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The blood–brain barrier (BBB) is an interface primarily comprised of brain endothelial cells (BECs), separating the central nervous system (CNS) from the systemic circulation while carefully regulating the transport of molecules and inflammatory cells, and maintaining the required steady-state environment. Inflammation modulates many BBB functions, but the ultrastructural cytoarchitectural changes of the BBB with inflammation are understudied. Inflammation was induced in male 8–10-week-old CD-1 mice with intraperitoneal lipopolysaccharide (LPS), using a regimen (3 mg/kg at 0, 6, and 24 h) that caused robust BBB disruption but had minimal lethality at the study timepoint of 28 h. Perfusion-fixed brains were collected and the frontal cortical layer III regions were analyzed using a transmission electron microscopy (TEM). The LPS-treated mice had pronounced ultrastructural remodeling changes in BECs that included plasma membrane ruffling, increased numbers of extracellular microvesicles, small exosome formation, aberrant BEC mitochondria, increased BEC transcytosis, while tight junctions appeared to be unaltered. Aberrant pericytes were contracted with rounded nuclei and a loss of their elongated cytoplasmic processes. Surveilling microglial cells were attracted to the neurovascular unit (NVU) of BECs, and astrocyte detachment and separation were associated with the formation of a perivascular space and pericapillary edema. The LPS treatment resulted in numerous ultrastructural aberrant remodeling changes to the neurovascular unit's BECs, microglia, pericytes, and astrocytes. In summary, a disturbance of the NVU morphology is a consequence of LPS treatment. [ABSTRACT FROM AUTHOR]

The internalization of solutes by macropinocytosis provides an essential route for nutrient uptake in many cells. Macrophages increase macropinocytosis in response to growth factors and other stimuli. To test the hypothesis that nutrient environments modulate solute uptake by macropinocytosis, this study analyzed the effects of extracellular amino acids on the accumulation of fluorescent fluidphase probes in murine macrophages. Nine amino acids, added individually or together, were capable of suppressingmacropinocytosis in murine bone marrow-derived macrophages stimulated with the growth factors colony stimulating factor 1 (CSF1) or interleukin 34, both ligands of the CSF1 receptor (CSF1R). The suppressive amino acids did not inhibit macropinocytosis in response to lipopolysaccharide, the chemokine CXCL12, or the tumor promoter phorbol myristate acetate. Suppressive amino acids promoted release of CSF1R from cells and resulted in the formation of smaller macropinosomes in response to CSF1. This suppression of growth factor-stimulated macropinocytosis indicates that different nutrient environments modulate CSF1R levels and bulk ingestion by macropinocytosis, with likely consequences for macrophage growth and function. [ABSTRACT FROM AUTHOR]

Macropinocytosis is increasingly recognized for its versatile adaptations and functions as a highly conserved, ubiquitous pathway for the bulk uptake of fluid, particulate cargo, and membranes. Innate immune cells and transformed cancer cells share the capacity for both constitutive and induced macropinocytosis, which is used for immune surveillance, ingestion of pathogens, immune response shaping, and enhancement of scavenging for nutrients as fuel for cell survival and proliferation. Immunology and cancer biology are leading a resurgence of interest in defining the molecular and physiological regulation of macropinocytosis, partly in pursuit of ways to control macropinocytic uptake in disease settings. New approaches, including high-resolution live imaging, screening of cell surface molecular inventories, biophysics, and exploration of cell microenvironments, have converged to provide new insights into macropinosome induction, formation, and maturation. Recent studies reveal mechanisms for fluid control in and by macrophage macropinosomes that impinge on membrane trafficking and cell migration. EGFR, PTEN, V-ATPase, syndecan 1, and galectin-3 have roles variably in the metabolic regulation of Ras or PI3K signaling for Rac1-mediated macropinocytosis in cancer. These molecular pathways and mechanisms contribute to the impressive adaptability of macropinocytosis in many cells and tissues and in disease. [ABSTRACT FROM AUTHOR]

Owing to its unique redox properties, cerium oxide (nanoceria) nanoparticles have been shown to confer either radiosensitization or radioprotection to human cells. We investigated nanoceria’s ability to modify cellular health and reactive oxygen species (ROS) at various absorbed doses (Gray) of ionizing radiation in MDA-MB231 breast carcinoma cells. We used transmission electron microscopy to visualize the uptake and compartmental localization of nanoceria within cells at various treatment concentrations. The effects on apoptosis and other cellular health parameters were assessed using confocal fluorescence imaging and flow cytometry without and with various absorbed doses of ionizing radiation, along with intracellular ROS levels. Our results showed that nanoceria were taken up into cells mainly by macropinocytosis and segregated into concentration-dependent large aggregates in macropinosomes. Confocal imaging and flow cytometry data showed an overall decrease in apoptotic cell populations in proportion to increasing nanoparticle concentrations. This increase in cellular health was observed with a corresponding reduction in ROS at all tested absorbed doses. Moreover, this effect appeared pronounced at lower doses compared to unirradiated or untreated populations. In conclusion, internalized nanoceria confers radioprotection with a corresponding decrease in ROS in MDA-MB231 cells, and this property confers significant perils and opportunities when utilized in the context of radiotherapy.

The neonatal Fc receptor (FcRn) rescues albumin and IgG following endocytosis and thereby extends the half-life of these plasma proteins. However, the pathways for the uptake of these soluble FcRn ligands, and the recycling itinerary of the FcRn-ligand complexes, have not been identified in primary cells. Here we have defined the recycling of human albumin and IgG in primary mouse macrophages selectively expressing the human FcRn. Albumin is internalised by macropinocytosis; in the absence of FcRn, internalised albumin is rapidly degraded, while in the presence of FcRn albumin co-localises to SNX5-positive-membrane domains and is partitioned into tubules emanating from early macropinosomes for delivery in transport carriers to the plasma membrane. Soluble monomeric IgG was also internalised by macropinocytosis and rapidly recycled by the same pathway. In contrast, the fate of IgG bound to surface Fcγ receptors differed from monomeric IgG endocytosed by macropinocytosis. Overall, our findings identify a rapid recycling pathway for FcRn ligands from early macropinosomes to the cell surface of primary cells. [ABSTRACT FROM AUTHOR]

In the previous research, we found that anticancer agent LS-1-2F could cause the vacuolation of tumor cells. Herein we investigated the effect of compound LS-1-2F on the endocytosis of macromolecules, including fluorescence quantum dots, human serum albumin, single-stranded RNA, and monoclonal antibody, into tumor cells. We found that LS-1-2F could accelerate the endocytosis of these large molecules by laser confocal microscope and flow cytometry. The effect of LS-1-2F on the improvement of the internalization efficiency of Herceptin biosimilar was particularly significant. Promoting endocytosis will help increase the efficiency of liquid-phase drug uptake in drug-resistant cancer cells and could potentially facilitate the use of drugs in nanoparticle delivery vehicles. [ABSTRACT FROM AUTHOR]

Macropinocytosis is a unique pathway of endocytosis characterised by the nonspecific internalisation of large amounts of extracellular fluid, solutes and membrane in large endocytic vesicles known as macropinosomes. Macropinocytosis is important in a range of physiological processes, including antigen presentation, nutrient sensing, recycling of plasma proteins, migration and signalling. It has become apparent in recent years from the study of specialised cells that there are multiple pathways of macropinocytosis utilised by different cell types, and some of these pathways are triggered by different stimuli. Understanding the physiological function of macropinocytosis requires knowledge of the regulation and fate of the macropinocytosis pathways in a range of cell types. Here, we compare the mechanisms of macropinocytosis in different primary and immortalised cells, identify the gaps in knowledge in the field and discuss the potential approaches to analyse the function of macropinocytosis in vivo.

Abstract: Salmonella enterica induces membrane ruffling and genesis of macropinosomes during its interactions with epithelial cells. This is achieved through the type three secretion system‐1, which first mediates bacterial attachment to host cells and then injects bacterial effector proteins to alter host behaviour. Next, Salmonella enters into the targeted cell within an early membrane‐bound compartment that matures into a slow growing, replicative niche called the Salmonella Containing Vacuole (SCV). Alternatively, the pathogen disrupts the membrane of the early compartment and replicate at high rate in the cytosol. Here, we show that the in situ formed macropinosomes, which have been previously postulated to be relevant for the step of Salmonella entry, are key contributors for the formation of the mature intracellular niche of Salmonella. We first clarify the primary mode of type three secretion system‐1 induced Salmonella entry into epithelial cells by combining classical fluorescent microscopy with cutting edge large volume electron microscopy. We observed that Salmonella, similarly to Shigella, enters epithelial cells inside tight vacuoles rather than in large macropinosomes. We next apply this technology to visualise rupturing Salmonella containing compartments, and we use extended time‐lapse microscopy to establish early markers that define which Salmonella will eventually hyper replicate. We show that at later infection stages, SCVs harbouring replicating Salmonella have previously fused with the in situ formed macropinosomes. In contrast, such fusion events could not be observed for hyper‐replicating Salmonella, suggesting that fusion of the Salmonella entry compartment with macropinosomes is the first committed step of SCV formation. [ABSTRACT FROM AUTHOR]

Macropinocytosis is an evolutionarily conserved process, which is characterized by the formation of membrane ruffles and the uptake of extracellular fluid. We recently demonstrated a role for CYFIP-related Rac1 Interactor (CYRI) proteins in macropinocytosis. High-molecular weight dextran (70kDa or higher) has generally been used as a marker for macropinocytosis because it is too large to fit in smaller endocytic vesicles, such as those of clathrin or caveolin-mediated endocytosis. Through the use of an image-based dextran uptake assay, we showed that cells lacking CYRI proteins internalise less dextran compared to their wild-type counterparts. Here, we will describe a step-by-step experimentation procedure to detect internalised dextran in cultured cells, and an image pipeline to analyse the acquired images, using the open-access software ImageJ/Fiji. This protocol is detailed yet simple and easily adaptable to different treatment conditions, and the analysis can also be automated for improved processing speed., Competing Interests: Competing InterestsThe authors declare no competing interests., (Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC.)

Simple Summary: Cerium oxide nanoparticles (nanoceria) influence its environment by donating or accepting electrons readily. We investigated nanoceria's ability to modify the health of treatment-resistant breast cancer cell type, with various doses of potentially damaging radiation. We used electron microscopy to determine the presence and location of various amounts of nanoceria in cells. We also used imaging modalities such as confocal fluorescence and flow cytometry to study cell-health, cell-death, and amounts of potentially damaging unstable molecules called reactive oxygen species, all without or with different doses of radiation. Our results showed that nanoceria were taken up by a cell-drinking process called macropinocytosis, and then got segregated into large compartments called macropinosomes. There was an overall decrease in cell-death with increasing nanoparticle concentrations. This increase in cell-health resulted in a reduction of the reactive oxygen species at all tested radiation doses. Moreover, this effect appeared prominent at lower radiation doses compared to populations not treated with radiation or nanoparticles. In conclusion, our discovery shows that internalized nanoceria provide protection from radiation with a corresponding decrease in reactive oxygen species in this type of breast cancer cells and this property confers significant perils and opportunities when utilized in the context of cancer radiation therapy. Owing to its unique redox properties, cerium oxide (nanoceria) nanoparticles have been shown to confer either radiosensitization or radioprotection to human cells. We investigated nanoceria's ability to modify cellular health and reactive oxygen species (ROS) at various absorbed doses (Gray) of ionizing radiation in MDA-MB231 breast carcinoma cells. We used transmission electron microscopy to visualize the uptake and compartmental localization of nanoceria within cells at various treatment concentrations. The effects on apoptosis and other cellular health parameters were assessed using confocal fluorescence imaging and flow cytometry without and with various absorbed doses of ionizing radiation, along with intracellular ROS levels. Our results showed that nanoceria were taken up into cells mainly by macropinocytosis and segregated into concentration-dependent large aggregates in macropinosomes. Confocal imaging and flow cytometry data showed an overall decrease in apoptotic cell populations in proportion to increasing nanoparticle concentrations. This increase in cellular health was observed with a corresponding reduction in ROS at all tested absorbed doses. Moreover, this effect appeared pronounced at lower doses compared to unirradiated or untreated populations. In conclusion, internalized nanoceria confers radioprotection with a corresponding decrease in ROS in MDA-MB231 cells, and this property confers significant perils and opportunities when utilized in the context of radiotherapy. [ABSTRACT FROM AUTHOR]

Macropinocytosis is a unique pathway of endocytosis characterised by the nonspecific internalisation of large amounts of extracellular fluid, solutes and membrane in large endocytic vesicles known as macropinosomes. Macropinocytosis is important in a range of physiological processes, including antigen presentation, nutrient sensing, recycling of plasma proteins, migration and signalling. It has become apparent in recent years from the study of specialised cells that there are multiple pathways of macropinocytosis utilised by different cell types, and some of these pathways are triggered by different stimuli. Understanding the physiological function of macropinocytosis requires knowledge of the regulation and fate of the macropinocytosis pathways in a range of cell types. Here, we compare the mechanisms of macropinocytosis in different primary and immortalised cells, identify the gaps in knowledge in the field and discuss the potential approaches to analyse the function of macropinocytosis in vivo. [ABSTRACT FROM AUTHOR]

Macropinocytosis has emerged as an important mechanism for non-selective route to internalize extracellular fluids and dissolved molecules in eukaryotic cell. As fundamental cellular behavior, macropinocytosis plays specific and distinct roles in many physiological and pathological processes, such as nutrients uptake, antigen presentation, pathogen capture, and tumorigenesis. It supports tumorigenesis by providing metabolic needs to dividing cells in Ras driven cancer. In recent years, macropinocytosis has gained considerable interest in physiology and various diseases, including cancer, neurodegenerative diseases and atherosclerosis, which in turn has led to the discovery of new endocytic recycling systems. Approaches to assess macropinocytosis will provide insight into its underlying regulatory molecular mechanisms and enable the physiological control of macropinocytosis for controlled drug delivery and targeted cancer therapy. Macropinocytosis is an important phenomenon in Ras-expressing cancer cells and, recently, we have revealed a functional role for macropinocytosis in cancer associated fibroblasts (CAFs) fueling cancer cell growth. Here, we describe a protocol for detection of macropinocytosis in prostatic fibroblasts in vitro by utilizing fluorescently-labeled, lysine-fixable, 70 kDa high molecular weight dextran. Macropinosomes are visualized as fluorescent intracellular puncta either by confocal or fluorescent microscopy. To follow, subsequent intracellular events and their underlying mechanisms after macropinosomes formation, we perform co-localization of quenched BSA (DQ™-BSA) along with dextran labeling in cancer associated fibroblasts. Our protocol provides a consistent way to understand macropinocytosis in wild type or genetic manipulated prostatic fibroblast., Competing Interests: The authors declare that they have no conflict of interest.

Dynamic remodelling of the cortical actin cytoskeleton is required for phagocytic uptake of pathogens and other particles by macrophages. Actin can also be nucleated de novo on membranes of nascent phagosomes, a process that can stimulate or inhibit phagosome fusion with lysosomes. Recently, phagosomes were shown to polymerize actin in transient pulses, called actin `flashing', whose function remains unexplained. Here, we investigated phagosomal actin dynamics in live macrophages expressing actin tagged with green fluorescent protein (GFP). We show that only immature phagosomes can transiently induce assembly of actin coat, which forms a barrier preventing phagosome-lysosome docking and fusion. The capacity of phagosomes to assemble actin is enhanced in cells exposed to increased phagocytic load, which also exhibit a delay in phagosome maturation. Parallel analysis indicated that polymerization of actin on macropinosomes also induces compression and propulsion. We show that dynamic interactions between membrane elastic tension and compression forces of polymerizing actin can also lead to macropinosome constriction and scission - a process that is obstructed on rigid phagosomes. We hypothesize that the rate of individual phagosome maturation, as well as the biogenesis and remodelling of macropinosomes, can be regulated by the extent and manner of actin assembly on their membrane. [ABSTRACT FROM AUTHOR]