-NC had the highest dissolution rate followed by 240 CsA-NC and finally 450 nm CsA-NC. The saturation of cell uptake of CsA-NCs was observed with the increase of incubation concentration and time. 240 nm and 450 nm CsA-NCs had the lowest and highest uptake efficiency at different time and drug concentration, respectively. The uptake of all three-sized CsA-NCs declined significantly in some different degree after the pre-treatment with different endocytosis inhibitors. 165 nm CsA-NC showed a highest transport capacity across monolayers at the same concentration and time. The results suggest that the size of CsA-NCs can not only affect the efficiency of cellular uptake, but also the type of endocytosis. Decreasing particle size of CsA-NCs can improve transport capacity of CsA through cell monolayer.

Piezo1 activation using Yoda1 inhibits macropinocytosis in A431 human epidermoid carcinoma cells

Macropinocytosis is a type of endocytosis accompanied by actin rearrangement-driven membrane deformation, such as lamellipodia formation and membrane ruffling, followed by the formation of large vesicles, macropinosomes. Ras-transformed cancer cells efficiently acquire exogenous amino acids for their survival through macropinocytosis. Thus, inhibition of macropinocytosis is a promising strategy for cancer therapy. To date, few specific agents that inhibit macropinocytosis have been developed. Here, focusing on the mechanosensitive ion channel Piezo1, we found that Yoda1, a Piezo1 agonist, potently inhibits macropinocytosis induced by epidermal growth factor (EGF). The inhibition of ruffle formation by Yoda1 was dependent on the extracellular Ca2+ influx through Piezo1 and on the activation of the calcium-activated potassium channel KCa3.1. This suggests that Ca2+ ions can regulate EGF-stimulated macropinocytosis. We propose the potential for macropinocytosis inhibition through the regulation of a mechanosensitive channel activity using chemical tools.

Endocytosis is a significant contributor to uranium(VI) uptake in tobacco (Nicotiana tabacum) BY-2 cells in phosphate-deficient culture

Endocytosis of metals in plants is a growing field of study involving metal uptake from the rhizosphere. Uranium, which is naturally and artificially released into the rhizosphere, is known to be taken up by certain species of plant, such as Nicotiana tabacum, and we hypothesize that endocytosis contributes to the uptake of uranium in tobacco. The endocytic uptake of uranium was investigated in tobacco BY-2 cells using an optimized setup of culture in phosphate-deficient medium. A combination of methods in biochemistry, microscopy and spectroscopy, supplemented by proteomics, were used to study the interaction of uranium and the plant cell. We found that under environmentally relevant uranium concentrations, endocytosis remained active and contributed to 14% of the total uranium bioassociation. Proteomics analyses revealed that uranium induced a change in expression of the clathrin heavy chain variant, signifying a shift in the type of endocytosis taking place. However, the rate of endocytosis remained largely unaltered. Electron microscopy and energy-dispersive X-ray spectroscopy showed an adsorption of uranium to cell surfaces and deposition in vacuoles. Our results demonstrate that endocytosis constitutes a considerable proportion of uranium uptake in BY-2 cells, and that endocytosed uranium is likely targeted to the vacuole for sequestration, providing a physiologically safer route for the plant than uranium transported through the cytosol.

Assimilation of synthetic plastic nanoparticles by the oomycete Pythium aquatile

Abstract. The degradation of synthetic plastics in the environment proceeds through the stages of micro- and nanoparticles. Freshwater fungi and fungus-like organisms are key decomposers of organic material. The most fungi (including the fungus-like oomycetes) feed by osmotrophy, which involves the release of exoenzymes into the environment to break down complex biological polymers, followed by cellular uptake of simplified monomers. This article aims to cultivate oomycete Pythium aquatile H hnk, 1953 in the presence of synthetic plastic nanoparticles as an only source of carbon compounds. The nanoparticles were bulk stained with water-insoluble fluorescent dye. We studied such widely used polymers as polystyrene (PS), poly(vinyl chloride), poly(methyl acrylate) (PMA) and poly(methyl methacrylate) (PMMA). This first experiment on exposure of plastic nanoparticles to oomycete hyphae showed the ability of these fungus-like organisms to capture these particles through a certain type of endocytosis. The fate of nanoplastics in the presence of P. aquatile depends on the chemical structure of the polymer. PMMA with a quaternary carbon atom is the most stable, PMA and PS containing the active -CH atom are rapidly destroyed by oomycetes.

Size-dependent cellular uptake and localization profiles of silver nanoparticles.

Purpose: Silver nanoparticles (AgNPs) have been widely applied in various fields as excellent antibacterial reagents over the past decades. Although the particle size is considered as the most crucial factor influencing cellular uptake, transportation, and accumulation behaviors, there are still many controversies regarding the correlation between size and uptake of AgNPs. In this study, size-dependent cellular uptake of AgNPs with different diameters was investigated in B16 cells.Methods: The uptake of AgNPs was investigated by inductively coupled plasma-mass spectrometry (ICP-MS) and transmission electron microscopic (TEM) imaging in B16 cells.Results: Twenty nanometer and 100 nm AgNPs had the lowest and highest uptake efficiency at both 12 hours and 24 hours, respectively. Smaller AgNPs crossed the plasma membrane faster with uniform distribution: 5 nm AgNPs were detected in both cytoplasm and nucleus at 0.5 hours after incubation. Larger AgNPs were extremely difficult to migrate: 100 nm AgNPs were detected in the nucleus at 12 hours after incubation. Internalization of AgNPs was directly observed, mainly within membrane-bound structures, such as intracellular vesicles and late endosomes. The uptake of all four-sized AgNPs (5 nm, 20 nm, 50 nm, 100 nm) decreased significantly after the pre-treatment with chlorpromazine hydrochloride, which can specifically inhibit the clathrin-mediated endocytosis. The internalization efficiencies of AgNPs (5 nm, 20 nm, 50 nm) were markedly reduced by methyl-β-cyclodextrin, a specific caveolin-mediated endocytosis inhibitor, whereas 5-(N-ethyl-N-isopropyl) amiloride as an inhibitor of macropinocytosis inhibited the uptake of larger sizes of AgNPs (50 nm and 100 nm).Conclusion: The results suggest that the size of AgNPs can not only affect the efficiency of cellular uptake, but also the type of endocytosis. The clathrin-mediated endocytosis may be the most common endocytic pathway for AgNPs in B16 cells, and AgNPs at each size were likely to enter cells by a major internalization pathway.

Calcium deprivation enhances non-selective fluid-phase endocytosis and modifies membrane lipid profiles in Arabidopsis roots

Ecological studies have revealed significant decreases in calcium (Ca) levels in various soils, and widely occurring physiological Ca deficits worldwide. These changes may cause decreases in plant diversity and increases in plant vulnerability to environmental stress, but the underlying cellular mechanism is not well understood. In this study, we found that in Arabidopsis thaliana roots, deprivation of Ca2+, but not other minerals, dramatically enhanced plasma membrane invagination, endosome formation, and trafficking to the vacuole through the trans-Golgi network and pre-vacuole compartment, a typical pathway of endocytosis. Antagonist and cellular tracing analyses using non-bioactive, membrane-impermeable fluorescent probes indicated that this type of endocytosis is not regulated by receptors, instead representing a non-selective, non-specific fluid phase-based process. We performed lipid-profiling analysis of roots in response to Ca2+ deprivation, finding increased phosphatidylcholine (PC), Lyso-PC, phosphatidylethanolamine (PE), Lyso-PE, phosphatidylinositol (PI) and triacylglycerols (TAG) biosynthesis but deceased phosphatidic acid (PA) and diacylglycerols (DAG) biosynthesis. The increased TAG contents and molar ratio of PC/PE in these roots might reflect a cellular response to maintain membrane stability and the balance between the membrane and storage lipids. This study demonstrates the essential role of Ca in maintaining plasma membrane stability and the selectivity of plant root cells, and it highlights the potential deleterious effect of decreased Ca levels in surface soil on plant growth.

Revisiting the Role of Clathrin-Mediated Endoytosis in Synaptic Vesicle Recycling.

Without robust mechanisms to efficiently form new synaptic vesicles (SVs), the tens to hundreds of SVs typically present at the neuronal synapse would be rapidly used up, even at modest levels of neuronal activity. SV recycling is thus critical for synaptic physiology and proper function of sensory and nervous systems. Yet, more than four decades after it was originally proposed that the SVs are formed and recycled locally at the presynaptic terminals, the mechanisms of endocytic processes at the synapse are heavily debated. Clathrin-mediated endocytosis, a type of endocytosis that capitalizes on the clathrin coat, a number of adaptor and accessory proteins, and the GTPase dynamin, is well understood, while the contributions of clathrin-independent fast endocytosis, kiss-and-run, bulk endocytosis and ultrafast endocytosis are still being evaluated. This review article revisits and summarizes the current knowledge on the SV reformation with a focus on clathrin-mediated endocytosis, and it discusses the modes of SV formation from endosome-like structures at the synapse. Given the importance of this topic, future advances in this active field are expected to contribute to better comprehension of neurotransmission, and to have general implications for neuroscience and medicine.

Mimicking Cell Pinocytosis: Lipid Vesicles Engulfment of Oil-in-Water Droplets

Pinocytosis, a type of endocytosis, is a process by which extracellular liquid droplets are taken up by cells. It involves droplet adhesion and engulfment by the membrane. We develop a synthetic system that reproduces the essential steps of pinocytosis at the level of a single interacting pair - the membrane and the droplet. The system consists of an engulfing giant unilamellar vesicle (GUV) and an engulfed polymer-stabilized oil-in-water emulsion droplet. The interaction between the GUV and the droplet is mediated by magnesium ions and is reversed by EDTA or Triton X-100. Adhesion energy |W|, measured from the contact angle between a GUV adhering to a droplet, either freestanding or manipulated with micropipettes, is affected by the magnesium concentration. In the weak adhesion regime (|W|∼0.4 mJ/m2, ≤ 0.5 mM Mg2+), GUVs adhere to the droplets and deform. Interaction is reversed by retracting the pipette-held GUV. In the strong adhesion regime (γ∼1.2 mJ/m2, 0.5 > mM Mg2+), the GUV completely engulfs the droplet and the interaction is irreversible. The droplet becomes wrapped by a single bilayer in a process that increases membrane tension and leads to vesicle poration. Both adhesion and engulfment lead to a decrease in lipid diffusion coefficient from 7 μm2/s on free-standing bilayers to 1 μm2/s on the droplet-supported bilayer. Interestingly, the contact line of the membrane-droplet adhesion area is often connected to many lipid nanotubes. The developed synthetic pinocytosis system of GUV-droplet pairs allows detailed investigation of the endocytosis processes of natural cells but with reduced complexity. Future surface modifications of the membrane/droplet interaction have the potential to mimic specific pathogen-recognition interactions. This work is part of the MaxSynBio consortium which is jointly funded by the Federal Ministry of Education and Research of Germany and the Max Planck Society.

How the stimulus defines the dynamics of vesicle pool recruitment, fusion mode, and vesicle recycling in neuroendocrine cells.

The pattern of stimulation defines important characteristics of the secretory process in neurons and neuroendocrine cells, including the pool of secretory vesicles being recruited, the type and amount of transmitters released, the mode of membrane retrieval, and the mechanisms associated with vesicle replenishment. This review analyzes the mechanisms that regulate these processes in chromaffin cells, as well as in other neuroendocrine and neuronal models. A common factor in these mechanisms is the spatial and temporal distribution of the Ca(2+) signal generated during cell stimulation. For instance, neurosecretory cells and neurons have pools of vesicles with different locations with respect to Ca(2+) channels, and those pools are therefore differentially recruited following different patterns of stimulation. In this regard, a brief stimulus will induce the exocytosis of a small pool of vesicles that is highly coupled to voltage-dependent Ca(2+) channels, whereas longer or more intense stimulation will provoke a global Ca(2+) increase, promoting exocytosis irrespective of vesicle location. The pattern of stimulation, and therefore the characteristics of the Ca(2+) signal generated by the stimulus also influence the mode of exocytosis and the type of endocytosis. Indeed, low-frequency stimulation favors kiss-and-run exocytosis and clathrin-independent fast endocytosis, whereas higher frequencies promote full fusion and clathrin-dependent endocytosis. This latter type of endocytosis is accelerated at high-frequency stimulation. Synaptotagmins, calcineurin, dynamin, complexin, and actin remodeling, appear to be involved in the mechanisms that determine the response of these processes to Ca(2+) . In chromaffin cells, a brief stimulus induces the exocytosis of a small pool of vesicles that is highly coupled to voltage-dependent Ca(2+) channels (A), whereas longer or high-frequency stimulation provokes a global Ca(2+) increase, promoting exocytosis irrespective of vesicle location (B). Furthermore, low-frequency stimulation favors kiss-and-run exocytosis (A), whereas higher frequencies promote full fusion (B). In this review, we analyze the mechanisms by which a given stimulation pattern defines the mode of exocytosis, and recruitment and recycling of neurosecretory vesicles. This article is part of a mini review series on Chromaffin cells (ISCCB Meeting, 2015).

Abstract 5257: Cancer cells have not only sweet tooth but also large mouths: Evidence for uptake of ATP into KRas oncogene-expressing cancer cells by macropinocytosis

Abstract Cancer cells are known to exhibit reprogrammed metabolism and prefer to use glycolygsis for their ATP synthesis even when oxygen is abundant, a phenomenon called the Warburg effect. However, cancer cells are also known for more rapid growth and proliferation, therefore requiring more ATP than normal cells of the same tissues. How cancer cells manage to use low efficiency glycolysis but still meet their higher ATP demands is enigmatic. In our previous study, we found that extracellular ATP induced large intracellular ATP increase and drug resistance to tyrosine kinase inhibitors (TKIs) that function as ATP analogs / competitors in cancer cells. The induced ATP increase was from neither OXPHOS nor glycolysis, and was not dependent on AMPK, the master ATP regulator. Furthermore, the increase did not involve new gene expression or new protein synthesis. Recently, it was reported that cells transformed with an oncogenic form of KRas gene (KRas+) exhibit a phenotype of macropinocytosis, a type of endocytosis. KRas-induced macropinocytosis nonspecifically takes up extracellular proteins. All these led us to hypothesize that the increased intracellular ATP induced by extracellular ATP is, at least in part, not produced intracellularly but is transported into cancer cells. To test this hypothesis, high molecular weight fluorescent dextran was use to determine if KRas+ human non-small cell lung cancer A549 cells exhibit macropinocytosis. Nonhydrolyzable fluorescent ATP analog was used to visualize if the ATP analog can be transported into A549 cells along with dextran. Fluorescence microscopy studies revealed that A549 cells exhibit the phenotype of macropinocytosis, which can be reduced by a macropinocytosis inhibitor EIPA. HMW dextran and the nonhydrolysable fluorescent ATP analog were found co-localized inside A549 cells, demonstrating that they were transported into A549 cells via macropinosomes. It was also found that the ATP increase cannot be explained by purinergic receptor signaling alone. In comparison, KRas- human breast cancer MCF7 cells do not take up HMW dextran, indicating that they do not exhibit macropinocytosis. All these provide strong and visual evidence that ATP can be transported into KRas+ A549 cancer cells by macropinocytosis. Other experiments using radioactive ATP or purinergic receptor inhibitors also strongly support this conclusion. This is the first time that ATP (ATP analog) is shown to be taken into cancer cells. This finding will have major impact on how we view cancer cells in terms of their energy exchange with other cancer or noncancer cells in the tumor and on the Warburg effect. It will also have profound implications on how to more effectively inhibiting KRas+ or KRas- cancer cells by targeting ATP transport and ATP metabolism. Citation Format: Yanrong Qian, Xuan Wang, Yi Liu, Yunsheng Li, Robert A. Colvin, Xiaozhuo Chen. Cancer cells have not only sweet tooth but also large mouths: Evidence for uptake of ATP into KRas oncogene-expressing cancer cells by macropinocytosis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5257. doi:10.1158/1538-7445.AM2014-5257

Abstract 3364: Extracellular ATP-induced intracellular ATP concentration elevation mediated by macropinocytosis promotes growth, survival, and drug resistance of cancer cells

Abstract Cancer cells switch from highly efficient mitochondrial oxidative phosphorylation (OXPHOS) to low efficiency glycolysis for ATP synthesis even when oxygen is abundant, a phenomenon called the Warburg effect. Because of the higher rates of cell growth and proliferation, cancer cells need more ATP than normal cells of the same tissues. However, the switch is counterintuitive since cancer cells need more ATP but cancer cells prefer to use a low efficiency pathway to produce ATP. How cancer cells secure all their ATP needs is not well understood. On the other hand, extracellular ATP concentration in cancer (intratumoral ATP concentration) are found to be in the range of several hundred μM, ∼10^3-10^4 times higher than those found in normal tissues. Huge differences exist between cancer and normal cells regarding extracellular ATP. Based on these observations, we hypothesized that cancer cells take up ATP from extracellular space to supplement their energy needs. Here we report that in multiple cancer cell lines belonging to multiple cancer types, 0.5-3 mM ATP, within the intratumoral ATP concentration range, elevated intracellular ATP levels by more than 50%. Extracellular ATP also reduced stress and promoted survival of cancer cells that were under OXPHOS and glucose metabolism inhibitions. Furthermore, extracellular ATP increased the viability of cancer cells treated with sunitinib or pazopanib, anticancer drugs working as ATP analogs by competing with ATP for the ATP binding site of targeted tyrosine kinases (TKs). In contrast, extracellular ATP did not affect the activity of paclitaxel, a drug unrelated to ATP. Inhibitor studies revealed that the extracellular ATP-induced intracellular ATP increase shows no dependence on OXPHOS, glycolysis, master ATP regulator AMPK, transcription or translation. These results strongly suggest that not all the increased intracellular ATP is synthesized inside of cancer cells. Fluorescence microscopy study showed that human lung cancer A549 cells, which are KRas oncogene mutation-positive (KRas+), exhibit macropinotcytosis, a type of endocytosis that non-specifically take in extracellular nutritional molecules by “fluid drinking”. Inhibition of macropinocytosis resulted in decrease in dextran take-up and intracellular ATP levels, and alleviated the drug resistance to TKIs in dose-dependent manners. These strongly suggest that macropinocytosis is responsible, at least in part, for transporting extracellular ATP into cancer cells, which led to drug resistance. All these results demonstrate, for the first time, unrecognized roles of extracellular ATP in contributing to the intracellular ATP pool and increase drug resistance in cancer cells and suggest a novel ATP transport mechanism. These findings challenge our traditional view on ATP and shed new lights on ATP homeostasis and ATP-induced drug resistance in cancer cells. Citation Format: Yanrong Qian, Xuan Wang, Yi Liu, Yunsheng Li, Robert A. Colvin, Lingying Tong, Shiyong Wu, Xiaozhuo Chen. Extracellular ATP-induced intracellular ATP concentration elevation mediated by macropinocytosis promotes growth, survival, and drug resistance of cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3364. doi:10.1158/1538-7445.AM2014-3364

Characterization of endocytic uptake of MK2‐inhibitor peptides

Cell penetrating peptides (CPP) have been widely used to increase the cellular delivery of their associated cargo. Multiple modes of uptake have been identified; however, they cannot be predicted a priori. Elucidating these mechanisms is important for understanding peptide function as well as further optimizing cellular delivery. We have developed a class of mitogen activated protein kinase activated protein kinase 2 (MK2) inhibitor peptides, named FAK and YARA that utilize CPP domains to gain cellular access. In this study, we investigate the mechanism of endocytosis of these MK2 inhibitors by examining the uptake of fluorescently labeled peptide in human monocyte (THP-1) and mesothelial cells, and looking for colocalization with known markers of endocytosis. Our results indicate that uptake of the MK2 inhibitors was minimally enhanced by the addition of the fluorescent label, and that the type of endocytosis used by the inhibitor depends on several factors including concentration, cell type, and which CPP was used. We found that in THP-1 cells, the uptake of YARA occurred primarily via macropinocytosis, whereas FAK entered via all three mechanisms of endocytosis examined in this study. In mesothelial cells, uptake of YARA occurred via caveolae-mediated endocytosis, but became less specific at higher concentrations; whereas uptake of FAK occurred through clathrin-mediated endocytosis. In all cases, the delivery resulted in active inhibition of MK2. In summary, the results support endocytic uptake of fluorescently labeled FAK and YARA in two different cell lines, with the mechanism of uptake dependent on extracellular concentration, cell type, and choice of CPP.

Proton-induced endocytosis is dependent on cell membrane fluidity, lipid-phase order and the membrane resting potential

Recently it has been shown that decreasing the extracellular pH of cells stimulates the formation of inward membrane invaginations and vesicles, accompanied by an enhanced uptake of macromolecules. This type of endocytosis was coined as proton-induced uptake (PIU). Though the initial induction of inward membrane curvature was rationalized in terms of proton-based increase of charge asymmetry across the membrane, the dependence of the phenomenon on plasma membrane characteristics is still unknown. The present study shows that depolarization of the membrane resting potential elevates PIU by 25%, while hyperpolarization attenuates it by 25%. Comparison of uptake in suspended and adherent cells implicates that the resting-potential affects PIU through remodeling the actin-cytoskeleton. The pH at the external interface of the cell membrane rather than the pH gradient across it determines the extent of PIU. PIU increases linearly upon temperature increase in the range of 4–36°C, in correlation with the membrane fluidity. The plasma membrane fluidity and the lipid phase order are modulated by enriching the cell's membrane with cholesterol, tergitol, dimethylsulfoxide, 6-ketocholestanol and phloretin and by cholesterol depletion. These treatments are shown to alter the extent of PIU and are better correlated with membrane fluidity than with the lipid phase order. We suggest that the lipid phase order and fluidity influence PIU by regulating the lipid order gradient across the perimeter of the lipid-condensed microdomains (rafts) and alter the characteristic tension line that separates the higher ordered lipid-domains from the lesser ordered ones.

Symbiotic nitrogen fixation in legume nodules: process and signaling. A review

The Green Revolution was accompanied by a huge increase in the application of fertilizers, particularly nitrogen. Recent studies indicate that a sizeable proportion of the human population depends on synthetic nitrogen (N) fertilizers to provide the 53 million t N that is harvested globally in food crops each year. Nitrogen fertilizers affect the balance of the global nitrogen cycle, pollute groundwater and increase atmospheric nitrous oxide (N2O), a potent “greenhouse” gas. The production of nitrogen fertilizer by industrial nitrogen fixation not only depletes our finite reserves of fossil fuels, but also generates large quantities of carbon dioxide, contributing to global warming. The process of biological nitrogen fixation offers an economically attractive and ecologically sound means of reducing external nitrogen input and improving the quality and quantity of internal resources. Recent studies show that in irrigated cropping systems, legume N is generally less susceptible to loss processes than fertilizers. Biological nitrogen fixation (BNF) has provided a number of useful paradigms for both basic and applied research. Establishing a fully functional symbiosis requires a successful completion of numerous steps that lead from recognition signals exchanged between the plant and bacteria to the differentiation and operation of root nodules, the plant organ in which nitrogen fixation takes place. The initial sensing of the two organisms by each other starts with the release of root exudates by the plant that include flavonoids and nutrients such as organic acids and amino acids. Flavonoids secreted by the host plant into the rhizosphere function as inducers of the rhizobial nod genes. nod gene induction results in the secretion of lipochitin oligosaccharides that are thought to bind to specific plant receptor kinases that contain LysM motifs, such as NFR1 and NFR5 in Lotus japonicus and LYK3 and LTK4 in Medicago truncatula. This initiates a complex signaling pathway involving calcium spiking in root hairs. The result is that the root hairs curl and trap the rhizobia, which then enter the root hair through tubular structures known as infection threads that are formed by the plant. The infection threads then grow into the developed nodule tissue. Ultimately, the invading bacteria are taken into the plant cell by a type of endocytosis in which they are surrounded by a plant-derived peribacteroid membrane (PBM). The resulting symbiosomes fill the plant cell cytoplasm and as plant and bacterial metabolism develops, the bacteria become mature bacteroids able to convert atmospheric nitrogen to ammonium. To increase knowledge of this system of particular importance in sustainable agriculture, major emphasis should be laid on the basic research. More work is needed on the genes responsible in rhizobia and legumes, the structural chemical bases of rhizobia/legume communication, and signal transduction pathways responsible for the finely orchestrated induction of the symbiosis-specific genes involved in nodule development and nitrogen fixation. This review unfolds the various events involved in the progression of symbiosis.

Mechanistic aspects of CPP-mediated intracellular drug delivery: Relevance of CPP self-assembly

In recent years, cell-penetrating peptides have proven to be an efficient intracellular delivery system. The mechanism for CPP internalisation, which first involves interaction with the extracellular matrix, is followed in most cases by endocytosis and finally, depending on the type of endocytosis, an intracellular fate is reached. Delivery of cargo attached to a CPP requires endosomal release, for which different methods have recently been proposed. Positively charged amino acids, hydrophobicity and/or amphipathicity are common to CPPs. Moreover, some CPPs can self-assemble. Herein is discussed the role of self assembly in the cellular uptake of CPPs. Sweet Arrow Peptide (SAP) CPP has been shown to aggregate by CD and TEM (freeze-fixation/freeze-drying), although the internalised species have yet to be identified as either the monomer or an aggregate.

Cell-penetrating peptides are excluded from the mitochondrial matrix

CPPs (cell-penetrating peptides) facilitate cellular uptake of covalently attached macromolecules, through an as yet controversial mechanism that either involves direct membrane passage or a type of endocytosis. We investigated the potential of the CPPs penetratin and Tat to act as mitochondria-targeting vectors by testing whether they were internalized by isolated mitochondria, and by mitochondria within cells in culture. We also tested peptides conjugated to the mitochondria-targeting moiety triphenylphosphonium. We found no evidence for mitochondrial uptake by penetratin, Tat or their triphenylphosphonium conjugates. This result suggests that CPPs are unsuitable as mitochondria-targeting vectors, and implies an endocytic mode of cellular uptake for CPPs.