A Cytoskeletal Linker Protein Involved in Axon Outgrowth

DESCRIPTION (provided by applicant): MACF (Microtubule actin crosslinking factor) is a large protein (608 kDa) that can associate with the actin and microtubule networks. MACF has a complex domain structure. The N-terminal part of MACF is made up of a calponin-type actin-binding domain and a plakin-like domain. This domain is common to the plakin family of proteins that has been shown to connect cytoskeletal elements to each other and to the junctional complexes at the plasma membrane. The plakin-like domain is followed by a central rod domain composed of spectrin repeats and calmodulinlike EF hand motifs, similar to members of the spectrin superfamily. Finally, the C-terminal domain contains a novel microtubule-binding domain. The N-terminal domain exhibits a striking homology to a proposed neuronal isoform of BPAG1, the mutated gene product of the mouse mutant. dystonia musculorum (dt). The rod domain and the EF hand motifs of MACF are highly homologous to dystrophin. MACF is ubiquitously expressed in the mouse embryo with high expression levels in the nervous system and moderate expression levels in muscles. Kakapo, also known as short stop, is the Drosophila homologue of MACF and is essential for neuronal growth and adhesion between and within cell layers. Mutations in the kakapo/short stop gene in Drosophila cause defects in muscle-tendon cell differentiation, local development of neuronal processes and axonal outgrowth. The properties of kakapo/short stop make MACF a potential key player in axonal outgrowth and we therefore propose to study the function and interaction partners of MACF in more detail. In this proposal, we will determine the expression pattern and localization of MACF: antibodies against MACF will be raised and used to study the expression pattern of MACF. We will investigate loss-of-function phenotypes in C. elegans with type-specific GFP-labeled neurons using double-stranded RNA interference to a C. elegans MACF homologue and we will carry out a series of rescue experiments and dominant negative experiments on cultured dt and wild type neurons. We will define the association partners of MACF: the microtubule-binding domain at the C-terminus of MACF will be dissected in vivo by transfection studies and in vitro by microtubule-binding assays. Other association partners to different domains of MACF will be isolated by using the yeast two-hybrid system and by co-immunoprecipitation experiments. Finally, we will characterize a novel splice variant of MACF.