Endocytosis and vesicular trafficking
John M. Graham, Joan A. Higgins in Membrane Analysis, 2020
This chapter considers methods for studying receptors, endocytosis and the small transfer vesicles which shuttle between organelles. Receptors are concentrated into specialized areas of the plasma membrane, which invaginate to form vesicles which move into the cytosol taking with them molecules bound to the receptors. Receptor-mediated endocytosis has a number of important roles in internalizing molecules which cannot cross the plasma membrane through transport proteins or channels. Nonspecific binding can be determined by addition of an excess of the true ligand or an agonist, which will compete for binding. If the receptor is involved in endocytosis the principles are the same; however, the incubation must be carried out at 0–4°C to prevent internalization. Conventional biochemical methods has therefore been necessary to develop novel systems to investigate the organization of cells and vesicular transfer between organelles. The factors involved in vesicular tranport have been identified using a cell-free reconstituted system based on that developed by Rothman and co-workers.
TRPML Subfamily of Endolysosomal Channels
Bruno Gasnier, Michael X. Zhu in Ion and Molecule Transport in Lysosomes, 2020
The mucolipin subgroup of the transient receptor potential superfamily of cation channels (TRPMLs) are evolutionarily conserved non-selective cation channels that function in endolysosomal membranes, and play key roles in the regulation of endocytosis, autophagy, and intracellular trafficking. The lack of effective antibodies against TRPMLs, which is exacerbated by the low expression levels of these proteins, has hindered detailed examination of their tissue and subcellular distribution. Consequently, most studies investigating the location of these channels across tissues rely on mRNA quantification. The endocytic pathway is initiated at the plasma membrane, where molecularly defined regions of the bilayer are pinched inwards to form early endosomes via a pathway orchestrated with exquisite spatiotemporal precision. The purpose of the following sections is to provide an overview of some of the tools and resources that may be used to study the localization, trafficking, and channel function of TRPMLs.
Chapter 2: Application of Microspheres to Measure Cell Surface- Associated Proteolytic Enzyme Activities
Zoltán A. Tökés in Micro spheres: Medical and Biological Applications, 2017
This chapter summarizes the experience obtained with the rolling- microsphere assay and illustrates its usefulness to measure enzyme activity at the cell surface. It explains the results obtained with suspensions of erythrocytes, human and murine lymphocytes, and with monolayers of endothelial cells, myocytes, normal and transformed hepatomas, fibroblasts, and epithelial cells. The chapter formulates an approach that meets the two criteria in measuring proteolytic activity available at the cell surface. The first is that the substrate should be accessible only to the outside of the cells and should not be internalized by endocytosis. The second criterion is that the substrate should be available in two configurations, either in direct contact with the cell surface, or adjacent to the cell without contact. The sensitivity of the assay with monolayers of cells was similar to the one with suspensions of cells. Endothelial cells serve as a model for regulated tissue invasion.
Development of a monoclonal anti-ADAMTS-5 antibody that specifically blocks the interaction with LRP1
Published in mAbs, 2017
Salvatore Santamaria, Oleg Fedorov, John McCafferty, Gillian Murphy, Jayesh Dudhia, Hideaki Nagase, Kazuhiro Yamamoto
The potent aggrecanase ADAMTS-5 is constitutively secreted by chondrocytes, but it is rapidly endocytosed in normal cartilage via the cell surface endocytic receptor LRP1. Therefore it is difficult to detect the total ADAMTS-5 activity produced. In this study, we isolated a monoclonal anti-ADAMTS-5 antibody 1B7 that blocks LRP1-mediated internalization without affecting the aggrecanolytic activity. Addition of 1B7 to cultured human chondrocytes revealed the full aggrecanolytic activity of ADAMTS-5 generated by the cells. 1B7 is a useful tool to estimate the ADAMTS-5 activity and to identify its potential roles in the tissues.
A bispecific antibody against two different epitopes on hepatitis B surface antigen has potent hepatitis B virus neutralizing activity
Published in mAbs, 2013
Wenlong Tan, Yanchun Meng, Hui Li, Yang Chen, Siqi Han, Jing Zeng, Ang Huang, Bohua Li, Yanyun Zhang, Yajun Guo
Treatment of chronic hepatitis B virus (HBV) infection with interferon and viral reverse transcriptase inhibitor regimens results in poor viral clearance, loss of response, and emergence of drug-resistant mutant virus strains. These problems continue to drive the development of new therapeutic approaches to combat HBV. Here, we engineered a bispecific antibody using two monoclonal antibodies cloned from hepatitis B surface antigen (HBsAg)-specific memory B cells from recombinant HBsAg-vaccinated healthy volunteers. Next, we evaluated its efficacy in neutralizing HBV in HepaRG cells. This bispecific antibody, denoted as C4D2-BsAb, had superior HBV-neutralizing activity compared with the combination of both parental monoclonal antibodies, possibly through steric hindrance or induction of HBsAg conformational changes. Moreover, C4D2-BsAb has superior endocytotic characteristics into hepatocytes, which inhibits the secretion of HBsAg. These results suggest that the anti-HBsAg bispecific antibody may be an effective treatment method against HBV infection.
Endocytosis at the blood–brain barrier: From basic understanding to drug delivery strategies
Published in Journal of Drug Targeting, 2006
Mathew W. Smith, Mark Gumbleton
The blood–brain barrier (BBB) protects the central nervous system (CNS) from potentially harmful xenobiotics and endogenous molecules. Anatomically, it comprises the brain microvasculature whose functionality is nevertheless influenced by associated astrocyte, pericyte and neuronal cells. The highly restrictive paracellular pathway within brain microvasculature restricts significant CNS penetration to only those drugs whose physicochemical properties afford ready penetration into hydrophobic cell membranes or are capable of exploiting endogenous active transport processes such as solute carriers or endocytosis pathways. Endocytosis at the BBB is an essential pathway by which the brain obtains its nutrients and affords communication with the periphery. The development of strategies to exploit these endocytic pathways for the purposes of drug delivery to the CNS is still an immature field although some impressive results have been documented with the targeting of particular receptors. This current article initially provides an overview of general endocytosis processes and pathways showing evidence of their functional existence within the BBB. Subsequent sections provide, in an entity-specific manner, comprehensive reviews on BBB transport investigations of endocytosis involving: transferrin and the targeting of the transferrin receptor; hormones; cytokines; cell penetrating peptides; microorganisms and toxins, and nanoparticles aimed at more effectively delivering drugs to the CNS.
Related Knowledge Centers
- Endosomes
- Phagocytes
- Immunoglobulin
- Exocytosis
- Cell Physiological Phenomena
- Ldl
- Transferrin