Abstract
BackgroundProcess 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.ResultsThe 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.ConclusionsExogenous 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.
Highlights
Process formation by glial cells is crucial to their function
Well over 50% of pMayven-EGFP transfectants had intense regions of EGFP activity positioned towards the process tips overlapping in part with lamellipodial actin (Fig. 3A) similar to the distribution pattern described in oligodendrocyte precursor cell (OPC) transfectants [7]
Mayven was clearly absent from the more fine filipodial extensions and does not define the membrane boundary of olfactory ensheathing cells (OECs) lamellipodium (Fig. 3A,B). This contrasted to the distribution of exogenous Lasp-1, an actin binding protein in which does demarcate the lamellipodial membrane as depicted in pMayven-HA and pLasp-EGFP cotransfectants (Fig. 3C)
Summary
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. A candidate protein, Mayven (KLHL2) has been reported to have a crucial role in the extension of oligodendrocyte precursor cell (OPC) processes [6,7] This actin binding protein [8] belongs to the BTB-BACKKelch family with around 50 members [9]. The Kelch domain at the carboxyl terminus (AAs 308-591) is associated F-actin binding [8] and comprises six contiguous units forming a characteristic βpropeller tertiary structure exposing a plethora of contact sites for interacting side chains of as yet unidentified binding partners [17] This domain organization may favour the formation of a multi-protein complex integral to cytoskeletal remodelling. Krp binds to the actin binding protein Lasp at the leading edge of the membrane interacting with an integrin CD44-adaptor protein Ezrin complex providing a molecular conduit between the ECM and intracellular signal transduction [18]
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