Endocytosis of wheat germ agglutinin binding sites from the cell surface into a tubular endosomal network

Abstract

By using fluorescence and electron microscopy, the endocytic pathway encountered by cell surface components after they had bound wheat germ agglutinin (WGA) was visualized. The majority of these components are thought to consist of sialylated glycoproteins (HMWAG) that represent a subpopulation of the total cell surface proteins but most of the externally disposed plasma membrane proteins of the cell. Examination of semi-thin sections by medium- and high-voltage electron microscopy revealed the three-dimensional organization of vesicular and tubular endosomes. Binding of either fluorescein isothiocyanate-, horseradish peroxidase-, or ferritin-conjugated WGA to cells at 4 degrees C showed that the HMWAG were distributed uniformly over the cell surface. Warming of surface-labeled cells to 37 degrees C resulted in the endocytosis of WGA into peripheral endosomes via invagination of regions of both coated and uncoated membrane. The peripheral endosome appeared as isolated complexes comprising a vesicular element (300-400 nm diam.) surrounded by and continuous with tubular cisternae (45-60 nm diam.), which did not interconnect the endosomes. After 30 min or more label also became localized in a network of anastomosing tubules (45-60 nm diam.) that were located in the centrosomal region of the cell. Endocytosed WGA-HMWAG complexes did not become associated with cisternae of the Golgi apparatus, although tubular and vesicular endosomes were noted in the vicinity of the trans-Golgi region. The accumulation of WGA-HMWAG in the endosomes within the centrosomal region was inhibited when cells were incubated at 18 degrees C. None of these compartments contained acid phosphatase activity, a result that is consistent with other data that the HMWAG do not pass through lysosomes initially. The kinetics of labeling were consistent with the interpretation that recycling of most of the WGA binding surface glycoproteins occurred rapidly from early peripheral endosomes followed by the late trans-Golgi compartment. In conclusion, a portion of cell surface glycoproteins are routed to a complex arrangement of tubular and vesicular compartments following endocytosis that includes a putative post-endosomal, tubular reticulum that appears to be separate from the trans-most Golgi saccule.

The endocytic process in CHO cells, a toxic pathway of the polyene antibiotic amphotericin B

“…Moreover, low temperature (18°C) rules out the fusion of endosomes (28,29), and the repartition of fluorescent tracers in cells is then equivalent to the repartition observed in the presence of AmB, supporting the fact that in the presence of the polyene the endocytic process is blocked at one step.…”

Opposing effects of Ndel1 and α1 or α2 on cytoplasmic dynein through competitive binding to Lis1

“…WGA recognizes glycoproteins at the plasma membrane, endosome, and trans-Golgi cisternae (Liang et al, 2004;Raub et al, 1990;Virtanen et al, 1980). As endosomes and Golgi cisternae are subjected to dynein-mediated transport towards MT minus-ends, they are usually enriched around the MT-organizing center (MTOC) (Hirokawa, 1998;Karki and Holzbaur, 1999).…”

Sorting of membrane components from endosomes and subsequent recycling to the cell surface occurs by a bulk flow process.

“…At present we do not know what proportion of C6-NBD-SM is internalized via coated pits since some cell surface molecules such as glycolipids and glycolipid-binding proteins, ricin, cholera, and tetanus toxin, are internalized via noncoated pit mediated endocytosis in addition to clathrin-mediated endocytosis (for review see van Deurs et al, 1989). These two pathways appear to merge at the level of the peripherally distributed sorting endosomes (Raub et al, 1990a;Tran et al, 1987), and our data show that in TRVb-1 cells, as early as 2 min, greater than 80% of the total internalized C6-NBD-SM fluorescence is colocalized with Tf. Cumulatively, these data show that C6-NBD-SM is internalized via a constitutive 'bulk' process into the endocytic system and can be used as a marker for bulk membrane flow through the endocytic compartments.…”

“…Sorting endosomes have been characterized in BHK cells (early endosomes; Griffiths et al, 1989;Marsh et al, 1986) and CHO cells (Raub et al, 1990a;Yamashiro et al, 1984). They consist of a few narrow diameter (•50-60 nm) tubules attached to spherical vesicles ~250-500 nm in diameter.…”