Characterization of NF-L and betaIISigma1-spectrin interaction in live cells.
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 alphaII/betaII-spectrin (nonerythroid spectrin or fodrin). It has been shown that alphaII/betaII-spectrin is closely associated with NFs in vivo and that betaII-spectrin subunit binds to NF-L filaments in vitro. In the present study we seek to elucidate the relationship between NF-L and betaII-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 alphaII/betaIISigma1-spectrin. Double-immunofluorescence and electron microscopy studies showed that a large portion of betaIISigma1-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 betaIISigma1-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.
Macioce
P
P
Laboratory of Cell Biology, Laboratory of Ultrastructure, Istituto Superiore di Sanità, Viale Regina Elena 299, Rome, 00161, Italy. macioce@iss.it
Gandolfi
N
N
Leung
C L
CL
Chin
S S
SS
Malchiodi-Albedi
F
F
Ceccarini
M
M
Petrucci
T C
TC
Liem
R K
RK
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D006801:Humans; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D016900:Neurofilament Proteins / Q000235:genetics / Q000378:metabolism*; D011233:Precipitin Tests; D051381:Rats; D013049:Spectrin / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D008840:Microfilament Proteins; D016900:Neurofilament Proteins; D013049:Spectrin; C044882:neurofilament protein L