Filamin repeat segments required for photosensory signalling in Dictyostelium discoideum.
BACKGROUND
Filamin is an actin binding protein which is ubiquitous in eukaryotes and its basic structure is well conserved - an N-terminal actin binding domain followed by a series of repeated segments which vary in number in different organisms. D. discoideum is a well established model organism for the study of signalling pathways and the actin cytoskeleton and as such makes an excellent organism in which to study filamin. Ddfilamin plays a putative role as a scaffolding protein in a photosensory signalling pathway and this role is thought to be mediated by the unusual repeat segments in the rod domain.


RESULTS
To study the role of filamin in phototaxis, a filamin null mutant, HG1264, was transformed with constructs each of which expressed wild type filamin or a mutant filamin with a deletion of one of the repeat segments. Transformants expressing the full length filamin to wild type levels completely rescued the phototaxis defect in HG1264, however if filamin was expressed at lower than wild type levels the phototaxis defect was not restored. The transformants lacking any one of the repeat segments 2-6 retained defective phototaxis and thermotaxis phenotypes, whereas transformants expressing filaminDelta1 exhibited a range of partial complementation of the phototaxis phenotype which was related to expression levels. Immunofluorescence microscopy showed that filamin lacking any of the repeat segments still localised to the same actin rich areas as wild type filamin. Ddfilamin interacts with RasD and IP experiments demonstrated that this interaction did not rely upon any single repeat segment or the actin binding domain.


CONCLUSIONS
This paper demonstrates that wild type levels of filamin expression are essential for the formation of functional photosensory signalling complexes and that each of the repeat segments 2-6 are essential for filamins role in phototaxis. By contrast, repeat segment 1 is not essential provided the mutated filamin lacking repeat segment 1 is expressed at a high enough level. The defects in photo/thermosensory signal transduction caused by the absence of the repeats are due neither to mislocalisation of filamin nor to the loss of RasD recruitment to the previously described photosensory signalling complex.
Annesley
Sarah J
SJ
Department of Microbiology, La Trobe University, Vic., Australia. S.Annesley@latrobe.edu.au
Bandala-Sanchez
Esther
E
Ahmed
Afsar U
AU
Fisher
Paul R
PR
D000199:Actins / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002465:Cell Movement / Q000502:physiology* / Q000528:radiation effects*; D003285:Contractile Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure*; D004023:Dictyostelium / Q000502:physiology* / Q000528:radiation effects*; D064448:Filamins; D017353:Gene Deletion; D008027:Light; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure*; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D050505:Mutant Proteins / Q000378:metabolism; D010641:Phenotype; D015800:Protozoan Proteins / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid / Q000502:physiology; D015398:Signal Transduction; D020744:rac GTP-Binding Proteins / Q000378:metabolism
D000199:Actins; D003285:Contractile Proteins; D064448:Filamins; D008840:Microfilament Proteins; D046912:Multiprotein Complexes; D050505:Mutant Proteins; D015800:Protozoan Proteins; D020744:rac GTP-Binding Proteins