Mammalian cells take up extracellular material by a variety of different mechanisms that are collectively termed endocytosis. Endocytic mechanisms serve many important cellular functions including the uptake of extracellular nutrients, regulation of cell-surface receptor expression, maintenance of cell polarity, and antigen presentation. Endocytic pathways are also utilized by viruses, toxins, and symbiotic microorganisms to gain entry into cells. One of the best-characterized endocytic mechanisms is receptor-mediated endocytosis via clathrin-coated pits. This type of endocytosis constitutes the major emphasis of this review, with a brief discussion of other endocytic mechanisms and their comparison with the receptor-mediated pathway. This review describes and evaluates critically current understanding of the mechanisms of entry of plasma membrane components such as the receptor-ligand complexes and membrane lipids as well as the extracellular fluid into cells. The intracellular sorting and trafficking of these molecules upon internalization are also described. The roles of endocytosis in physiological and pathological processes are discussed. These include maintenance of cell polarization, antigen presentation, glucose transport, atherosclerosis, Alzheimer's disease, and the endocytosis of toxins and viruses.
Microglia are immune system cells associated with Alzheimer's disease plaques containing beta-amyloid (A beta). Murine microglia internalize microaggregates of fluorescently labeled or radioiodinated A beta peptide 1-42. Uptake was confirmed using aggregates of unlabeled A beta detected by immunofluorescence. Uptake of A beta was reduced by coincubation with excess acetyl-low density lipoprotein (Ac-LDL) or other scavenger receptor (SR) ligands, and Dil-labeled Ac-LDL uptake by microglia was blocked by excess A beta. CHO cells transfected with class A or B SRs showed significantly enhanced uptake of A beta. These results show that microglia express SRs that may play a significant role in the clearance of A beta plaques. Binding to SRs could activate inflammation responses that contribute to the pathology of Alzheimer's disease.
To examine TGN38 trafficking from the cell surface to the TGN, CHO cells were stably transfected with a chimeric transmembrane protein, TacTGN38. We used fluorescent and 125I-labeled anti-Tac IgG and Fab fragments to follow TacTGN38's postendocytic trafficking. At steady-state, anti-Tac was mainly in the TGN, but shortly after endocytosis it was predominantly in early endosomes. 11% of cellular TacTGN38 is on the plasma membrane. Kinetic analysis of trafficking of antibodies bound to TacTGN38 showed that after short endocytic pulses, 80% of internalized anti-Tac returned to the cell surface (t 1/2 = 9 min), and the remainder trafficked to the TGN. When longer filling pulses and chases were used to load anti-Tac into the TGN, it returned to the cell surface with a t 1/2 of 46 min. Quantitative confocal microscopy analysis also showed that fluorescent anti-Tac fills the TGN with a 46-min t 1/2. Using the measured rate constants in a simple kinetic model, we predict that 82% of TacTGN38 is in the TGN, and 7% is in endosomes. TacTGN38 leaves the TGN slowly, which accounts for its steady-state distribution despite the inefficient targeting from the cell surface to the TGN.
Highly fluorescent copper nanoclusters (Cu NCs) have been synthesized using single-step reduction of copper sulfate by hydrazine in the presence of lysozyme. The fluorescence quantum yield was measured to be as high as 18%. The emission was also found to be dependent on the excitation wavelength. Mass spectrometric analyses indicated the presence of species corresponding to Cu2 to Cu9. Transmission electron microscopic analyses indicated the formation of agglomerated particles of average diameter of 2.3 nm, which were constituted of smaller particles of average diameter of 0.96 nm. They were found to be stable between pH 4 and 10 and in addition having excellent chemical and photostability. The noncytotoxic NCs were used to successfully label cervical cancer HeLa cells.
We have developed a technique to detect, recognize, and track each individual low density lipoprotein receptor (LDL-R) molecule and small receptor clusters on the surface of human skin fibroblasts. Molecular recognition and high precision (30 nm) simultaneous automatic tracking of all of the individual receptors in the cell surface population utilize quantitative time-lapse low light level digital video fluorescence microscopy analyzed by purpose-designed algorithms executed on an image processing work station. The LDL-Rs are labeled with the biologically active, fluorescent LDL derivative dil-LDL. Individual LDL-Rs and unresolved small clusters are identified by measuring the fluorescence power radiated by the sub-resolution fluorescent spots in the image; identification of single particles is ascertained by four independent techniques. An automated tracking routine was developed to track simultaneously, and without user intervention, a multitude of fluorescent particles through a sequence of hundreds of time-lapse image frames. The limitations on tracking precision were found to depend on the signal-to-noise ratio of the tracked particle image and mechanical drift of the microscope system. We describe the methods involved in (i) time-lapse acquisition of the low-light level images, (ii) simultaneous automated tracking of the fluorescent diffraction limited punctate images, (iii) localizing particles with high precision and limitations, and (iv) detecting and identifying single and clustered LDL-Rs. These methods are generally applicable and provide a powerful tool to visualize and measure dynamics and interactions of individual integral membrane proteins on living cell surfaces.
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