Coronin and vacuolin identify consecutive stages of a late, actin-coated endocytic compartment in Dictyostelium.

Cells of the unicellular eukaryote Dictyostelium discoideum take up all their nutrients by endocytosis. Both particle- and fluid-containing vacuoles are transiently surrounded by a cytoskeletal coat [1] [2]. When this coat has dissociated, acidification and digestion of the vesicle contents occur, followed by exocytosis of the indigestible remnants after 60-90 minutes. At least nine compartments are needed for mathematical modelling of endocytic transit [3], suggesting that markers associate for only a few minutes with a specific endocytic compartment. Among the proteins that have been identified as components of endocytic vesicles are actin, subunits of the V-H+ ATPase and small GTP-binding proteins of the Rab family [4] [5] [6] [7]. Using a monoclonal antibody produced against Dictyostelium endocytic vesicles, we have isolated a cDNA corresponding to a novel protein that we have named vacuolin. In order to determine the precise step along the endocytic pathway that involves vacuolin, we generated a fusion protein of the green fluorescent protein (GFP) and vacuolin. GFP-vacuolin-decorated vesicles were identified as a post-lysosomal compartment that acquires endocytic markers shortly before exocytosis. At earlier stages, this post-lysosomal compartment was identified by the binding of a tagged cytoskeletal protein, coronin-GFP. Vacuoles were coated with filamentous actin along the entire post-lysosomal pathway, and the integrity of the actin coat was required for exocytosis.

Dictyostelium discoideum protein disulfide isomerase, an endoplasmic reticulum resident enzyme lacking a KDEL-type retrieval signal.

The primary activity of protein disulfide isomerase (PDI), a multifunctional resident of the endoplasmic reticulum (ER), is the isomerization of disulfide bridges during protein folding. We isolated a cDNA encoding Dictyostelium discoideum PDI (Dd-PDI). Phylogenetic analyses and basic biochemical properties indicate that it belongs to a subfamily called P5, many members of which differ from the classical PDIs in many respects. They lack an intervening inactive thioredoxin module, a C-terminal acidic domain involved in Ca2+ binding and a KDEL-type retrieval signal. Despite the absence of this motif, the ER is the steady-state location of Dd-PDI, suggesting the existence of an alternative retention mechanism for P5-related enzymes.

High-throughput generation and engineering of recombinant human antibodies.

The first version of the Human Combinatorial Antibody Library (HuCAL) is a single-chain Fv-based phage display library (HuCAL-scFv) with 2x10(9) members optimised for high-throughput generation and targeted engineering of human antibodies. 61% of the library genes code for functional scFv as judged by sequencing. We show here that since HuCAL-scFv antibodies are expressed in high levels in Escherichia coli, automated panning and screening in miniaturised settings (96- and 384-well format) have now become feasible. Additionally, the unique modular design of HuCAL-genes and -vectors allows the distinctly facilitated conversion of scFv into Fab, miniantibody and immunoglobulin formats, and the fusion with a variety of effector functions and tags not only convenient for therapeutic applications but also for high-throughput purification and detection. Thus, the HuCAL principle enables the rapid and high-throughput development of human antibodies by optimisation strategies proven useful in classical low molecular weight drug development. We demonstrate in this report that HuCAL is a very convenient source of human antibodies for various applications.

Coronin involved in phagocytosis: dynamics of particle-induced relocalization visualized by a green fluorescent protein Tag.

Coronin is a protein involved in cell locomotion and cytokinesis of Dictyostelium discoideum. Here we show that coronin is strongly enriched in phagocytic cups formed in response to particle attachment. A fusion of coronin with green fluorescent protein (GFP) accumulates in the cups within less than 1 min upon attachment of a particle and is gradually released from the phagosome within 1 min after engulfment is completed. Phagocytic cup formation competes with leading edge formation and can be interrupted at any stage. When the cup regresses, coronin dissociates from the site of accumulation. TRITC-labeled yeast cells have been used to assay phagocytosis quantitatively in wild-type and coronin-null cells. In the mutant, the rate of uptake is reduced to about one third, which shows that coronin contributes to the efficiency of phagocytosis to about the same extent as it improves the speed of cell locomotion.

Targeted gene disruption reveals a role for vacuolin B in the late endocytic pathway and exocytosis.

Cells of Dictyostelium discoideum take up fluid by macropinocytosis. The contents of macropinosomes are acidified and digested by lysosomal enzymes. Thereafter, an endocytic marker progresses in an F-actin dependent mechanism from the acidic lysosomal phase to a neutral post-lysosomal phase. From the post-lysosomal compartment indigestible remnants are released by exocytosis. This compartment is characterised by two isoforms of vacuolin, A and B, which are encoded by different genes. Fusions of the vacuolin isoforms to the green fluorescent protein associate with the cytoplasmic side of post-lysosomal vacuoles in vivo. Vacuolin isoforms also localise to patches at the plasma membrane. Since vacuolins have no homologies to known proteins and do not contain domains of obvious function, we investigated their role by knocking out the genes separately. Although the sequences of vacuolins A and B are about 80% identical, only deletion of the vacuolin B gene results in a defect in the endocytic pathway; the vacuolin A knock-out appeared to be phenotypically normal. In vacuolin B- mutants endocytosis is normal, but the progression of fluid-phase marker from acidic to neutral pH is impaired. Furthermore, in the mutants post-lysosomal vacuoles are dramatically increased in size and accumulate endocytic marker, suggesting a role for vacuolin B in targeting the vacuole for exocytosis.