Drosophila development requires spectrin network formation.
The head-end associations of spectrin give rise to tetramers and make it possible for the molecule to form networks. We analyzed the head-end associations of Drosophila spectrin in vitro and in vivo. Immunoprecipitation assays using protein fragments synthesized in vitro from recombinant DNA showed that interchain binding at the head end was mediated by segment 0-1 of alpha-spectrin and segment 18 of beta-spectrin. Point mutations equivalent to erythroid spectrin mutations that are responsible for human hemolytic anemias diminished Drosophila spectrin head-end interchain binding in vitro. To test the in vivo consequence of deficient head-end interchain binding, we introduced constructs expressing head-end interchain binding mutant alpha-spectrin into the Drosophila genome and tested for rescue of an alpha-spectrin null mutation. An alpha-spectrin minigene lacking the codons for head-end interchain binding failed to rescue the lethality of the null mutant, whereas a minigene with a point mutation in these codons overcame the lethality of the null mutant in a temperature-dependent manner. The rescued flies were viable and fertile at 25 degrees C, but they became sterile because of defects in oogenesis when shifted to 29 degrees C. At 29 degrees C, egg chamber tissue disruption and cell shape changes were evident, even though the mutant spectrin remained stably associated with cell membranes. Our results show that spectrin's capacity to form a network is a crucial aspect of its function in nonerythroid cells.
Deng
H
H
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138.
Lee
J K
JK
Goldstein
L S
LS
Branton
D
D
D000595:Amino Acid Sequence; D000743:Anemia, Hemolytic / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D002918:Chymotrypsin; D003001:Cloning, Molecular; D004330:Drosophila / Q000502:physiology*; D004912:Erythrocytes / Q000378:metabolism; D004926:Escherichia coli; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009866:Oogenesis; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013049:Spectrin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D046911:Macromolecular Substances; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013049:Spectrin; D002918:Chymotrypsin