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1.
J Vis Exp ; (174)2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34487110

RESUMO

In recent years, the field of macropinocytosis has grown rapidly. Macropinocytosis has emerged as a central mechanism by which innate immune cells maintain organismal homeostasis and immunity. Simultaneously, and in contrast to its homeostatic role, it can also drive various pathologies, including cancer and viral infections. Unlike other modes of endocytosis, the tools developed for studying the maturation of macropinosomes remain underdeveloped. Here the protocol describes newly developed tools for studying the redox environment within the lumen of early and maturing macropinosomes. Methodologies for using ratiometric fluorescence microscopy in assessing the pH, production of reactive oxygen species, and the degradative capacity within the lumen of individual macropinosomes in live cells are described. Single organelle measurements offer the advantage of revealing spatiotemporal heterogeneity, which is often lost with population-based approaches. Emphasis is placed on the basic principles of dual fluorophore ratiometric microscopy, including probe selection, instrumentation, calibration, and single-cell versus population-based methods.


Assuntos
Endossomos , Pinocitose , Endossomos/metabolismo , Concentração de Íons de Hidrogênio , Microscopia de Fluorescência , Oxirredução
2.
J Vis Exp ; (174)2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34515683

RESUMO

Macropinocytosis is a non-specific fluid-phase uptake pathway that allows cells to internalize large extracellular cargo, such as proteins, pathogens, and cell debris, through bulk endocytosis. This pathway plays an essential role in a variety of cellular processes, including the regulation of immune responses and cancer cell metabolism. Given this importance in biological function, examining cell culture conditions can provide valuable information by identifying regulators of this pathway and optimizing conditions to be employed in the discovery of novel therapeutic approaches. The study describes an automated imaging and analysis technique using standard laboratory equipment and a cell imaging multi-mode plate reader for the rapid quantification of the macropinocytic index in adherent cells. The automated method is based on the uptake of high molecular weight fluorescent dextran and can be applied to 96-well microplates to facilitate assessments of multiple conditions in one experiment or fixed samples mounted onto glass coverslips. This approach is aimed at maximizing reproducibility and reducing experimental variation while being both time-saving and cost-effective.


Assuntos
Endossomos , Pinocitose , Endocitose , Microscopia de Fluorescência , Reprodutibilidade dos Testes
3.
J Vis Exp ; (174)2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34398147

RESUMO

Macropinocytosis is a highly conserved, actin-dependent endocytic process that allows the uptake of extracellular material, including proteins and lipids. In proliferating cells, macropinocytosis can deliver extracellular nutrients to the lysosome, processed into critical macromolecule building blocks. Recent studies have highlighted the dependence of multiple cancers on macropinocytosis, including breast, colorectal and pancreatic cancer. Ras mutations are thought to be the driver events behind macropinocytosis initiation, leading to the activation of cellular anabolic processes via the mTORC1 signaling pathway. Interestingly, mTORC1 can also be activated by macropinocytosis independently of Ras. Therefore, macropinocytosis represents a metabolic vulnerability that can be leveraged to target macropinocytic tumors by limiting their access to nutrients therapeutically. In Tuberous Sclerosis Complex (TSC) and Lymphangioleiomyomatosis (LAM), mTORC1-hyperactivation leads to enhanced macropinocytosis and metabolic reprogramming. Here, we describe a flow cytometry-based protocol to assess macropinocytosis in mammalian cells quantitatively. TSC2-deficient MEFs are employed, which exhibit aberrant activation of mTORC1 and have been shown to have increased macropinocytosis compared to TSC2-expressing cells. Cells treated with pharmacologic inhibitors of macropinocytosis are incubated with fluorescently labeled, lysine-fixable, 70 kDa dextran, or fluorescently labeled bovine serum albumin (BSA) assayed by flow cytometry. To date, robust image-based techniques have been developed to quantitatively assess macropinocytosis in tumor cells in vitro and in vivo. This analysis provides a quantitative assessment of macropinocytosis in multiple experimental conditions and complements existing image-based techniques.


Assuntos
Linfangioleiomiomatose , Pinocitose , Animais , Citometria de Fluxo , Lisossomos , Alvo Mecanístico do Complexo 1 de Rapamicina
4.
Molecules ; 26(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34361779

RESUMO

Delivering nucleic acids into the endothelium has great potential in treating vascular diseases. However, endothelial cells, which line the vasculature, are considered as sensitive in nature and hard to transfect. Low transfection efficacies in endothelial cells limit their potential therapeutic applications. Towards improving the transfection efficiency, we made an effort to understand the internalization of lipoplexes into the cells, which is the first and most critical step in nucleic acid transfections. In this study, we demonstrated that the transient modulation of caveolae/lipid rafts mediated endocytosis with the cholesterol-sequestrating agents, nystatin, filipin III, and siRNA against Cav-1, which significantly increased the transfection properties of cationic lipid-(2-hydroxy-N-methyl-N,N-bis(2-tetradecanamidoethyl)ethanaminium chloride), namely, amide liposomes in combination with 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (AD Liposomes) in liver sinusoidal endothelial cells (SK-Hep1). In particular, nystatin was found to be highly effective with 2-3-fold enhanced transfection efficacy when compared with amide liposomes in combination with Cholesterol (AC), by switching lipoplex internalization predominantly through clathrin-mediated endocytosis and macropinocytosis.


Assuntos
Cavéolas/efeitos dos fármacos , Colesterol/química , Células Endoteliais/efeitos dos fármacos , Lipossomos/química , Microdomínios da Membrana/efeitos dos fármacos , Transfecção/métodos , Animais , Cavéolas/química , Cavéolas/metabolismo , Caveolina 1/antagonistas & inibidores , Caveolina 1/genética , Caveolina 1/metabolismo , Linhagem Celular Transformada , Colesterol/metabolismo , Clatrina/metabolismo , DNA/química , DNA/metabolismo , Endocitose/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Filipina/química , Filipina/farmacologia , Expressão Gênica , Lipossomos/metabolismo , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Nistatina/química , Nistatina/farmacologia , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/farmacologia , Pinocitose/efeitos dos fármacos , Plasmídeos/química , Plasmídeos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos
5.
J Vis Exp ; (174)2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34424243

RESUMO

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase nutrients and other material in cells. The dramatic extension of cell surface ruffles, their closure to form macropinosomes, and the maturation of internalized macropinosomes are key events in this pathway that can be difficult to capture using conventional confocal imaging based on tracking a bolus of fluorescent cargo. Fluorescent dextrans are commonly used experimentally as fluid phase markers for macropinosomes and for other endocytic pathways. A method the lab has adopted to optimize the imaging of dextran uptake involves using live imaging of cells bathed in high concentrations of fluorescent dextran in the medium, with the unlabeled cells appearing in relief (as black). The cell ruffles are highlighted to visualize ruffle closure, and internalized macropinosomes appear as fluorescent vacuoles in the cell interior. This method is optimal for visualizing macropinosome features and allows for easy segmentation and quantification. This paper describes dual-labeling of pathways with different sized dextrans and the co-expression of lipid probes and fluorescent membrane proteins to demark macropinosomes and other endosomes. The detection of internalized dextran at an ultrastructural level using correlative light and electron microscopy (CLEM) is also demonstrated. These cell processes can be imaged using multiple live imaging modalities, including in 3D. Taken together, these approaches optimize macropinosome imaging for many different settings and experimental systems.


Assuntos
Endossomos , Pinocitose , Membrana Celular , Microscopia Eletrônica , Vacúolos
6.
Nat Commun ; 12(1): 4838, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376698

RESUMO

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.


Assuntos
Membrana Celular/metabolismo , Microscopia de Fluorescência/métodos , Fosfatidilinositol 3-Quinases/metabolismo , Pinocitose/fisiologia , Animais , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Células HEK293 , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Macrófagos/ultraestrutura , Camundongos , Microscopia Eletrônica de Varredura , Morfolinas/farmacologia , Fosfatidilinositóis/metabolismo , Pinocitose/efeitos dos fármacos , Células RAW 264.7
7.
FASEB J ; 35(9): e21742, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34403506

RESUMO

Withdrawal from contact inhibition is necessary for epithelial cancer precursor cells to initiate cell growth and motility. Nevertheless, little is understood about the mechanism for the sudden initiation of cell growth under static conditions. We focused on cellular junctions as one region where breaking out of contact inhibition occurs. In well-differentiated endometrial cancer cells, Sawano, the ligand administration for tricellular tight junction protein LSR, which transiently decreased the robust junction property, caused an abrupt increase in cell motility and consequent excessive multilayered cell growth despite being under contact inhibition conditions. We observed that macropinocytosis essentially and temporarily occurred as an antecedent event for the above process at intercellular junctions without disruption of the junction apparatus but not at the apical plasma membrane. Collectively, we concluded that the formation of macropinocytosis, which is derived from tight junction-mediated signaling, was triggered for the initiation of cell growth in static precancerous epithelium.


Assuntos
Adesão Celular , Inibição de Contato , Pinocitose , Receptores de Lipoproteínas/metabolismo , Fatores de Transcrição/metabolismo , Toxinas Bacterianas/farmacologia , Sítios de Ligação , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Humanos , Junções Intercelulares/efeitos dos fármacos , Junções Intercelulares/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Fenótipo , Pinocitose/efeitos dos fármacos , Transporte Proteico , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo
8.
J Vis Exp ; (172)2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34279488

RESUMO

Adenosine triphosphate (ATP), including extracellular ATP (eATP), has been shown to play significant roles in various aspects of tumorigenesis, such as drug resistance, epithelial-mesenchymal transition (EMT), and metastasis. Intratumoral eATP is 103 to 104 times higher in concentration than in normal tissues. While eATP functions as a messenger to activate purinergic signaling for EMT induction, it is also internalized by cancer cells through upregulated macropinocytosis, a specific type of endocytosis, to perform a wide variety of biological functions. These functions include providing energy to ATP-requiring biochemical reactions, donating phosphate groups during signal transduction, and facilitating or accelerating gene expression as a transcriptional cofactor. ATP is readily available, and its study in cancer and other fields will undoubtedly increase. However, eATP study remains at an early stage, and unresolved questions remain unanswered before the important and versatile activities played by eATP and internalized intracellular ATP can be fully unraveled. These authors' laboratories' contributions to these early eATP studies include microscopic imaging of non-hydrolysable fluorescent ATP, coupled with high- and low-molecular weight fluorescent dextrans, which serve as macropinocytosis and endocytosis tracers, as well as various endocytosis inhibitors, to monitor and characterize the eATP internalization process. This imaging modality was applied to tumor cell lines and to immunodeficient mice, xenografted with human cancer tumors, to study eATP internalization in vitro and in vivo. This paper describes these in vitro and in vivo protocols, with an emphasis on modifying and finetuning assay conditions so that the macropinocytosis-/endocytosis-mediated eATP internalization assays can be successfully performed in different systems.


Assuntos
Trifosfato de Adenosina , Pinocitose , Animais , Linhagem Celular Tumoral , Endocitose , Humanos , Camundongos , Microscopia de Fluorescência
9.
Acc Chem Res ; 54(14): 2916-2927, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34232016

RESUMO

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.


Assuntos
Membrana Celular/metabolismo , Nanopartículas/metabolismo , Transporte Biológico/fisiologia , Cavéolas/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Microdomínios da Membrana/metabolismo , Nanopartículas/química , Tamanho da Partícula , Pinocitose/fisiologia , Propriedades de Superfície
10.
Int J Biol Macromol ; 185: 907-916, 2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34242647

RESUMO

The present study was to investigate the mechanisms involved in macrophage activation by polysaccharides from the fruits of Rubus chingii Hu (RFPs). The results showed that RFPs enhanced pinocytic and phagocytic activity, promoted the expression and secretion of inflammatory factors (ROS, PTGS2, iNOS, IL-6, IL-10 and TNF-α) and chemokines (CCL2 and CXCL10), and boosted the expression of accessory and costimulatory molecules (CD40, CD80, CD86, MHC-I and MHC-II). RNA-Seq analysis identified 2564 DEGs, 1710 GO terms and 101 KEGG pathways. TNF was identified as the core gene via analysis of pathway information integration and PPI network. The western blot analysis combined with functional verification assay confirmed that MAPK, NF-κB and Jak-STAT pathways were essential to RFPs-mediated macrophage activation. TLR2 was revealed to be the functional receptor and involved in the early recognition of RFPs. These results indicated that RFPs modulated macrophage immune response mainly through TLR2-dependent MAPK, NF-κB and Jak-STAT pathways.


Assuntos
Adjuvantes Imunológicos/farmacologia , Redes Reguladoras de Genes/efeitos dos fármacos , Macrófagos/citologia , Polissacarídeos/farmacologia , Rubus/química , Animais , Sobrevivência Celular/efeitos dos fármacos , Perfilação da Expressão Gênica , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Fagocitose , Pinocitose , Células RAW 264.7 , Análise de Sequência de RNA
11.
J Vis Exp ; (171)2021 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-34125102

RESUMO

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.


Assuntos
Citoesqueleto de Actina , Membrana Celular , Microscopia Eletrônica de Varredura , Pinocitose , Extensões da Superfície Celular
12.
Arch Biochem Biophys ; 709: 108967, 2021 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-34157295

RESUMO

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.


Assuntos
Citoesqueleto de Actina/metabolismo , Membrana Celular/metabolismo , Movimento Celular/fisiologia , Extensões da Superfície Celular/metabolismo , Neoplasias/metabolismo , Fator 1 de Ribosilação do ADP/metabolismo , Citoesqueleto de Actina/química , Actinas/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Membrana Celular/química , Membrana Celular/ultraestrutura , Extensões da Superfície Celular/química , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Neoplasias/patologia , Pinocitose/fisiologia , Polimerização , Proteínas Serina-Treonina Quinases/metabolismo , Estresse Mecânico
13.
Front Immunol ; 12: 649600, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34135890

RESUMO

Using the optogenetic photo-manipulation of photoactivatable (PA)-Rac1, remarkable cell surface ruffling and the formation of a macropinocytic cup (premacropinosome) could be induced in the region of RAW264 macrophages irradiated with blue light due to the activation of PA-Rac1. However, the completion of macropinosome formation did not occur until Rac1 was deactivated by the removal of the light stimulus. Following PA-Rac1 deactivation, some premacropinosomes closed into intracellular macropinosomes, whereas many others transformed into long Rab10-positive tubules without forming typical macropinosomes. These Rab10-positive tubules moved centripetally towards the perinuclear Golgi region along microtubules. Surprisingly, these Rab10-positive tubules did not contain any endosome/lysosome compartment markers, such as Rab5, Rab7, or LAMP1, suggesting that the Rab10-positive tubules were not part of the degradation pathway for lysosomes. These Rab10-positive tubules were distinct from recycling endosomal compartments, which are labeled with Rab4, Rab11, or SNX1. These findings suggested that these Rab10-positive tubules may be a part of non-degradative endocytic pathway that has never been known. The formation of Rab10-positive tubules from premacropinosomes was also observed in control and phorbol myristate acetate (PMA)-stimulated macrophages, although their frequencies were low. Interestingly, the formation of Rab10-positive premacropinosomes and tubules was not inhibited by phosphoinositide 3-kinase (PI3K) inhibitors, while the classical macropinosome formation requires PI3K activity. Thus, this study provides evidence to support the existence of Rab10-positive tubules as a novel endocytic pathway that diverges from canonical macropinocytosis.


Assuntos
Endocitose/imunologia , Macrófagos/imunologia , Microtúbulos/metabolismo , Pinocitose/imunologia , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Endocitose/efeitos dos fármacos , Endocitose/efeitos da radiação , Complexo de Golgi/metabolismo , Microscopia Intravital , Luz , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Microtúbulos/imunologia , Microtúbulos/efeitos da radiação , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Optogenética , Pinocitose/efeitos dos fármacos , Pinocitose/efeitos da radiação , Células RAW 264.7 , Acetato de Tetradecanoilforbol/farmacologia , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo
14.
J Cell Sci ; 134(14)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34109410

RESUMO

Macropinocytosis allows cells to take up extracellular material in a non-selective manner into large vesicles called macropinosomes. After internalization, macropinosomes acquire phosphatidylinositol 3-phosphate (PtdIns3P) on their limiting membrane as they mature into endosomal-like vesicles. The molecular mechanisms that underlie recycling of membranes and transmembrane proteins from these macropinosomes still need to be defined. Here, we report that JIP4 (officially known as SPAG9), a protein previously described to bind to microtubule motors, is recruited to tubulating subdomains on macropinosomes by the PtdIns3P-binding protein Phafin2 (officially known as PLEKHF2). These JIP4-positive tubulating subdomains on macropinosomes contain F-actin, the retromer recycling complex and the retromer cargo VAMP3. Disruption of the JIP4-Phafin2 interaction, deletion of Phafin2 or inhibition of PtdIns3P production by VPS34 impairs JIP4 recruitment to macropinosomes. Whereas knockout of JIP4 suppresses tubulation, its overexpression enhances tubulation from macropinosomes. JIP4-knockout cells display increased retention of macropinocytic cargo in both early and late macropinosomes. Collectively, these data identify JIP4 and Phafin2 as components of a tubular recycling pathway that operates from macropinosomes. This article has an associated First Person interview with the first author of the paper.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte , Fosfatidilinositóis , Proteínas de Transporte Vesicular , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Endossomos/metabolismo , Humanos , Fosfatidilinositóis/metabolismo , Pinocitose , Ligação Proteica , Transporte Proteico , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
15.
J Cell Biol ; 220(7)2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34128957

RESUMO

Actin organization underpins conserved functions at the leading edge of cells. In this issue, Yang et al. (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202010096) characterize Leep1 as a bi-functional regulator of migration and macropinocytosis through PIP3 and the Scar/WAVE complex.


Assuntos
Actinas , Pinocitose , Actinas/genética
16.
Cell Physiol Biochem ; 55(S1): 171-184, 2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34156175

RESUMO

BACKGROUND/AIMS: Trypan blue is routinely used in cell culture experiments to distinguish between dead cells, which are labelled by trypan blue, and viable cells, which are apparently free of any staining. The assumption that trypan blue labelling is restricted to dead cells derives from the observation that rupture of the plasma membrane correlates with intense trypan blue staining. However, decades ago, trypan blue has been used to trace fluid uptake by viable macrophage-like cells in animals. These studies contributed to the concept of the reticuloendothelial system in vertebrates. Trypan blue itself does not show a fluorescence signal, but trypan blue-labelled proteins do. Therefore, intracellular localization of trypan blue-labelled proteins could give a clue to the entrance pathway of the dye in viable cells. METHODS: We used fluorescence microscopy to visualize trypan blue positive structures and to evaluate whether the bactericide, silver, enhances cellular trypan blue uptake in the brain macrophage-like cell line, BV-2. The pattern of chromatin condensation, visualized by DAPI staining, was used to identify the cell death pathway. RESULTS: We observed that silver nitrate at elevated concentrations (≥ 10 µM) induced in most cells a necrotic cell death pathway. Necrotic cells, identified by pycnotic nuclei, showed an intense and homogenous trypan blue staining. Apoptotic cells, characterized by crescent-like nuclear chromatin condensations, were not labelled by trypan blue. At lower silver nitrate concentrations, most cells were viable, but they showed trypan blue labelling. Viable cells showed a cell-type specific distribution of heterochromatin and revealed a perinuclear accumulation of bright trypan blue-labelled vesicles and, occasionally, a faint homogenous trypan blue labelling of the cytoplasm and nucleus. Amiloride, which prevents macropinocytosis by blocking the Na+ / H+ exchange, suppressed perinuclear accumulation of dye-labelled vesicles. Swelling of cells in a hypotonic solution induced an intense intracellular accumulation of trypan blue. Cells exposed to a hypotonic solution in the presence of 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), which blocks volume-regulated ion channels, prevented labelling of the cytoplasm and nucleus but did not affect labelling of perinuclear vesicles. CONCLUSION: In viable cells trypan blue-labelled vesicles indicate trypan blue uptake by macropinocytosis and trypan blue-labelled cytosol could indicate a further entry pathway for the dye, like activated volume-regulated channels. Accordingly, fluorescence microscopic analysis of trypan blue-labelled cells allows not only a discrimination between necrotic and apoptotic cell death pathway but also a discrimination between the mode of trypan blue uptake in viable cells - via pinocytosis or via activated volume-regulated ion channels - in the same preparation at the single cell level.


Assuntos
Corantes/análise , Microglia/citologia , Pinocitose , Azul Tripano/análise , Animais , Morte Celular , Linhagem Celular , Sobrevivência Celular , Camundongos , Microscopia de Fluorescência/métodos , Coloração e Rotulagem/métodos
17.
Commun Biol ; 4(1): 718, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34112916

RESUMO

Recently, we involved the carbohydrate-binding protein Galectin-3 (Gal-3) as a druggable target for KRAS-mutant-addicted lung and pancreatic cancers. Here, using glioblastoma patient-derived stem cells (GSCs), we identify and characterize a subset of Gal-3high glioblastoma (GBM) tumors mainly within the mesenchymal subtype that are addicted to Gal-3-mediated macropinocytosis. Using both genetic and pharmacologic inhibition of Gal-3, we showed a significant decrease of GSC macropinocytosis activity, cell survival and invasion, in vitro and in vivo. Mechanistically, we demonstrate that Gal-3 binds to RAB10, a member of the RAS superfamily of small GTPases, and ß1 integrin, which are both required for macropinocytosis activity and cell survival. Finally, by defining a Gal-3/macropinocytosis molecular signature, we could predict sensitivity to this dependency pathway and provide proof-of-principle for innovative therapeutic strategies to exploit this Achilles' heel for a significant and unique subset of GBM patients.


Assuntos
Proteínas Sanguíneas/metabolismo , Neoplasias Encefálicas/metabolismo , Galectinas/metabolismo , Glioblastoma/metabolismo , Células-Tronco Neoplásicas/metabolismo , Animais , Proteínas Sanguíneas/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Feminino , Galectinas/genética , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Camundongos , Células-Tronco Neoplásicas/patologia , Pinocitose , Mapas de Interação de Proteínas , Transcriptoma , Células Tumorais Cultivadas
18.
Methods Mol Biol ; 2304: 193-205, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34028718

RESUMO

Macropinocytosis and phagocytosis are the processes by which eukaryotic cells use their plasma membrane to engulf liquid or a large particle and give rise to an internal compartment called the macropinosomes or phagosome, respectively. Dictyostelium discoideum provides a powerful system to understand the molecular mechanism of these two fundamental cellular processes that impact human health and disease. Recent developments in fluorescence microscopy allow direct visualization of intracellular signaling events with high temporal and spatial resolution. Here, we describe methods to visualize temporospatial activation or localization of key signaling components that are crucial for macropinocytosis and phagocytosis using confocal fluorescence microscopy.


Assuntos
Dictyostelium/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas ras/metabolismo , Ativação Enzimática , Regulação da Expressão Gênica , Humanos , Microscopia Confocal , Microscopia de Fluorescência , Fagocitose , Pinocitose , Proteínas de Protozoários/metabolismo , Transdução de Sinais
19.
J Cell Biol ; 220(7)2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-33978708

RESUMO

Polarity is essential for diverse functions in many cell types. Establishing polarity requires targeting a network of specific signaling and cytoskeleton molecules to different subregions of the cell, yet the full complement of polarity regulators and how their activities are integrated over space and time to form morphologically and functionally distinct domains remain to be uncovered. Here, by using the model system Dictyostelium and exploiting the characteristic chemoattractant-stimulated translocation of polarly distributed molecules, we developed a proteomic screening approach, through which we identified a leucine-rich repeat domain-containing protein we named Leep1 as a novel polarity regulator. We combined imaging, biochemical, and phenotypic analyses to demonstrate that Leep1 localizes selectively at the leading edge of cells by binding to PIP3, where it modulates pseudopod and macropinocytic cup dynamics by negatively regulating the Scar/WAVE complex. The spatiotemporal coordination of PIP3 signaling, Leep1, and the Scar/WAVE complex provides a cellular mechanism for organizing protrusive structures at the leading edge.


Assuntos
Actinas/economia , Polaridade Celular/genética , Pinocitose/genética , Proteínas de Protozoários/genética , Actinas/genética , Movimento Celular/genética , Quimiotaxia/genética , Citoplasma/genética , Dictyostelium/genética , Pseudópodes/genética , Transdução de Sinais/genética
20.
Nat Nanotechnol ; 16(7): 830-839, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33958764

RESUMO

Nanoparticulate albumin bound paclitaxel (nab-paclitaxel, nab-PTX) is among the most widely prescribed nanomedicines in clinical use, yet it remains unclear how nanoformulation affects nab-PTX behaviour in the tumour microenvironment. Here, we quantified the biodistribution of the albumin carrier and its chemotherapeutic payload in optically cleared tumours of genetically engineered mouse models, and compared the behaviour of nab-PTX with other clinically relevant nanoparticles. We found that nab-PTX uptake is profoundly and distinctly affected by cancer-cell autonomous RAS signalling, and RAS/RAF/MEK/ERK inhibition blocked its selective delivery and efficacy. In contrast, a targeted screen revealed that IGF1R kinase inhibitors enhance uptake and efficacy of nab-PTX by mimicking glucose deprivation and promoting macropinocytosis via AMPK, a nutrient sensor in cells. This study thus shows how nanoparticulate albumin bound drug efficacy can be therapeutically improved by reprogramming nutrient signalling and enhancing macropinocytosis in cancer cells.


Assuntos
Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mutação , Nanopartículas , Neoplasias Experimentais/tratamento farmacológico , Paclitaxel , Proteínas Proto-Oncogênicas p21(ras)/genética , Albumina Sérica Humana , Animais , Linhagem Celular Tumoral , Glucose/deficiência , Glucose/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Nanopartículas/química , Nanopartículas/uso terapêutico , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Paclitaxel/farmacocinética , Paclitaxel/farmacologia , Pinocitose , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Células RAW 264.7 , Albumina Sérica Humana/química , Albumina Sérica Humana/farmacologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética
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