Characterization of NF-L and βIIΣ1-Spectrin Interaction in Live Cells

Abstract

Neurofilaments (NFs) are neuron-specific intermediate filaments (IFs) composed of three different subunits, NF-L, NF-M, and NF-H. NFs move down the axon with the slow component of axonal transport, together with microtubules, microfilaments, and αII/βII-spectrin (nonerythroid spectrin or fodrin). It has been shown that αII/βII-spectrin is closely associated with NFs in vivo and that βII-spectrin subunit binds to NF-L filaments in vitro. In the present study we seek to elucidate the relationship between NF-L and βII-spectrin in vivo. We transiently transfected full-length NF-L and carboxyl-terminal deleted NF-L mutants in SW13 Cl.2 Vim− cells, which lack an endogenous IF network and express αII/βIIΣ1-spectrin. Double-immunofluorescence and electron microscopy studies showed that a large portion of βIIΣ1-spectrin colocalizes with the structures formed by NF-L proteins. We found a similar association between NF-L proteins and actin. However, coimmunoprecipitation experiments in transfected cells and the yeast two-hybrid system results failed to demonstrate a direct interaction of NF-L with βIIΣ1-spectrin in vivo. The presence of another protein that acts as a bridge between the membrane skeleton and neurofilaments or modulating their association may therefore be required.