Subcellular localization of Mayven following expression of wild type and mutant EGFP tagged cDNAs.
BACKGROUND
Process formation by glial cells is crucial to their function. Mayven, an actin binding, multi-domain polypeptide, and member of the BTB-BACK-Kelch family have been shown to be important in oligodendrocyte process extension. To assess the role of Mayven in neural cell process extension we have tracked the subcellular distribution of exogenous Mayven following expression of a rat Mayven -EGFP cDNA in a variety of neural cell backgrounds and specifically in OEC tranfectants following drug treatment to disrupt the integrity of the cytoskeleton. A comparison was made between the subcellular localization following transient transfection of OECs with full-length Mayven cDNA and a series of mutant domain constructs.


RESULTS
The subcellular location of Mayven in OEC transfectants showed a characteristic distribution with intense foci of staining towards the process tips corresponding to regions of accumulated Mayven overlapping in part with lammelipodial actin and was absent from the filipodia and the outer membrane. This signature pattern was also observed in Schwann cells, Oli-Neu cells, astrocytes and the neuroblastoma cell line B104 transfectants and resembled the exogenous and endogenous Mayven distribution in oligodendrocytes. This contrasted with the localization pattern in non-neural cells. There was a re-localization of Mayven in OEC transfectants following drug treatment to challenge the integrity of the actin cytoskeleton while breakdown of the microtubular component had no discernible impact on the accumulation of Mayven in the process tips. Deletion of the first three amino acids of the SH3 motif of the putative Fyn Kinase binding domain at the amino terminus significantly compromised this signature pattern as did the removal of the last Kelch repeat unit of six unit Kelch domain comprising the carboxyl terminus. In addition, there was a reduction in process length in mutant transfectants. Co-expression studies with a haemagglutinin (HA) tagged wild type Mayven cDNA and EGFP tagged mutant cDNAs suggested a homomeric interaction mediated by the BTB/POZ domain.


CONCLUSIONS
Exogenous Mayven is transported to the lamellipodia in neural transfectants associating with the actin cytoskeletal network. In addition to the importance of the internal BTB/POZ domain, this subcellular distribution pattern is dependent on the presence of an intact amino and carboxyl terminus.
Montague
Paul
P
Division of Clinical Neuroscience, Glasgow Biomedical Research Centre, Room 4B17, 120 University Place, University of Glasgow, Glasgow G12 8TA, UK.
Kennedy
Peter G E
PG
Barnett
Susan C
SC
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D007150:Immunohistochemistry; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009457:Neuroglia / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D051381:Rats; D014162:Transfection
D018076:DNA, Complementary; C552071:Klhl2 protein, rat; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins