Role Of Macropinocytosis Research Articles

The Expression of Macropinocytosis-Related Genes in Ovarian Cancer and Their Relationships with Prognosis

Background: The mechanism of macropinocytosis has been reported in receptor sorting used by motile cells. Besides, the role of macropinocytosis was previously recognized in cancer progression. We evaluated the prognostic value of macropinocytosis gene expression in ovarian cancer (OC).

Abstract PO-130: Macropinocytosis at the nexus of crosstalk in the pancreatic tumor microenvironment

Abstract A feature of tumor cells is the acquisition of nutrients via macropinocytosis, an endocytic form of protein scavenging that functions to support metabolism and proliferation in the context of a nutrient-depleted tumor microenvironment. Here, we provide evidence that macropinocytosis is also operational in the tumor stroma. We find that glutamine deficiency triggers macropinocytic uptake in patient-derived cancer-associated fibroblasts (CAFs) from pancreatic ductal adenocarcinoma (PDAC) tumors. Mechanistically, stromal macropinocytosis is dependent on a CaMKK2-AMPK signaling axis that activates Rac1. We find that these signaling events are potentiated via an enhancement of cytosolic Ca2+ caused by glutamine limitation. Interestingly, glucose deprivation, which robustly enhances AMPK activation, does not activate Rac1 and fails to induce uptake in CAFs. We determine that the selectivity of the observed macropinocytic response is mediated by the glutamine depletion-induced upregulation of ARHGEF2, a Rho/Rac guanine nucleotide exchange factor. We elucidate that macropinocytosis has dual function in CAFs – it serves as a source of intracellular amino acids that sustain CAF viability, and it provides extracellular amino acids that promote tumor cell survival. By specifically ablating macropinocytosis in the stroma, we demonstrate that macropinocytosis-deficient CAFs are unable to enhance PDAC tumor growth. Current efforts are focused on how different metabolic inputs in the tumor microenviroment can differentially regulate the CaMKK2-AMPK signaling axis in stromal cells. These results highlight the functional role of macropinocytosis in the tumor stroma and provide a mechanistic understanding of how nutrient deficiency can control stromal protein scavenging. Citation Format: Yijuan Zhang, M. Victoria Recouvreux, Michael Jung, Koen Galencamp, Yunbo Li, Olga Zagnitko, David Scott, Andrew Lowy, Cosimo Commisso. Macropinocytosis at the nexus of crosstalk in the pancreatic tumor microenvironment [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-130.

KRAS-Mutated Myeloma Cells Exploit Autophagy and Macropinocytosis to Tolerate Glutamine-Deprived Conditions

Introduction: Sustained cancer cell growth requires up-regulation of nutrient acquisition mechanisms. Novel approaches can identify “tumor fuel”, the way it is obtained and then altered for cellular usage. Autophagy is a conserved catabolic process that is thought to replenish nutrient pool and thereby starvation survival by providing bioenergetic and biosynthetic substrates. Some KRAS-mutated cancers show heightened dependency to glutamine as a source of energy. Since a large proportion of myeloma tumors are KRAS-mutated, we investigated in the present study the contribution of macropinocytosis and autophagy as the cellular mechanisms involved in protein uptake and degradation, respectively, to growth and survival of myeloma cells under glutamine-deprived condition.Methods: Macropinosomes were visualized using confocal microscopy in KRAS-mutated and WT human myeloma cells utilising TMR-dextran and 5-[N-ethyl-N-isopropyl] amiloride (EIPA) as a marker and specific inhibitor of macropinocytosis, respectively. Internalization and degradation of labelled albumin and its co-localization with TMR-dextran were evaluated in parallel. Bafilomycin was employed to evaluate the dependency of protein degradation on autophagy. Cell sensitivity to glutamine deprivation was examined with an ATP-based viability test. To evaluate the possible role of macropinocytosis and autophagy at glutamine deprivation conditions, the effect of albumin supplementation on cell growth and viability was studied in the presence or absence of EIPA or chloroquine. To test whether the degradation of macropinocytosed albumin results in the production of intracellular glutamine, the intracellular concentrations of glutamine was directly measured.Results: KRAS-mutated KMS28-PE, KMS18 and MM1S cells showed higher levels of EIPA-sensitive macropinocytosis compared to WT-KRAS KMS34, KMS12-BM and TK1 cells. Macropinocytosis led to the internalization of albumin which subsequently underwent bafilomycin-sensitive proteolytic degradation in KRAS-mutated cells. The viability of WT-KRAS cells was reduced in sub-physiological concentrations of glutamine when compared with KRAS-mutated cells that not only remained viable but could continue to slowly proliferate over a period of 7 days (p˂0.05). The compromised growth rate of KRAS-mutated cells at very low concentrations of glutamine was rescued by albumin and this effect was abrogated by EIPA or chloroquine (p˂0.05). The KRAS-mutated cells demonstrated an EIPA-sensitive increase in intracellular concentration of glutamine following albumin supplementation (p˂0.05) that was abrogated following the treatment with chloroquine. In WT cells, in contrast, the level of intracellular glutamine remained almost unchanged following albumin supplementation.Conclusions: Autophagy and macropinocytosis maintain cellular growth and survival in the context of nutrient deprivation by provision of glutamine, and potentially other amino acids, in RAS-mutated myeloma cancer. These data suggest that novel anti-cancer strategies interrupting these cellular mechanisms may represent a promising therapeutic approach to myeloma. DisclosuresSpencer:Janssen: Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding.

Visualizing Membrane Ruffle Formation using Scanning Electron Microscopy.

Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may form spontaneously or in response to growth factors, inflammatory cytokines, and phorbol esters. Some of the membrane protrusions may reorganize into circular membrane ruffles that fuse at their distal margins and form cups that close and separate into the cytoplasm as large, heterogeneous vacuoles called macropinosomes. During the process, ruffles trap extracellular fluid and solutes that internalize within macropinosomes. High-resolution scanning electron microscopy (SEM) is a commonly used imaging technique to visualize and quantify membrane ruffle formation, circular protrusions, and closed macropinocytic cups on the cell surface. The following protocol describes the cell culture conditions, stimulation of the membrane ruffle formation in vitro, and how to fix, dehydrate, and prepare cells for imaging using SEM. Quantification of membrane ruffling, data normalization, and stimulators and inhibitors of membrane ruffle formation are also described. This method can help answer key questions about the role of macropinocytosis in physiological and pathological processes, investigate new targets that regulate membrane ruffle formation, and identify yet uncharacterized physiological stimulators as well as novel pharmacological inhibitors of macropinocytosis.

Autophagy-Associated Proteins Control Ebola Virus Internalization Into Host Cells.

Ebola virus (EBOV) enters host cells by macropinocytosis, a poorly understood process. Recent studies have suggested that cell factors involved in autophagy, an evolutionally conserved pathway leading to the lysosomal degradation of protein aggregates and organelles during cellular stress, also have roles in macropinocytosis. Here, we demonstrate that autophagy-associated proteins are required for trafficking of EBOV into the cell body. Depleting cells of beclin 1, autophagy-related protein 7, or microtubule-associated protein 1A/B light chain 3B (LC3B) abolished EBOV uptake, owing to a block in vesicle formation at the cell surface. Both LC3B-I and LC3B-II interacted with macropinocytic structures. Our work indicates that, although various forms of LC3B possess an inherent ability to associate with forming macropinosomes, LC3B-II is critical for internalization of macropinocytic vesicles and, therefore, EBOV from the cell surface.

Chlamydia exploits filopodial capture and a macropinocytosis-like pathway for host cell entry.

Pathogens hijack host endocytic pathways to force their own entry into eukaryotic target cells. Many bacteria either exploit receptor-mediated zippering or inject virulence proteins directly to trigger membrane reorganisation and cytoskeletal rearrangements. By contrast, extracellular C. trachomatis elementary bodies (EBs) apparently employ facets of both the zipper and trigger mechanisms and are only ~400 nm in diameter. Our cryo-electron tomography of C. trachomatis entry revealed an unexpectedly diverse array of host structures in association with invading EBs, suggesting internalisation may progress by multiple, potentially redundant routes or several sequential events within a single pathway. Here we performed quantitative analysis of actin organisation at chlamydial entry foci, highlighting filopodial capture and phagocytic cups as dominant and conserved morphological structures early during internalisation. We applied inhibitor-based screening and employed reporters to systematically assay and visualise the spatio-temporal contribution of diverse endocytic signalling mediators to C. trachomatis entry. In addition to the recognised roles of the Rac1 GTPase and its associated nucleation-promoting factor (NPF) WAVE, our data revealed an additional unrecognised pathway sharing key hallmarks of macropinocytosis: i) amiloride sensitivity, ii) fluid-phase uptake, iii) recruitment and activity of the NPF N-WASP, and iv) the localised generation of phosphoinositide-3-phosphate (PI3P) species. Given their central role in macropinocytosis and affinity for PI3P, we assessed the role of SNX-PX-BAR family proteins. Strikingly, SNX9 was specifically and transiently enriched at C. trachomatis entry foci. SNX9-/- cells exhibited a 20% defect in EB entry, which was enhanced to 60% when the cells were infected without sedimentation-induced EB adhesion, consistent with a defect in initial EB-host interaction. Correspondingly, filopodial capture of C. trachomatis EBs was specifically attenuated in SNX9-/- cells, implicating SNX9 as a central host mediator of filopodial capture early during chlamydial entry. Our findings identify an unanticipated complexity of signalling underpinning cell entry by this major human pathogen, and suggest intriguing parallels with viral entry mechanisms.

Abstract 100: High-throughput Screening of FDA-approved Drugs Identifies Novel Inhibitors of Macropinocytosis

Aims: Macropinocytosis has been implicated in atherosclerosis, cancer, allergic disorders, and other pathologies. Unfortunately, most currently available pharmacological inhibitors of macropinocytosis interrupt other endocytic processes and have non-specific endocytosis-independent effects. The goal of the present study was to perform a high-throughput screen (HTS) of FDA-approved drugs to identify new, clinically relevant inhibitors of macropinocytosis. Methods: In the present study, 640 FDA-approved compounds were tested for their ability to inhibit macropinocytosis. A series of secondary assays were performed to confirm inhibitory activity, determine IC 50 values, and investigate cell toxicity. The ability of identified hits to inhibit phagocytosis, clathrin- and caveolin-mediated endocytosis was also investigated. Scanning electron microscopy and molecular biology techniques were utilized to investigate the mechanisms by which selected compounds inhibit macropinocytosis. Results: The HTS campaign identified 14 compounds that at ~10 μM concentration inhibit >95% of macropinocytotic solute internalization. Our results demonstrated that three lead compounds, namely imipramine, phenoxybenzamine, and vinblastine, potently inhibit (IC 50 ≤ 130 nM) macropinocytosis without exerting cytotoxic effects or inhibiting other endocytic pathways. Mechanistically, we found that imipramine inhibits translocation of ADP-ribosylation factor 6 to the plasma membrane and prevents membrane ruffle formation, a critical early step leading to macropinocytosis. Imipramine inhibited macropinocytosis in multiple cell types, including cancer cells, dendritic cells, and macrophages. Finally, incubation of macrophages with imipramine inhibited nLDL macropinocytosis and foam cell formation in vitro . Innovation and Conclusion: The identified macropinocytosis inhibitors may prove useful as new pharmacological tools to more fully discern the role of macropinocytosis in pathological processes and as therapeutic agents in various disorders involving macropinocytosis.

Blebbishields and mitotic cells exhibit robust macropinocytosis.

Cancer stem cells can survive and undergo transformation after apoptosis by initiating robust endocytosis. Endocytosis in-turn drives formation of serpentine filopodia, which promote construction of blebbishields from apoptotic bodies. However, the status and role of macropinocytosis in blebbishields is not known. Here, we show by scanning electron microscopy and by macropinocytosis assays that blebbishields exhibit robust macropinocytosis. Inhibiting dynamin-mediated endocytosis does not affect macropinocytosis in blebbishields or in mitotic cells. In addition, inhibiting macropinocytosis did not inhibit construction of blebbishields from apoptotic bodies. Thus, although apoptotic cancer stem cells exhibit robust macropinocytosis, macropinocytosis is not essential to generate blebbishields, although it may play other roles in blebbishield biology. © 2016 BioFactors, 43(2):181-186, 2017.

The role of macropinocytosis in the propagation of protein aggregation associated with neurodegenerative diseases.

With the onset of the rapidly aging population, the impact of age related neurodegenerative diseases is becoming a predominant health and economic concern. Neurodegenerative diseases such as Alzheimer's disease, Creutzfeldt-Jakob disease (CJD), Parkinson's disease, Huntington's disease, frontotemporal dementia (FTD), and amyotrophic lateral sclerosis (ALS) result from the loss of a specific subsets of neurons, which is closely associated with accumulation and deposition of specific protein aggregates. Protein aggregation, or fibril formation, is a well-studied phenomenon that occurs in a nucleation-dependent growth reaction. Recently, there has been a swell of literature implicating protein aggregation and its ability to propagate cell-to-cell in the rapid progression of these diseases. In order for protein aggregation to be kindled in recipient cells it is a requisite that aggregates must be able to be released from one cell and then taken up by others. In this article we will explore the relationship between protein aggregates, their propagation and the role of macropinocytosis in their uptake. We highlight the ability of neurons to undergo stimulated macropinocytosis and identify potential therapeutic targets.

Delivery of CSF-1R to the lumen of macropinosomes promotes its destruction in macrophages.

Activation of the macrophage colony stimulating factor-1 receptor (CSF-1R) by CSF-1 stimulates pronounced macropinocytosis and drives proliferation of macrophages. Although the role of macropinocytosis in CSF-1R signaling remains unknown, we show here that, despite internalizing large quantities of plasma membrane, macropinosomes contribute little to the internalization of the CSF-1-CSF-1R complex. Rather, internalization of the CSF-1R in small endocytic vesicles that are sensitive to clathrin disruption, outcompetes macropinosomes for CSF-1R endocytosis. Following internalization, small vesicles carrying the CSF-1R underwent homotypic fusion and then trafficked to newly formed macropinosomes bearing Rab5. As these macropinosomes matured, acquiring Rab7, the CSF-1R was transported into their lumen and degraded. Inhibition of macropinocytosis delayed receptor degradation despite no disruption to CSF-1R endocytosis. These data indicate that CSF-1-stimulated macropinosomes are sites of multivesicular body formation and accelerate CSF-1R degradation. Furthermore, we demonstrate that macropinocytosis and cell growth have a matching dose dependence on CSF-1, suggesting that macropinosomes might be a central mechanism coupling CSF-1R signaling and macrophage growth.

Rabankyrin‐5 Interacts with EHD1 and Vps26 to Regulate Endocytic Trafficking and Retromer Function

Rabankyrin-5 (Rank-5) has been implicated as an effector of the small GTPase Rab5 and plays an important role in macropinocytosis. We have now identified Rank-5 as an interaction partner for the recycling regulatory protein, Eps15 homology domain 1 (EHD1). We have demonstrated this interaction by glutathione S-transferase-pulldown, yeast two-hybrid assay, isothermal calorimetry and co-immunoprecipitation, and found that the binding occurs between the EH domain of EHD1 and the NPFED motif of Rank-5. Similar to EHD1, we found that Rank-5 colocalizes and interacts with components of the retromer complex such as vacuolar protein sorting 26 (Vps26), suggesting a role for Rank-5 in retromer-based transport. Indeed, depletion of Rank-5 causes mislocalization of Vps26 and affects both the retrieval of mannose 6-phosphate receptor transport to the Golgi from endosomes and biosynthetic transport. Moreover, Rank-5 is required for normal retromer distribution, as overexpression of a wild-type Rank-5-small interfering RNA-resistant construct rescues retromer mislocalization. Finally, we show that depletion of either Rank-5 or EHD1 impairs secretion of vesicular stomatitis virus glycoprotein. Overall, our data identify a new interaction between Rank-5 and EHD1, and novel endocytic regulatory roles that include retromer-based transport and secretion.

Dictyostelium discoideum Nucleoside Diphosphate Kinase C Plays a Negative Regulatory Role in Phagocytosis, Macropinocytosis and Exocytosis

Nucleoside diphosphate kinases (NDPKs) are ubiquitous phosphotransfer enzymes responsible for producing most of the nucleoside triphosphates except for ATP. This role is important for the synthesis of nucleic acids and proteins and the metabolism of sugars and lipids. Apart from this housekeeping role NDPKs have been shown to have many regulatory functions in diverse cellular processes including proliferation and endocytosis. Although the protein has been shown to have a positive regulatory role in clathrin- and dynamin-mediated micropinocytosis, its roles in macropinocytosis and phagocytosis have not been studied. The additional non-housekeeping roles of NDPK are often independent of enzyme activity but dependent on the expression level of the protein. In this study we altered the expression level of NDPK in the model eukaryotic organism Dictyostelium discoideum through antisense inhibition and overexpression. We demonstrate that NDPK levels affect growth, endocytosis and exocytosis. In particular we find that Dictyostelium NDPK negatively regulates endocytosis in contrast to the positive regulatory role identified in higher eukaryotes. This can be explained by the differences in types of endocytosis that have been studied in the different systems - phagocytosis and macropinocytosis in Dictyostelium compared with micropinocytosis in mammalian cells. This is the first report of a role for NDPK in regulating macropinocytosis and phagocytosis, the former being the major fluid phase uptake mechanism for macrophages, dendritic cells and other (non dendritic) cells exposed to growth factors.

Integrin Stimulation Favors Uptake of Macromolecules by Cardiomyocytes In Vitro

Previously, our research group showed that integrin stimulation induces release of cardiac troponin I from viable neonatal rat ventricular cardiomyocytes (NRCMs), but would it also stimulate uptake of exogenous macromolecules? For this purpose, beating NRCMs were incubated without or with an RGD motif-containing peptide (GRGDS) to stimulate integrins in the presence of Texas Red-conjugated ovalbumin (OTR; 45 kDa) or dextran (DTR; 70 kDa). After incubation periods of 8, 16 and 24 hours endocytosis of red label was quantified by fluorescence microscopy. Uptake of OTR and DTR by NRCMs was intensified by GRGDS treatment (p for trend <0.001 and 0.019, respectively) and increased with duration of incubation (p<0.001 for both). The GRGDS-induced uptake of OTR by NRCMs correlated positively with OTR concentration (p<0.001). Experiments with pharmacological inhibitors of endocytosis indicated that in the absence of GRGDS, NRCMs take up OTR by the clathrin-mediated pathway of endocytosis while the GRGDS-dependent OTR uptake occurs by macropinocytosis. Cultures of NRCMs that were stretched cyclically showed ≈4-fold increased uptake of OTR compared to stationary NRCM cultures. Immunofluorescence microscopy revealed that the dysferlin-positive plasma membrane (PM) areas in beating GRGDS-treated NRCMs were ≈3-fold larger than in contracting NRCMs incubated with vehicle (p<0.001). However, in non-beating NRCMs exposure to GRGDS did not induce larger dysferlin-positive PM areas, nor did it stimulate uptake of OTR. After inhibition of dysferlin expression by short hairpin RNA-mediated RNA interference, OTR uptake by contracting NRCMs could no longer be stimulated via GRGDS treatment. We conclude that in NRCMs, stimulation of integrins by RGD motif-containing peptides or stretch cause uptake of labeled macromolecules. The latter process appears to depend on the contractile behavior of the NRCMs and on the PM repair protein dysferlin, probably because of its role in macropinocytosis.

The mechanism of cell uptake for luminescent lanthanide optical probes: the role of macropinocytosis and the effect of enhanced membrane permeability on compartmentalisation

Inhibition studies and co-localisation experiments with several emissive lanthanide complexes reveal cell uptake by macropinocytosis.

An amino acid substitution in PBP-3 in Haemophilus influenzae associate with the invasion to bronchial epithelial cells

Haemophilus influenzae is a common pathogen of respiratory infections. We examined whether beta-lactamase-negative ampicillin-resistant (BLNAR) strains that are known to have ampicillin resistance due to a substitution of amino acid of penicillin binding protein (PBP)-3, differ from beta-lactamase-negative ampicillin-susceptible strains with regard to invasion of bronchial epithelium. After 3h incubation of each of 34 beta-lactamase-negative ampicillin-susceptible and 57 BLNAR strains in the presence of BEAS-2B cells, a human bronchial epithelium cell line, extracellular bacteria were killed using gentamicin and intracellular bacteria numbered. All nine strains in which the efficiency of invasion was 1% or higher were BLNAR strains. The rate of invasion was significantly greater in strains with PBP-3 amino acid substitution (Met377 to Ile, Ser385 to Thr, Leu389 to Phe, and Asn526 to Lys) (n=34) than in those with no amino acid substitution. Electron microscopy showed that high invasive BLNAR strains were observed in cytoplasm of BEAS-2B cell layer. The injured cells were 9.44+/-1.76% among attaching cells examined by trypan blue staining after 6h. These data may suggest that the amino acid substitution of the PBP in BLNAR strains may at least partly play roles in macropinocytosis, leading to the invasion and injury to epithelial cells.

IQ-domain GTPase-activating Protein 1 Regulates β-Catenin at Membrane Ruffles and Its Role in Macropinocytosis of N-cadherin and Adenomatous Polyposis Coli

Beta-catenin is an integral component of E-cadherin dependent cell-cell junctions. Here we show that beta-catenin co-localizes with IQ-domain GTPase-activating protein 1 (IQGAP1), adenomatous polyposis coli (APC), and N-cadherin at actin-positive membrane ruffles in NIH 3T3 fibroblasts. We used deletion mapping to identify the membrane ruffle-targeting region of beta-catenin, localizing it to amino acids 47-217, which overlap the IQGAP1 binding site. Knockdown by small interference RNA (siRNA) revealed IQGAP1-dependent membrane targeting of beta-catenin, APC, and N-cadherin. Transient overexpression of IQGAP1 or N-cadherin increased beta-catenin at membrane ruffles. IQGAP1/APC regulates cell migration, and using a wound healing assay we demonstrate that siRNA-mediated loss of beta-catenin also caused a modest reduction in the rate of cell migration. More significantly, we discovered that beta-catenin is internalized by Arf6-dependent macropinocytosis near sites of membrane ruffling. The beta-catenin macropinosomes co-stained for APC, N-cadherin, and to a lesser extent IQGAP1, and internalization of each binding partner was abrogated by siRNA-dependent knockdown of beta-catenin. In addition, beta-catenin macropinosomes co-localized with the lysosomal marker, lysosome associated membrane protein 1, consistent with their recycling by the late endosomal machinery. Our findings expand on current knowledge of beta-catenin function. We propose that in motile cells beta-catenin is recruited by IQGAP1 and N-cadherin to active membrane ruffles, wherein beta-catenin mediates the internalization and possible recycling of the membrane-associated proteins N-cadherin and APC.

Novel Dictyostelium unconventional myosin, MyoM, has a putative RhoGEF domain

We have cloned a novel unconventional myosin gene myoM in Dictyostelium. Phylogenetic analysis of the motor domain indicated that MyoM does not belong to any known subclass of the myosin superfamily. Following the motor domain, two calmodulin-binding IQ motifs, a putative coiled-coil region, and a Pro, Ser and Thr-rich domain, lies a combination of dbl homology and pleckstrin homology domains. These are conserved in Rho GDP/GTP exchange factors (RhoGEFs). We have identified for the first time the RhoGEF domain in the myosin sequences. The growth and terminal developmental phenotype of Dictyostelium cells were not affected by the myoM − mutation. Green fluorescent protein-tagged MyoM, however, accumulated at crown-shaped projections and membranes of phase lucent vesicles in growing cells, suggesting its possible roles in macropinocytosis.