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First published online 31 May 2005
doi: 10.1242/jcs.02408

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Basigin (EMMPRIN/CD147) interacts with integrin to affect cellular architecture

¹ Department of Molecular, Cellular and Developmental Biology, Yale University, 266 Whitney Avenue, New Haven, CT 06511, USA
² Life Sciences Centre, Dalhousie University, 1355 Oxford Street, Nova Scotia, B3H 4J1, Canada
³ Department of Biological Sciences, Fulbright College of Arts & Sciences, University of Arkansas, Fayetteville, AR 72701, USA

View larger version (34K): [in a new window]   Fig. 1. Basigin gene structure and developmental northern blots. (A) Structure of the bsg gene encodes nine transcripts according to data from the Drosophila genome project. Transcribed portions of the gene are shown as blue boxes. Transcripts fall into classes 1a-d, 2a-d and 3. Transcripts 1a-d and 2a-d encode the same protein, D-basigin 265 (sequence shown in Fig. 2). Transcript 3 encodes a slightly longer protein, D-basigin 298. The P elements, P1096 and P1478, inserted at precisely the same location (marked by arrow) 1145 bp from the ATG for D-basigin 265 and 981 bp from the start of the first coding exon. The location of the start codon for transcript 3 is also indicated. The excision line 19 (black bar) contains a 4 kb deletion that removes the first coding exon. (B) Northern blot showing expression of bsg in embryos at stages 5, 9, 13, and 16, first-, second- and third-instar larvae and adult heads (A). The major band of 2.5 kb is consistent with the length of class 1 and 2 bsg transcripts.

View larger version (69K): [in a new window]   Fig. 2. Sequence alignment of basigin protein family members. Identical residues are marked in black and similar residues in yellow. Only the extreme C-terminus of D-basigin 298 is shown (last line). The first 50 amino acids (aa) of D-basigin 298 are not homologous to the first 24 aa of D-basigin 265. Then, the two proteins are identical for the 240 aa from residue 25 in D-basigin 265 (corresponding to residue 50 in basigin 298) through to the end of D-basigin 265. There are eight additional aa at the C-terminus of D-basigin 298. D-basigin 298 accession number CG31605-PG can be found at NCBI or flybase{at}bio.indiana.edu.

View larger version (111K): [in a new window]   Fig. 3. D-basigin expressing cells spread and form lamellipodia. (A) Actin cytoskeleton of High Five cell labeled with Alexa-568 phalloidin. (B) The same cell labeled with anti-tubulin antibody and visualized with Alexa-488 conjugated secondary antibody. (C) High Five cell expressing basigin labeled with Alexa-568 phalloidin. (D) The same cell as in C labeled with antibody to tubulin. High Five cells have a spherical morphology (A and B) whereas High Five cells expressing D-basigin form lamellipodia (C and D). Bar, 40 µm.

View larger version (69K): [in a new window]   Fig. 4. D-basigin partially colocalizes with actin and integrin. (A-F) High Five cells expressing D-basigin with a C-terminal V5 tag. (A and C) Cells labeled with anti-V5 antibody and visualized with Alexa 488-conjugated secondary antibody. (B,D-F) Cells labeled with Alexa 568-phalloidin to visualize F-actin. (A) D-basigin is expressed diffusely and in numerous vesicles within the cell. There is strong D-basigin expression at cell-cell contacts (arrow). (B) Actin colocalizes with D-basigin at cell-cell contacts (arrow). (C,D) High magnification view of the edge of a basigin-expressing cell (ca. 15 µm total width). There is significant colocalization of D-basigin (C) and actin filaments (D) as shown for marked filaments (asterisks). Some actin filaments do not colocalize with D-basigin (red arrowhead in D). D-basigin-mediated cell spreading requires integrin activity. (E) D-basigin-expressing High Five cells cultured under normal conditions. (F) Cells cultured in the presence of 200 µg/ml GRGDS peptide which competes for integrin binding sites. (G) S2 cell expressing Drosophila PS1ßPS integrins labeled with antibody against D-basigin and visualized with Alexa 568-conjugated secondary. (H) The same cell as in G labeled with a mixture of antibodies to PS1 and ßPS integrin and visualized with Alexa 488-conjugated secondary antibody. D-basigin and integrin colocalize at many sites. Bar, 20 µm (A,B); 60 µm (E,F); 10 µm (G,H).

View larger version (44K): [in a new window]   Fig. 5. D-basigin partially colocalizes with integrin in the visual system. (A) Frozen section of retina (re) and lamina (la, bracket) of fly heterozygous for P1096 enhancer trap. This expresses nuclear ß-gal and is labeled with anti-ß-gal and visualized with Vector ABC kit. Expression was seen in the nuclei of photoreceptor neurons R1-6 (downward pointing arrow; these nuclei occur in a tight distal cluster in each ommatidium) and R7 (upward pointing arrow) and in the migratory retinal basal glia (RBG, arrowhead) (Choi and Benzer, 1994). The RBG lie just beneath the retinal basement membrane. (B) White-eyed animal labeled with anti-D-basigin and visualized with Alexa 568-conjugated secondary antibody. (C) The same section labeled with anti-ßPS1 integrin monoclonal antibody, visualized with Alexa 488-conjugated secondary antibody. D-basigin and integrin colocalize to several places, see arrowheads. However, integrin is expressed in a thin line at the basement membrane, which lacks D-basigin expression (arrows in C). Bar, 25 µm.

View larger version (95K): [in a new window]   Fig. 6. D-basigin expression in the retinal photoreceptor neurons is necessary for the proper placement of their nuclei. Frozen sections through the retina (brackets in B and C); la, lamina. The normal locations of the R1-8 nuclei are marked in C. (A) Section labeled with anti-ß-gal expressed in R1-R6 nuclei. (B-G) Sections labeled with antibody to the neural nuclear protein, elav. (A) Mutant for the hypomorphic bsg allele, P1096. A few photoreceptor nuclei are displaced (arrow). (B) Mutant for bsg265. Nuclei are scattered throughout the retina (arrows). (C) Mutant for bsg265 rescued by a GMR-bsg265 transgene that drives expression of D-basigin 265 in all photoreceptor neurons. The nuclei of the R7 photoreceptors are just proximal to those of R1-R6 and the R8 nuclei lie near the basement membrane of the retina. (D) Mutant for bsg265 rescued by a GMR-mouse basigin transgene (Bsg). D-basigin and integrin interact to affect nuclear placement. (E) Male carrying a viable allele of integrin, ßPS1 (mysb45). (F) integrin bsg265 double mutant male carrying mysb45 also mutant in the eye for the P1096 bsg. Arrows indicate some of the misplaced nuclei. The number of misplaced nuclei is much greater than the sum of the two mutations independently (A and E). (G) Fly heterozygous for both the PS1 integrin allele, mewM6 and the ßPS integrin allele mysb45. Examples of misplaced nuclei are marked with arrows. (H) Fly mutant in the eye for Mmp2w307, which shows no abnormality in nuclear placement. Bar, 30 µm.

View larger version (204K): [in a new window]   Fig. 7. R1-R6 in bsg265 mutant terminals exhibit a mutant ultrastructural phenotype. (A) Cross section of a control cartridge innervated by non-mutant axons (control animals were generated by recombining a wild-type chromosome arm using the same basic procedure used to create mutant terminals as already described). This control exhibits a wild-type structure (Meinertzhagen and O'Neil, 1991) in which a ring of photoreceptor terminals (R) surrounds the axon profiles of lamina cells L1 and L2. The entire cartridge is surrounded by lamina epithelial glia (*). Terminals contain normal mitochondria (m) and synaptic profiles composed of synaptic vesicles, capitate projections (arrowheads) and T-bar synaptic ribbon release sites (arrow). (B) Cartridge innervated by bsg265 photoreceptors; the terminals are of variable sizes, most are larger than controls in A, which are shown at the same magnification, although one terminal is very small (*). At this distal section plane in the lamina, close to the eye, there are more mitochondrial profiles than normal. (C) In the same lamina as B, but cut at a proximal level, R1-R6 terminals lack mitochondrial profiles. Such profiles disappear in the distal third of the lamina's depth, with a cut-off that is sharply localized. At that level, cartridge cross-sections, as here, have some terminals with (m) and some without (*) mitochondrial profiles. (D) Individual R1-R6 terminal from C, exhibits misplaced rough ER (long arrows), never normally seen in either control or wild-type terminals and pleiomorphic profiles of synaptic vesicles (short arrow); capitate projections are mostly lacking, except those that are shallow (arrowhead), whereas T-bar ribbons are normal (double arrowhead). Bar, 1 µm.