Uptake Of Extracellular Materials Research Articles

Membrane Trafficking Mechanisms and Their Biological Relevance.

Most chemicals expressed in mammalian cells have complex delivery and transport mechanisms to get to the right intracellular sites. One of these mechanisms transports most transmembrane proteins, as well as almost all secreted proteins, from the endoplasmic reticulum, where they are formed, to their final location. Nearly all eukaryotic cells have a membrane trafficking mechanism that is both a prominent and critical component. This system, which consists of dynamically coupled compartments, supports the export and uptake of extracellular material, remodeling and signaling at the cellular interface, intracellular alignment, and maintenance of internal compartmentalization (organelles). In animal cells, this system enables both regular cellular activities and specialized tasks, such as neuronal transmission and hormone control. Human diseases, including neurodegenerative diseases, such as Alzheimer's disease, heart disease, and cancer, are associated with the dysfunction or dysregulation of the membrane trafficking system. Treatment and cure of human diseases depends on understanding the cellular and molecular principles underlying membrane trafficking pathways. A single gene mutation or mutations that result in impaired membrane trafficking cause a range of clinical disorders that are the result of changes in cellular homeostasis. Other eukaryotic organisms with significant economic and agricultural value, such as plants and fungi, also depend on the membrane trafficking system for their survival. In this review, we focused on the major human diseases associated with the process of membrane trafficking, providing a broad overview of membrane trafficking.

Membrane Trafficking Mechanisms and Their Biological Relevance

Most chemicals expressed in mammalian cells have complex delivery and transport mechanisms to get to the right intracellular sites. One of these mechanisms transports most transmembrane proteins, as well as almost all secreted proteins, from the endoplasmic reticulum, where they are formed, to their final location. Nearly all eukaryotic cells have a membrane trafficking mechanism that is both a prominent and critical component. This system, which consists of dynamically coupled compartments, supports the export and uptake of extracellular material, remodeling and signaling at the cellular interface, intracellular alignment, and maintenance of internal compartmentalization (organelles). In animal cells, this system enables both regular cellular activities and specialized tasks, such as neuronal transmission and hormone control. Human diseases, including neurodegenerative diseases, such as Alzheimer's disease, heart disease, and cancer, are associated with the dysfunction or dysregulation of the membrane trafficking system. Treatment and cure of human diseases depends on understanding the cellular and molecular principles underlying membrane trafficking pathways. A single gene mutation or mutations that result in impaired membrane trafficking cause a range of clinical disorders that are the result of changes in cellular homeostasis. Other eukaryotic organisms with significant economic and agricultural value, such as plants and fungi, also depend on the membrane trafficking system for their survival. In this review, we focused on the major human diseases associated with the process of membrane trafficking, providing a broad overview of membrane trafficking.

Early Endosomes Act as Local Exocytosis Hubs to Repair Endothelial Membrane Damage.

The plasma membrane of a cell is subject to stresses causing ruptures that must be repaired immediately to preserve membrane integrity and ensure cell survival. Yet, the spatio-temporal membrane dynamics at the wound site and the source of the membrane required for wound repair are poorly understood. Here, it is shown that early endosomes, previously only known to function in the uptake of extracellular material and its endocytic transport, are involved in plasma membrane repair in human endothelial cells. Using live-cell imaging and correlative light and electron microscopy, it is demonstrated that membrane injury triggers a previously unknown exocytosis of early endosomes that is induced by Ca2+ entering through the wound. This exocytosis is restricted to the vicinity of the wound site and mediated by the endosomal soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) VAMP2, which is crucial for efficient membrane repair. Thus, the newly identified Ca2+ -evoked and localized exocytosis of early endosomes supplies the membrane material required for rapid resealing of a damaged plasma membrane, thereby providing the first line of defense against damage in mechanically challenged endothelial cells.

Quantitative Assessment of Macropinocytosis in mTORC1-Hyperactive Cells using Flow Cytometry.

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.

Abstract I22: New players and unique features of cancer lysosomes

Abstract Pancreatic ductal adenocarcinoma (PDA) cells and tumors constitutively activate nutrient scavenging pathways, namely, autophagy (cellular self-catabolism) and macropinocytosis (bulk uptake of extracellular material) in order to maintain metabolic homeostasis. Importantly, both of these pathways converge on the lysosome, an acidic organelle that harbors within its lumen a complex series of enzymatic reactions that enable degradation of incoming cargo material. Our prior work has shown that the lysosome is greatly expanded and qualitatively different in PDA cells compared to normal tissue. PDA tumors are effectively “addicted” to enhanced catabolic activity as blocking lysosome function specifically inhibits in vitro and in vivo growth of PDA cell lines and tumor xenografts with minimal effects on normal cells. How key changes in lysosome composition and content contribute to the aggressive nature of PDA remains an open question. We have optimized methodologies to isolate and purify lysosomes from normal human pancreatic epithelial cells and several established patient-derived PDA cancer cell lines, using affinity-based capture techniques that isolate 50-80% intact lysosomes free of cytoplasm and other contaminating organelles. By systematically implementing lysosome purification from normal, primary, and metastatic PDA tumors combined with mass spectrometry-based proteomics, we have identified both resident and substrate proteins that are uniquely associated with PDA lysosomes. Several cancer-specific lysosomal membrane proteins emerged from this analysis that may endow cancer lysosomes with enhanced functionality, including rapid membrane repair in the face of sustained mechanical and chemical insults. Similarly, we find that PDA lysosomes degrade select cellular proteins to enable dynamic remodeling of the cellular proteome in a manner that favors tumor growth and progression. Collectively, our data provide strong evidence in support of the lysosome as a central mediator of cellular adaptation to stress and promoter of tumorigenesis. Citation Format: Rushika M Perera. New players and unique features of cancer lysosomes [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr I22.

Mouse and Human Neuronal Pentraxin 1 (NPTX1): Conservation, Genomic Structure, and Chromosomal Localization

We have previously identified novel members of the pentraxin family (neuronal pentraxin 1 and 2) that are expressed in the nervous system. Neuronal pentraxin 1 (NP1) was identified as a rat protein that may mediate the uptake of synaptic material and the presynaptic snake venom toxin, taipoxin. NP2 was identified as a separate gene discovered by screening for a human homolog for NP1. Here, we report human cDNA and mouse genomic DNA sequences for NP1 (gene symbol NPTX1). Human NP1 and mouse NP1 show 95 and 99% amino acid identity, respectively, with rat NP1 and conserve all potential glycosylation sites. Like rat NP1, human NP1 message is large (6.5 kb) and is exclusively localized to the nervous system. The mouse NP1 gene is 13 kb in length and contains four introns that break the coding sequence of NP1 in the same positions as the introns of the human NP2 gene. The human and mouse NP1 genes are localized to chromosome 17q25.1–q25.2 and chromosome 11e2–e1.3, respectively. These data demonstrate the existence of a separate family of pentraxin proteins that are expressed in the human brain and other tissues and that may play important roles in the uptake of extracellular material.

Human Neuronal Pentraxin II (NPTX2): Conservation, Genomic Structure, and Chromosomal Localization

We have previously identified a novel rat neuronal member of the pentraxin family (neuronal pentraxin) that may mediate the uptake of synaptic material and the presynaptic snake venom toxin, taipoxin. Here we report human cDNA and genomic sequences of a second neuronal pentraxin. This pentraxin, which we propose to name neuronal pentraxin II (NPII; gene symbol NPTX2), shows 54% amino acid identity to rat neuronal pentraxin (NPI) with 69% identity over the carboxyl-terminal half of NPI and is 88% identical to a newly identified sperm acrosomal pentraxin p50/apexin. Northern blot analysis reveals that NPII message is present in brain, testis, pancreas, liver, heart, and skeletal muscle, so, unlike NPI, NPII is not exclusively localized to neurons. Like NPI, NPII has potential N-linked glycosylation sites. The human NPII gene is 11 kb in length, contains four introns, and is localized to chromosome 7q21.3-q22.1. These data demonstrate the existence of a family of pentraxin proteins that are expressed in the brain and other tissues and that may play important roles in the uptake of extracellular material.

Structure of the endodermal epithelium of the chick yolk sac during early stages of development

The structure of the areas pellucida and vasculosa of the early chick embryo (stages 11-29) was examined by light, transmission and scanning electron microscopy. The most striking feature of the endodermal cells of these areas is the presence of large intracellular yolk drops which are characteristic of the regions in which they are found; lipid-like homogeneous drops in the area pellucida, heterogeneously composed pleomorphic drops in the mid-region of the area vasculosa and granular drops at the periphery of the area vasculosa in the region of the sinus terminalis. On morphological criteria it is postulated that granular drops may arise by direct engulfment of extracellular yolk, but this does not appear to be true for pleomorphic or homogeneous drops. Since the apical junctions between endodermal cells across the yolk sac are tight, they seal off the extraembryonic compartment from the vitelline circulation and presumably prevent intercellular passage of the yolk constituents. Thus the endodermal epithelium must mediate the transport of nutrients from the yolk mass to the developing embryo. Endodermal cells exhibit a variation across the yolk sac in the presence and number of structures associated with uptake of extracellular materials. The mid-region of the area vasculosa appears to be the most endocytotically active region with an abundance of microvilli, bristle-coated pits and vesicles and apical canaliculi and vacuoles. There is a close association between the endoderm and vitelline blood vessels and this association is maintained, as the yolk sac develops, by the formation of small vessels juxtaposed between the vascular surface of the endoderm and the walls of the large vitelline vessels.

Effects of metabolic inhibitors on the release of glutamate from the retina.

Abstract—The superfused, isolated retina of the chicken was used to investigate the mechanisms responsible for the increase in retinal transparency and the release of glutamate associated with stimuli known to elicit spreading depression (SD). We sought to distinguish between (1) mechanisms involving glutamate‐induced increase in Na+ permeability and consequent uptake of extracellular material into the intracellular compartment and (2) mechanisms involving interference with operation of the Na+ pump that would result in a similar uptake of extracellular materials. Tetrodotoxin (which inhibits inward movements of Na+) depressed the transparency increase caused by stimulation with glutamate but not that elicited by application of KCl. Ouabain (which inhibits the Na+ pump) caused a marked increase in tissue transparency. The application of inhibitors of the aerobic metabolism, such as DNP or cyanide, or deprivation of O2 had no effect on the retinal transparency; results suggesting that the energy for the Na+ pump could be supplied by glycolysis. Indeed iodoacetate (which inhibits glycolysis) caused a marked change in transparency. Furthermore we found evidence for a compound in the superfusion fluid supplemented with iodoacetate that may be a reaction product of glutamate and iodoacetate. In some preparations superfusion with glucose‐free solutions caused a slowly developing increase in transparency and release of glutamate; in others the increase in transparency was more sudden and there was a larger release of glutamate. Seemingly, interference with the tissue metabolism can cause an uptake of extracellular material either by arrest of the Na+ pump or by the release of glutamate, depending on the conditions of the experiment.