A transcription factor complex in Dictyostelium enables adaptive changes in macropinocytosis during the growth-to-development transition.

Macropinocytosis, an evolutionarily conserved endocytic pathway, mediates nonselective bulk uptake of extracellular fluid. It is the primary route for axenic Dictyostelium cells to obtain nutrients and has also emerged as a nutrient-scavenging pathway for mammalian cells. How cells adjust macropinocytic activity in various physiological or developmental contexts remains to be elucidated. We discovered that, in Dictyostelium cells, the transcription factors Hbx5 and MybG form a functional complex in the nucleus to maintain macropinocytic activity during the growth stage. In contrast, during starvation-induced multicellular development, the transcription factor complex undergoes nucleocytoplasmic shuttling in response to oscillatory cyclic adenosine 3',5'-monophosphate (cAMP) signals, which leads to increased cytoplasmic retention of the complex and progressive downregulation of macropinocytosis. Therefore, by coupling macropinocytosis-related gene expression to the cAMP oscillation system, which facilitates long-range cell-cell communication, the dynamic translocation of the Hbx5-MybG complex orchestrates a population-level adjustment of macropinocytic activity to adapt to changing environmental conditions.

Rai14 is a novel interactor of Invariant chain that regulates macropinocytosis.

Invariant chain (Ii, CD74) is a type II transmembrane glycoprotein that acts as a chaperone and facilitates the folding and transport of MHC II chains. By assisting the assembly and subcellular targeting of MHC II complexes, Ii has a wide impact on the functions of antigen-presenting cells such as antigen processing, endocytic maturation, signal transduction, cell migration, and macropinocytosis. Ii is a multifunctional molecule that can alter endocytic traffic and has several interacting molecules. To understand more about Ii's function and to identify further Ii interactors, a yeast two-hybrid screening was performed. Retinoic Acid-Induced 14 (Rai14) was detected as a putative interaction partner, and the interaction was confirmed by co-immunoprecipitation. Rai14 is a poorly characterized protein, which is believed to have a role in actin cytoskeleton and membrane remodeling. In line with this, we found that Rai14 localizes to membrane ruffles, where it forms macropinosomes. Depletion of Rai14 in antigen-presenting cells delays MHC II internalization, affecting macropinocytic activity. Intriguingly, we demonstrated that, similar to Ii, Rai14 is a positive regulator of macropinocytosis and a negative regulator of cell migration, two antagonistic processes in antigen-presenting cells. This antagonism is known to depend on the interaction between myosin II and Ii. Here, we show that Rai14 also binds to myosin II, suggesting that Ii, myosin II, and Rai14 work together to coordinate macropinocytosis and cell motility.

Macropinoscope: Real-Time Simultaneous Tracking of pH and Cathepsin B Activity in Individual Macropinosomes.

A fluorescent sensor that allows simultaneous analysis of environmental factors in a limited cellular space is useful for understanding precise molecular interactions in live cells and their biological responses. Macropinocytosis is a ubiquitous endocytic pathway for massive uptake of extracellular fluids, resulting in the formation of macropinosomes. Although macropinocytosis may impact intracellular delivery and cancer proliferation, information on the intracellular behaviors of macropinosomes is limited. Here, we aimed to develop a macropinoscope, a sensor that simultaneously detects pH and cathepsin B activity in individual macropinosomes. A macropinosome-specific marker, dextran (70 kDa), was employed as a platform, onto which fluorescein, Oregon Green, and tetramethylrhodamine were loaded for ratiometric pH sensing and imaging. A cathepsin-B-cleavable peptide sequence bearing sulfo-Cy5 and the quencher BHQ-3 was also mounted; cleavage of the sequence was detected as an increase in sulfo-Cy5 fluorescence. A steep decrease in pH was observed 5-10 min after macropinosome formation, which was accompanied by an immediate increase in cathepsin B activity. Our design concept will lead to the development of other macropinoscopes for the simultaneous detection of other parameters in individual macropinosomes.

Porcine Enteric Alphacoronavirus Entry through Multiple Pathways (Caveolae, Clathrin, and Macropinocytosis) Requires Rab GTPases for Endosomal Transport.

Porcine enteric alphacoronavirus (PEAV) is a new bat HKU2-like porcine coronavirus, and its endemic outbreak has caused severe economic losses to the pig industry. Its broad cellular tropism suggests a potential risk of cross-species transmission. A limited understanding of PEAV entry mechanisms may hinder a rapid response to potential outbreaks. This study analyzed PEAV entry events using chemical inhibitors, RNA interference, and dominant-negative mutants. PEAV entry into Vero cells depended on three endocytic pathways: caveolae, clathrin, and macropinocytosis. Endocytosis requires dynamin, cholesterol, and a low pH. Rab5, Rab7, and Rab9 GTPases (but not Rab11) regulate PEAV endocytosis. PEAV particles colocalize with EEA1, Rab5, Rab7, Rab9, and Lamp-1, suggesting that PEAV translocates into early endosomes after internalization, and Rab5, Rab7, and Rab9 regulate trafficking to lysosomes before viral genome release. PEAV enters porcine intestinal cells (IPI-2I) through the same endocytic pathway, suggesting that PEAV may enter various cells through multiple endocytic pathways. This study provides new insights into the PEAV life cycle. IMPORTANCE Emerging and reemerging coronaviruses cause severe human and animal epidemics worldwide. PEAV is the first bat-like coronavirus to cause infection in domestic animals. However, the PEAV entry mechanism into host cells remains unknown. This study demonstrates that PEAV enters into Vero or IPI-2I cells through caveola/clathrin-mediated endocytosis and macropinocytosis, which does not require a specific receptor. Subsequently, Rab5, Rab7, and Rab9 regulate PEAV trafficking from early endosomes to lysosomes, which is pH dependent. The results advance our understanding of the disease and help to develop potential new drug targets against PEAV.

C2GAP2 is a common regulator of Ras signaling for chemotaxis, phagocytosis, and macropinocytosis.

Phagocytosis, macropinocytosis, and G protein coupled receptor-mediated chemotaxis are Ras-regulated and actin-driven processes. The common regulator for Ras activity in these three processes remains unknown. Here, we show that C2GAP2, a Ras GTPase activating protein, highly expressed in the vegetative growth state in model organism Dictyostelium. C2GAP2 localizes at the leading edge of chemotaxing cells, phagosomes during phagocytosis, and macropinosomes during micropinocytosis. c2gapB- cells lacking C2GAP2 displayed increased Ras activation upon folic acid stimulation and subsequent impaired chemotaxis in the folic acid gradient. In addition, c2gaB- cells have elevated phagocytosis and macropinocytosis, which subsequently results in faster cell growth. C2GAP2 binds multiple phospholipids on the plasma membrane and the membrane recruitment of C2GAP2 requires calcium. Taken together, we show a shared negative regulator of Ras signaling that mediates Ras signaling for chemotaxis, phagocytosis, and macropinocytosis.

Macropinocytosis and Cancer: From Tumor Stress to Signaling Pathways.

Macropinocytosis is an evolutionarily conserved endocytic pathway that mediates the nonselective acquisition of extracellular material via large endocytic vesicles known as macropinosomes. In addition to other functions, this uptake pathway supports cancer cell metabolism through the uptake of nutrients. Cells harboring oncogene or tumor suppressor mutations are known to display heightened macropinocytosis, which confers to the cancer cells the ability to survive and proliferate despite the nutrient-scarce conditions of the tumor microenvironment. Thus, macropinocytosis is associated with cancer malignancy. Macropinocytic uptake can be induced in cancer cells by different stress stimuli, acting as an adaptive mechanism for the cells to resist stresses in the tumor milieu. Here, we review the cellular stresses that are known to promote macropinocytosis, as well as the underlying molecular mechanisms that drive this process.

Macropinocytosis and Cell Migration: Don't Drink and Drive .

Macropinocytosis is a nonspecific mechanism by which cells compulsively "drink" the surrounding extracellular fluids in order to feed themselves or sample the molecules therein, hence gaining information about their environment. This process is cell-intrinsically incompatible with the migration of many cells, implying that the two functions are antagonistic. The migrating cell uses a molecular switch to stop and explore its surrounding fluid by macropinocytosis, after which it employs the same molecular machinery to start migrating again to examine another location. This cycle of migration/macropinocytosis allows cells to explore tissues, and it is key to a range of physiological processes. Evidence of this evolutionarily conserved antagonism between the two processes can be found in several cell types-immune cells, for example, being particularly adept-and ancient organisms (e.g., the social amoeba Dictyostelium discoideum). How macropinocytosis and migration are negatively coupled is the subject of this chapter.

Signaling Pathways that Regulate Macropinocytosis in Mammalian Cells.

Macropinocytosis is an evolutionarily conserved endocytic pathway that mediates non-selective uptake of extracellular fluid in bulk. Macropinocytosis is initiated by localized polymerization of the actin cytoskeleton, which generates plasma membrane protrusions that enclose part of the environment into large endocytic vesicles. From amoebae to mammalian cells, the actin dynamics that drive macropinosome formation are regulated by a conserved set of intracellular signaling proteins including Ras superfamily GTPases and PI3-kinases. In mammalian cells, multiple upstream signaling pathways control activity of these core regulators in response to cell-extrinsic and cell-intrinsic stimuli. Growth factor signaling pathways play a central role in macropinocytosis induction. In addition, an increasing number of functionally diverse processes has been identified as macropinocytosis regulators, including several nutrient-sensing and developmental signaling pathways. Many of these signaling pathways have proto-oncogenic properties, and their dysregulation drives the high macropinocytic activity that is commonly observed in cancer cells. These regulatory principles illustrate how macropinocytosis is controlled by complex upstream inputs to exert diverse cellular functions in physiological and pathological contexts.

KRAS Addiction Promotes Cancer Cell Adaptation in Harsh Microenvironment Through Macropinocytosis.

KRAS is the most frequently mutated oncogene in cancer and despite intensive studies, attempts to develop effective therapies targeting KRAS or its downstream signaling have failed mostly due to the complexity of KRAS activation and function in cancer initiation and progression. Over the years, KRAS has been involved in several biological processes including cell survival, proliferation, and metabolism by promoting not only a favorable tumor environment but also a cell-microenvironment dialog to allow cancer cells to adapt to tumor microenvironment scarcity. One of the mechanisms involved in this adaption is KRAS-mediated macropinocytosis. Macropinocytosis is an evolutionarily conserved, large-scale, and nonselective form of endocytosis involving actin-driven cell membrane remodeling to engulf large amounts of extracellular fluids and proteins from the local environment. While macropinocytosis process has been known for decades, recent gain interest due to its regulation of KRAS-driven tumor growth in adverse microenvironments. By promoting extracellular protein and other macromolecules internalization, macropinocytosis provides a survival mechanism under nutrient scarce conditions and the potential for unrestricted tumor growth. Thus, a better understanding of macropinocytotic process is needed to develop alternative therapeutic strategies.

KRASG12R-Independent Macropinocytosis in Pancreatic Cancer.

Macropinocytosis is a critical route of nutrient acquisition in pancreatic cancer cells. Constitutive macropinocytosis is promoted by mutant KRAS, which activates the PI3Kα lipid kinase and RAC1, to drive membrane ruffling, macropinosome uptake and processing. However, our recent study on the KRASG12R mutant indicated the presence of a KRAS-independent mode of macropinocytosis in pancreatic cancer cell lines, thereby increasing the complexity of this process. We found that KRASG12R-mutant cell lines promote macropinocytosis independent of KRAS activity using PI3Kγ and RAC1, highlighting the convergence of regulation on RAC signaling. While macropinocytosis has been proposed to be a therapeutic target for the treatment of pancreatic cancer, our studies have underscored how little we understand about the activation and regulation of this metabolic process. Therefore, this review seeks to highlight the differences in macropinocytosis regulation in the two cellular subtypes while also highlighting the features that make the KRASG12R mutant atypical.

Microtopographical guidance of macropinocytic signaling patches.

In fast-moving cells such as amoeba and immune cells, dendritic actin filaments are spatiotemporally regulated to shape large-scale plasma membrane protrusions. Despite their importance in migration, as well as in particle and liquid ingestion, how their dynamics are affected by micrometer-scale features of the contact surface is still poorly understood. Here, through quantitative image analysis of Dictyostelium on microfabricated surfaces, we show that there is a distinct mode of topographical guidance directed by the macropinocytic membrane cup. Unlike other topographical guidance known to date that depends on nanometer-scale curvature sensing protein or stress fibers, the macropinocytic membrane cup is driven by the Ras/PI3K/F-actin signaling patch and its dependency on the micrometer-scale topographical features, namely PI3K/F-actin-independent accumulation of Ras-GTP at the convex curved surface, PI3K-dependent patch propagation along the convex edge, and its actomyosin-dependent constriction at the concave edge. Mathematical model simulations demonstrate that the topographically dependent initiation, in combination with the mutually defining patch patterning and the membrane deformation, gives rise to the topographical guidance. Our results suggest that the macropinocytic cup is a self-enclosing structure that can support liquid ingestion by default; however, in the presence of structured surfaces, it is directed to faithfully trace bent and bifurcating ridges for particle ingestion and cell guidance.

The phosphoinositide coincidence detector Phafin2 promotes macropinocytosis by coordinating actin organisation at forming macropinosomes.

Uptake of large volumes of extracellular fluid by actin-dependent macropinocytosis has an important role in infection, immunity and cancer development. A key question is how actin assembly and disassembly are coordinated around macropinosomes to allow them to form and subsequently pass through the dense actin network underlying the plasma membrane to move towards the cell center for maturation. Here we show that the PH and FYVE domain protein Phafin2 is recruited transiently to newly-formed macropinosomes by a mechanism that involves coincidence detection of PtdIns3P and PtdIns4P. Phafin2 also interacts with actin via its PH domain, and recruitment of Phafin2 coincides with actin reorganization around nascent macropinosomes. Moreover, forced relocalization of Phafin2 to the plasma membrane causes rearrangement of the subcortical actin cytoskeleton. Depletion of Phafin2 inhibits macropinosome internalization and maturation and prevents KRAS-transformed cancer cells from utilizing extracellular protein as an amino acid source. We conclude that Phafin2 promotes macropinocytosis by controlling timely delamination of actin from nascent macropinosomes for their navigation through the dense subcortical actin network.

The structural dynamics of macropinosome formation and PI3-kinase-mediated sealing revealed by lattice light sheet microscopy.

Macropinosomes are formed by shaping actin-rich plasma membrane ruffles into large intracellular organelles in a phosphatidylinositol 3-kinase (PI3K)-coordinated manner. Here, we utilize lattice lightsheet microscopy and image visualization methods to map the three-dimensional structure and dynamics of macropinosome formation relative to PI3K activity. We show that multiple ruffling morphologies produce macropinosomes and that the majority form through collisions of adjacent PI3K-rich ruffles. By combining multiple volumetric representations of the plasma membrane structure and PI3K products, we show that PI3K activity begins early throughout the entire ruffle volume and continues to increase until peak activity concentrates at the base of the ruffle after the macropinosome closes. Additionally, areas of the plasma membrane rich in ruffling had increased PI3K activity and produced many macropinosomes of various sizes. Pharmacologic inhibition of PI3K activity had little effect on the rate and morphology of membrane ruffling, demonstrating that early production of 3'-phosphoinositides within ruffles plays a minor role in regulating their morphology. However, 3'-phosphoinositides are critical for the fusogenic activity that seals ruffles into macropinosomes. Taken together, these data indicate that local PI3K activity is amplified in ruffles and serves as a priming mechanism for closure and sealing of ruffles into macropinosomes.

Chemically Designed Nanoscale Materials for Controlling Cellular Processes.

Nanoparticles are widely used in various biomedical applications as drug delivery carriers, imaging probes, single-molecule tracking/detection probes, artificial chaperones for inhibiting protein aggregation, and photodynamic therapy materials. One key parameter of these applications is the ability of the nanoparticles to enter into the cell cytoplasm, target different subcellular compartments, and control intracellular processes. This is particularly the case because nanoparticles are designed to interact with subcellular components for the required biomedical performance. However, cells are protected from their surroundings by the cell membrane, which exerts strict control over entry of foreign materials. Thus, nanoparticles need to be designed appropriately so that they can readily cross the cell membrane, target subcellular compartments, and control intracellular processes.In the past few decades there have been great advancements in understanding the principles of cellular uptake of foreign materials. In particular, it has been shown that internalization of foreign materials (small molecules, macromolecules, nanoparticles) is size-dependent: endocytotic uptake of materials requires sizes greater than 10 nm, and materials with sizes of 10-100 nm usually enter into cells by energy-dependent endocytosis via biomembrane-coated vesicles. Direct access to the cytosol is limited to very specific conditions, and endosomal escape of material appears to be the most practical approach for intracellular processing.In this Account, we describe how cellular uptake and intracellular processing of nanoscale materials can be controlled by appropriate design of size and surface chemistry. We first describe the cell membrane structure and principles of cellular uptake of foreign materials followed by their subcellular trafficking. Next, we discuss the designed surface chemistry of a 5-50 nm particle that offers preferential lipid-raft/caveolae-mediated endocytosis over clathrin-mediated endocytosis with minimum endosomal/lysosomal trafficking or energy-independent direct cell membrane translocation (without endocytosis) followed by cytosolic delivery without endosomal/lysosomal trafficking. In particular, we emphasize that the zwitterionic-lipophilic surface property of a nanoparticle offers preferential interaction with the lipid raft region of the cell membrane followed by lipid raft uptake, whereas a lower number of affinity biomolecules (<25) on the nanoparticle surface offers caveolae/lipid-raft uptake, while an arginine/guanidinium-terminated surface along with a size of <10 nm offers direct cell membrane translocation. Finally, we discuss how nanoprobes can be designed by adapting these surface chemistry and size preference principles so that they can readily enter into the cell, label different subcellular compartments, and control intracellular processes such as trafficking kinetics, exocytosis, autophagy, amyloid aggregation, and clearance of toxic amyloid aggregates. The Account ends with a Conclusions and Outlook where we discuss a vision for the development of subcellular targeting nanodrugs and imaging nanoprobes by adapting to these surface chemistry principles.

Oncogenic RAS drives the CRAF-dependent extracellular vesicle uptake mechanism coupled with metastasis.

Oncogenic RAS impacts communication between cancer cells and their microenvironment, but it is unclear how this process influences cellular interactions with extracellular vesicles (EVs). This is important as intercellular EV trafficking plays a key role in cancer invasion and metastasis. Here we report that overexpression of mutant RAS drives the EV internalization switch from endocytosis (in non-transformed cells) to macropinocytosis (in cancer cells) resulting in enhanced EV uptake. This process depends on the surface proteoglycan, fibronectin and EV engulfment mechanism regulated by CRAF. Both mutant RAS and activated CRAF expression is associated with formation of membrane ruffles to which they colocalize along with actin, sodium-hydrogen exchangers (NHEs) and phosphorylated myosin phosphatase (pMYPT). RAS-transformed cells internalize EVs in the vicinity of ruffled structures followed by apparent trafficking to lysosome and degradation. NHE inhibitor (EIPA) suppresses RAS-driven EV uptake, along with adhesion-independent clonal growth and experimental metastasis in mice. Thus, EV uptake may represent a targetable step in progression of RAS-driven cancers.

Shear stress triggered circular dorsal ruffles formation to facilitate cancer cell migration.

Circular dorsal ruffles (CDRs) are a kind of special ring-shaped membrane structure rich in F-actin, it is highly involved in the invasion-metastasis of tumor. Shear stress is one of the biophysical elements that affects the fate of tumor cells. However, how shear stress contributes to the CDRs formation is still unclear. In this study, we found that shear stress stimulated the formation of CDRs and promoted the migration of human breast MDA-MB-231 carcinoma cells. Integrin-linked kinase (ILK) mediated the recruiting of ADP-ribosylation factors (ARAP1/Arf1) to CDRs. Meanwhile, the transfection of ARAP1 or Arf1 mutant decreased the number of cells with CDRs, the CDRs areas and perimeters, thus blocked the cancer cell migration. This indicated that the ARAP1/Arf1 were necessary for the CDRs formation and cancer cell migration. Further study revealed that shear stress could stimulate the formation of intracellular macropinocytosis (MPS) thus promoted the ARAP1/Arf1 transportation to early endosome to regulate cancer cell migration after the depolymerization of CDRs. Our study elucidates that the CDRs formation is essential in shear stress-induced breast cancer cell migration, which provides a new research target for exploring the cytoskeletal mechanisms of breast cancer malignance.

Unconjugated p-cresol activates macrophage macropinocytosis leading to increased LDL uptake.

Patients with chronic kidney disease (CKD) and end-stage renal disease suffer from increased cardiovascular events and cardiac mortality. Prior studies have demonstrated that a portion of this enhanced risk can be attributed to the accumulation of microbiota-derived toxic metabolites, with most studies focusing on the sulfonated form of p-cresol (PCS). However, unconjugated p-cresol (uPC) itself was never assessed due to rapid and extensive first-pass metabolism that results in negligible serum concentrations of uPC. These reports thus failed to consider the host exposure to uPC prior to hepatic metabolism. In the current study, not only did we measure the effect of altering the intestinal microbiota on lipid accumulation in coronary arteries, but we also examined macrophage lipid uptake and handling pathways in response to uPC. We found that atherosclerosis-prone mice fed a high-fat diet exhibited significantly higher coronary artery lipid deposits upon receiving fecal material from CKD mice. Furthermore, treatment with uPC increased total cholesterol, triglycerides, and hepatic and aortic fatty deposits in non-CKD mice. Studies employing an in vitro macrophage model demonstrated that uPC exposure increased apoptosis whereas PCS did not. Additionally, uPC exhibited higher potency than PCS to stimulate LDL uptake and only uPC induced endocytosis- and pinocytosis-related genes. Pharmacological inhibition of varying cholesterol influx and efflux systems indicated that uPC increased macrophage LDL uptake by activating macropinocytosis. Overall, these findings indicate that uPC itself had a distinct effect on macrophage biology that might have contributed to increased cardiovascular risk in patients with CKD.

Cancer cells escape autophagy inhibition via NRF2-induced macropinocytosis.

Many cancers, including pancreatic ductal adenocarcinoma (PDAC), depend on autophagy-mediated scavenging and recycling of intracellular macromolecules, suggesting that autophagy blockade should cause tumor starvation and regression. However, until now autophagy-inhibiting monotherapies have not demonstrated potent anti-cancer activity. We now show that autophagy blockade prompts established PDAC to upregulate and utilize an alternative nutrient procurement pathway: macropinocytosis (MP) that allows tumor cells to extract nutrients from extracellular sources and use them for energy generation. The autophagy to MP switch, which may be evolutionarily conserved and not cancer cell restricted, depends on activation of transcription factor NRF2 by the autophagy adaptor p62/SQSTM1. NRF2 activation by oncogenic mutations, hypoxia, and oxidative stress also results in MP upregulation. Inhibition of MP in autophagy-compromised PDAC elicits dramatic metabolic decline and regression of transplanted and autochthonous tumors, suggesting the therapeutic promise of combining autophagy and MP inhibitors in the clinic.

Therapeutic Targeting of DGKA-Mediated Macropinocytosis Leads to Phospholipid Reprogramming in Tuberous Sclerosis Complex.

Lymphangioleiomyomatosis is a rare destructive lung disease affecting primarily women and is the primary lung manifestation of tuberous sclerosis complex (TSC). In lymphangioleiomyomatosis, biallelic loss of TSC1/2 leads to hyperactivation of mTORC1 and inhibition of autophagy. To determine how the metabolic vulnerabilities of TSC2-deficient cells can be targeted, we performed a high-throughput screen utilizing the "Repurposing" library at the Broad Institute of MIT and Harvard (Cambridge, MA), with or without the autophagy inhibitor chloroquine. Ritanserin, an inhibitor of diacylglycerol kinase alpha (DGKA), was identified as a selective inhibitor of proliferation of Tsc2-/- mouse embryonic fibroblasts (MEF), with no impact on Tsc2+/+ MEFs. DGKA is a lipid kinase that metabolizes diacylglycerol to phosphatidic acid, a key component of plasma membranes. Phosphatidic acid levels were increased 5-fold in Tsc2-/- MEFs compared with Tsc2+/+ MEFs, and treatment of Tsc2-/- MEFs with ritanserin led to depletion of phosphatidic acid as well as rewiring of phospholipid metabolism. Macropinocytosis is known to be upregulated in TSC2-deficient cells. Ritanserin decreased macropinocytic uptake of albumin, limited the number of lysosomes, and reduced lysosomal activity in Tsc2-/- MEFs. In a mouse model of TSC, ritanserin treatment decreased cyst frequency and volume, and in a mouse model of lymphangioleiomyomatosis, genetic downregulation of DGKA prevented alveolar destruction and airspace enlargement. Collectively, these data indicate that DGKA supports macropinocytosis in TSC2-deficient cells to maintain phospholipid homeostasis and promote proliferation. Targeting macropinocytosis with ritanserin may represent a novel therapeutic approach for the treatment of TSC and lymphangioleiomyomatosis. SIGNIFICANCE: This study identifies macropinocytosis and phospholipid metabolism as novel mechanisms of metabolic homeostasis in mTORC1-hyperactive cells and suggest ritanserin as a novel therapeutic strategy for use in mTORC1-hyperactive tumors, including pancreatic cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/8/2086/F1.large.jpg.

Uptake and Transport of Ultrafine Nanoparticles (Quantum Dots) in the Nasal Mucosa.

A wide variety of colloidal delivery systems, including polymeric nanoparticles, metal colloids, liposomes, and microemulsions have been reported to enhance the delivery of therapeutic agents across the nasal mucosa. The mechanisms involved in the uptake of these nanomaterials, especially ultrafine nanomaterials (diameters < 20 nm) through the nasal mucosa are not well understood. Fluorescent quantum dots (QDs) were used to investigate the uptake of ultrafine nanoparticles by bovine respiratory and olfactory mucosal tissues following in vitro exposure, and an inductively coupled plasma optical emission spectroscopy method was developed to quantify the amount of QDs localized within the tissues. QDs do not biodegrade or release their core materials and, as a result, this method allowed for the direct quantification of the nanoparticles themselves, rather than the measurement of a potentially dissociated drug or label. The results demonstrated that carboxylate-modified QDs (COOH-QDs) showed ∼2.5-fold greater accumulation in the epithelial and submucosal regions of olfactory tissues compared to that in respiratory tissues. Endocytic inhibitory studies showed that clathrin-dependent endocytosis, macropinocytosis, and caveolae-dependent endocytic process are all involved in the uptake of COOH-QDs into the respiratory tissues. In olfactory tissues, clathrin-dependent endocytosis is the major endocytic pathway involved in the uptake of COOH-QDs. Additional energy-independent pathways also appeared to allow the transfer of COOH-QDs within the olfactory mucosa. When polyethylene glycol-modified QDs known as PEGylated QDs (PEG-QDs) of similar size, ∼15 nm, were investigated, no nanoparticles were detected in the tissues suggesting that the PEG corona limits the interactions with endocytic and other uptake processes in the nasal epithelium. The capacity for nanoparticle uptake observed in the nasal mucosa, along with the ability of significant numbers of nanoparticles to enter the olfactory tissues using nonenergy-dependent pathways show that the pathways for ultrafine nanoparticle uptake in the nasal tissues have both drug delivery and toxicologic consequences. This places an increased importance on the careful selection of nanoparticle components and drugs intended for intranasal administration.