Developmental function of Nm23/awd: a mediator of endocytosis

Abstract

The metastasis suppressor gene Nm23 is highly conserved from yeast to human, implicating a critical developmental function. Studies in cultured mammalian cells have identified several potential functions, but many have not been directly verified in vivo. Here, we summarize the studies on the Drosophila homolog of the Nm23 gene, named a bnormal w ing d iscs (awd), which shares 78% amino acid identity with the human Nm23-H1 and H2 isoforms. These studies confirmed that awd gene encodes a nucleoside diphosphate kinase, and provided strong evidence of a role for awd in regulating cell differentiation and motility via regulation of growth factor receptor signaling. The latter function is mainly mediated by control of endocytosis. This review provides a historical account of the discovery and subsequent analyses of the awd gene. We will also discuss the possible molecular function of the Awd protein that underlies the endocytic function.

Fig. 1. Genetic cross that uncovered K-pn

The diagram follows the conventional designation of Drosophila genotype: X genes/X genes (or X/Y); 2nd chromosome genes/2nd chromosome genes; 3rd chromosome genes/3rd chromosome genes. Note that the 4th chromosome contains very few genes and is commonly omitted in genotyping. (a) In the original experiment conducted by Sturtevant to show the X-linkage of pn mutant, pn homozygous females were crossed with presumed wild-type males. The expected progenies would be that all females were phenotypically normal (red eyes) and all males had pn eyes. (b) Since the presumably normal fathers were actually K-pn homozygous mutant, all female progenies were pn and K-pn double heterozygotes while all male progenies were pn hemizygous and K-pn heterozygous. Since all female progenies were normal and viable, but no male progenies were recovered, the conclusion was that K-pn is a dominant, gain-of-function (or neomorphic) conditional lethal mutation in the pn genetic background.

Fig. 2. awd mutation results in ectopic tracheal tubule migration and accumulation of FGFR

(a) Wild-type and awd mutants embryos carrying a trachea-specific lacZ transgene were stained for LacZ to visualize tracheal cells. Wild-type embryos show organized array of tracheal tubules. In severe awd mutants, the tubule structure was completely disrupted and randomly motile tracheal cells can be seen (arrowheads point to some examples). Bars are 100 µm. (b) Wild-type and awd mutant embryos carrying the trachea-specific lacZ transgene were transformed with another transgene expressing FGFR-GFP fusion protein. The embryos were double stained for LacZ (red) and GFP (green). Zoom-in view of the migrating tips is shown. The tips migrate toward the top in this view. FGFR-GFP in wild-type embryo shows some membrane localization (arrow) but mostly is internalized. In awd mutants, FGFR-GFP is seen accumulated on the cell surface. Bars are 5 µm.

Fig. 3. awd expression inhibits border cell migration

(a) Schematic presentation of stage 8 and stage 9 Drosophila egg chambers. Anterior of the eggs are on the left. At stage 8, the germ cell complex consisting of nurse cells (NC) and the oocyte was enveloped by a single layer of somatic follicle cells (FC). At stage 9, a group of 6–10 follicle cells named border cells (BC, colored orange) originally located at the anterior end of the follicular epithelium delaminate from the epithelium and invade through the nurse cell complex. Arrow indicates the direction of border cell movement. (b) Stage 8 and stage 9 egg chambers from wild-type (left and middle panels, wt) and transgenics re-expressing awd in the border cells (right panel, +awd) were stained for β-catenin (red) and Awd (green). β-catenin stains follicle cell apical-lateral membrane and marks the border cells (arrows). In wild-type egg chambers, border cells delaminate from the follicle cell layer and move into the nurse cell proper (middle panel). These invading border cells lost Awd expression as compared to stage 8 when they remain in the follicle cell layer at the anterior (left panel). When Awd is re-expressed in the border cells (right panel), the border cells fail to move from the anterior tip.