Barbara M. Mullock

Combinatorial SNARE complexes with VAMP7 or VAMP8 define different late endocytic fusion events

Both heterotypic and homotypic fusion events are required to deliver endocytosed macromolecules to lysosomes and remodel late endocytic organelles. A trans‐SNARE complex consisting of Q‐SNAREs syntaxin 7, Vti1b and syntaxin 8 and the R‐SNARE VAMP8 has been shown by others to be responsible for homotypic fusion of late endosomes. Using antibody inhibition experiments in rat liver cell‐free systems, we confirmed this result, but found that the same Q‐SNAREs can combine with an alternative R‐SNARE, namely VAMP7, for heterotypic fusion between late endosomes and lysosomes. Co‐immunoprecipitation demonstrated separate syntaxin 7 complexes with either VAMP7 or VAMP8 in solubilized rat liver membranes. Additionally, overexpression of the N‐terminal domain of VAMP7, in cultured fibroblastic cells, inhibited the mixing of a preloaded lysosomal content marker with a marker delivered to late endosomes. These data show that combinatorial interactions of SNAREs determine whether late endosomes undergo homotypic or heterotypic fusion events.

Membrane dynamics and the biogenesis of lysosomes (Review)

Lysosomes are dynamic organelles receiving membrane traffic input from the biosynthetic, endocytic and autophagic pathways. They may be regarded as storage organelles for acid hydrolases and are capable of fusing with late endosomes to form hybrid organelles where digestion of endocytosed macromolecules occurs. Reformation of lysosomes from the hybrid organelles involves content condensation and probably removal of some membrane proteins by vesicular traffic. Lysosomes can also fuse with the plasma membrane in response to cell surface damage and a rise in cytosolic Ca 2+ concentration. This process is important in plasma membrane repair. The molecular basis of membrane traffic pathways involving lysosomes is increasingly understood, in large part because of the identification of many proteins required for protein traffic to vacuoles in the yeast Saccharomyces cerevisiae. Mammalian orthologues of these proteins have been identified and studied in the processes of vesicular delivery of newly synthesized lysosomal proteins from the trans-Golgi network, fusion of lysosomes with late endosomes and sorting of membrane proteins into lumenal vesicles. Several multi-protein oligomeric complexes required for these processes have been identified. The present review focuses on current understanding of the molecular mechanisms of fusion of lysosomes with both endosomes and the plasma membrane and on the sorting events required for delivery of newly synthesized membrane proteins, endocytosed membrane proteins and other endocytosed macromolecules to lysosomes.

EFFECT OF COLCHICINE ON THE TRANSFER OF IgA ACROSS HEPATOCYTES INTO BILE IN ISOLATED PERFUSED RAT LIVERS

In hepatocytes a stream of endocytic vesicles moves rapidly from the sinusoidal surface to the cyto- plasm in the region of the bile canaliculus. The vesi- cles carry polymeric IgA or the haptoglobin-haemo- globin complex if these are available in the blood [l-4]. The rapid concentration in a particular region of cytoplasm suggests that cytoskelktal elements may be involved. Accordingly we have examined the effects of the microtubule-disruptive drug, colchicine, on the transport of IgA across hepatocytes. Transportation of IgA across hepatocytes involves at least 4 separate processes which could require participation of microtubules. (1) Before uptake of IgA can take place, the receptor for IgA, the glycoprotein secretory component, which is synthesised in hepatocytes [5], must itself reach the sinusoidal plasma membrane [6]. Such movement of glycoproteins from the Golgi apparatus to the plasma membrane is inhibited by colchicine although glycoprotein synthesis is unaffected [7]. (2) However, if this were the only point of interfer- ence, the secretory component already on the membrane before the addition of colchicine would be expected to continue to transfer any IgA available to it, and also continue to move to the bile on its own [4]. The actual formation of endocytic vesicles is unlikely to be inhibited since in [8] the movement of the plasma mem- brane enzyme 5’-nucleotidase into the interior of the cell was unaffected by colchicine. t

The polymeric immunoglobulin A receptor is present on hepatocytes in human liver.

A monoclonal antibody raised against human colostrum secretory component produced even staining of hepatocyte plasma membranes, as well as bile duct lining cells, in all sections examined from eight normal and three abnormal human livers. Human bile samples incubated with free secretory component degraded it to varying extents, probably proteolytically; true levels of free secretory component will therefore often be higher than those reported. It seems likely that human liver resembles that of other mammals in transferring polymeric IgA through hepatocytes to the bile by means of the polymeric IgA receptor.

Immune responses to chlorpromazine in rats: Detection and relation to hepatotoxicity

It has frequently been suggested that the jaundice which occurs in a small percentage of human patients following treatment with chlorpromazine is due to a hypersensitivity reaction. It has, however, proved impossible to obtain an animal model for this condition. We now show that oral administration of chlorpromazine at 25 mg/kg per day to Wistar albino rats results in formation of both humoral and secretory antibodies to chlorpromazine. We also demonstrate that the severity of the hepatic changes observed in chlorpromazine-fed animals (periportal glycogen loss and centrilobular fatty change) is enhanced by preimmunization of the rats via the gut-associated lymphoid tissue with a chlorpramizine-protein conjugate. There was, however, no correlation between the titre of either serum or biliary antibodies in individual animals and the degree of liver damage. Our results therefore suggest than an immune mechanism is indeed implicated in chlorpromazine toxicity but show clearly that toxic symptoms are not a simple consequence of the formation of anti-chlorpromazine antibodies.

The presence and measurement of secretory component in human bile and blood

Five monoclonal antibodies which recognized three separate epitopes on the free secretory component molecule were produced using free secretory component obtained from human colostrum. Two-site immunoradiometric assays were developed to measure free secretory component and secretory IgA. Monoclonal antibody M9 was used on coated plates as the capture antibody. Monoclonal antibody M7 was used as the labelled signal antibody for the assay of free secretory component and a commercially available monoclonal anti-IgA antibody was used as the labelled signal antibody for the assay of secretory IgA. Free secretory component was found in human serum and bile. In serum, its concentration was raised in patients with high serum alkaline phosphatase due to liver disorders but not in patients with high serum alkaline phosphatase due to non-liver disorders. In bile from bile duct drains collected during the first week after liver transplantation, free secretory component was found in concentrations of up to 33 mg/l, in vast excess of that found in bile from gallstone patients (up to 0.3 mg/l). Bile from gallstone patients but not from liver transplant patients produced proteolytic degradation of free secretory component when incubated in vitro. The finding of large amounts of free secretory component, the free cleaved fragment of the polymeric IgA receptor in human bile, further supports the existence of the blood to bile transhepatocytic pathway in humans.

The Interaction of Late Endosomes with Lysosomes in a Cell-Free System

The passage of asialofetuin (ASF) through the endocytic pathway of rat hepatocytes is well described and involves binding to asialoglycoprotein receptors, internalisation via coated pits, appearance in an early, peripheral endosomal compartment(s) where the ligand becomes dissociated from receptor, appearance in a late, deep-lying endosomal compartment(s) and finally digestion in lysosomes (for references see Branch et al., 1987; Mullock et al., 1988). 125I-ASF movement through the various endosomal compartments in rat hepatocytes has been demonstrated by isopycnic centrifugation on vertical density gradients of Ficoll (1 – 22% w/v) and Nycodenz (0.25M sucrose – 45% Nycodenz), which respectively separate early from late endosomal compartments and endosomes from lysosomes (Branch et al., 1987; Perez et al., 1988).

Theory of Organelle Biogenesis

Organelles, defined as intracellular membrane-bound structures in eukaryotic cells, were described from the early days of light microscopy and the development of cell theory in the 19th century. During the 20th century, electron microscopy and subcellular fractionation enabled the discovery of additional organelles and, together with radiolabel-ling, allowed the first modern experiments on their biogenesis. Over the past 30 years, the development of cell-free systems and the use of yeast genetics have together established the major pathways of delivery of newly synthesised proteins to organelles and the vesicular traffic system used to transfer cargo between organelles in the secretory and endocytic pathways. Mechanisms of protein sorting, retrieval and retention have been described and give each organelle its characteristic composition. Insights have been gained into the mechanisms by which complex organelle morphology can be established. Organelle biogenesis includes the process of organelle inheritance by which organelles are divided between daughter cells during mitosis. Two inheritance strategies have been described, stochastic and ordered, which are not mutually exclusive. Among the major challenges of the future are the need to understand the role of self-organization in ensuring structural stability and the mechanisms by which a cell senses the status of its organelles and regulates their biogenesis.

Signals for Transport from Endosomes to Lysosomes

It has been proposed either that endocytosed ligands are delivered to lysosomes by a process of endosome maturation (Murphy, 1991) or by vesicular transport (Griffiths and Gruenberg, 1991). The latter mechanism implies the possibility of generating cell-free systems in which endosome membrane vesicles fuse with lysosomes and which can be used to identify factors required for targeting and fusion. In our studies we have investigated the interactions between rat liver endosome and lysosome fractions previously shown to be involved in the endocytic pathway for asialoglycoproteins in hepatocytes.