Using ferritin-labeled protein A and colloidal gold-labeled anti-rabbit IgG, the fate of the sheep transferrin receptor has been followed microscopically during reticulocyte maturation in vitro. After a few minutes of incubation at 37°C, the receptor is found on the cell surface or in simple vesicles of 100-200 nm, in which the receptor appears to line the limiting membrane of the vesicles. With time (60 min or longer), large multivesicular elements (MVEs) appear whose diameter may reach 1-1.5/~m. Inside these large MVEs are round bodies of ~50-nm diam that bear the receptor at their external surfaces. The limiting membrane of the large MVEs is relatively free from receptor. When the large MVEs fuse with the plasma membrane, their contents, the 50-nm bodies, are released into the medium. The 50-nrn bodies appear to arise by budding from the limiting membrane of the intracellular vesicles. Removal of surface receptor with pronase does not prevent exocytosis of internalized receptor. It is proposed that the exocytosis of the ~50-nm bodies represents the mechanism by which the transferrin receptor is shed during reticulocyte maturation.It is well known that the transferrin receptor is lost during the maturation of the reticulocyte into the erythrocyte (1-4). Recent studies have shown that the maturation process can be followed in vitro (4, 5-7). The loss of the transferrin receptor can be used as a marker of maturation (4-6), the transferrin receptor being released, undegraded, to the medium in vesicular form (6, 7). The transferrin receptor is known to recycle many times during the course of Fe 3÷ delivery without degradation of either the receptor or the natural ligand, transferrin (8-l 1). It has been shown in several systems (8,(12)(13)(14)(15)(16)(17)(18)(19)(20), using either labeled transferrin or antibody against the transferrin receptor, that the ligand and the receptor are internalized into vesicles (endosomes) during incubation at temperatures above 10*C and that this internalization may constitute part of the iron delivery mechanism.Since the transferrin receptor is largely lost from sheep reticulocytes during 24 h of incubation in vitro (5-7), intermediate stages associated with vesicle externalization during receptor elimination might become apparent during the longterm incubation of reticulocytes. No studies to date have addressed the question of the processing that may occur to prepare the receptor for externalization during reticulocyte maturation. We have previously shown (6) that a polyclonal 942 antibody against the transferrin receptor is internalized (as judged by resistance to acid treatment), and that during the course of long-term incubation (several hours at 37"C), this internalized antibody and the surface-bound antibody are lost from the cells along with the antibody-binding capacity of the cells. Since no degraded antibody was found, both internal and surface antibody appear to be eliminated from the cells without degradation.The rate of loss of internalized 125I-antibody as we...

In most human cancers, only a few genes are mutated at high frequencies; most are mutated at low frequencies. The functional consequences of these recurrent but infrequent “long tail” mutations are often unknown. We focused on 484 long tail genes in head and neck squamous cell carcinoma (HNSCC) and used in vivo CRISPR to screen for genes that, upon mutation, trigger tumor development in mice. Of the 15 tumor-suppressor genes identified, ADAM10 and AJUBA suppressed HNSCC in a haploinsufficient manner by promoting NOTCH receptor signaling. ADAM10 and AJUBA mutations or monoallelic loss occur in 28% of human HNSCC cases and are mutually exclusive with NOTCH receptor mutations. Our results show that oncogenic mutations in 67% of human HNSCC cases converge onto the NOTCH signaling pathway, making NOTCH inactivation a hallmark of HNSCC.

We have assessed whether exosome formation is a significant route for loss of plasma membrane functions during sheep reticulocyte maturation in vitro. Although the recovery of transferrin binding activity in exosomes is at best approximately 25-30% of the lost activity, recoveries of over 50% of the lost receptor can be obtained if 125I-labelled transferrin receptor is measured using an that receptor instability may contribute to the less than quantitative recovery of the transferrin receptor. Significantly higher (75-80%) levels of the nucleoside transporter can be recovered in exosomes during red cell maturation using 3H-nitrobenzylthioinosine binding to measure the nucleoside transporter. These data suggest that exosome formation is a major route for removal of plasma membrane proteins during reticulocyte maturation and plasma membrane remodelling. We have also shown that both in vivo and in vitro, embryonic chicken reticulocytes form exosomes which contain the transferrin receptor. Thus, exosome formation is not restricted to mammalian red cells, but also occurs in red cells, which retain organelles, such as nuclei and mitochondria, into the mature red cell stage.

Vesicles (exosomes) released during sheep reticulocyte maturation contain a number of plasma membrane functions. Using an antibody coated, magnetic core bead, it has been shown unequivocally that vesicles that contain the transferrin receptor also contain other plasma membrane activities, such as the nucleoside transporter and acetylcholinesterase. Lysosomal activities, normally found in the same pellet, are excluded from the transferrin receptor-containing exosomes, suggesting that there is a common mechanism to segregate and externalize specific plasma membrane proteins. In addition to the sheep, electron micrographic studies show that exosomes can be retrieved from the circulation of anemic pigs, rats, and rabbits.