Subcellular localization of Mayven following expression of wild type and mutant EGFP tagged cDNAs

Abstract

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.

Figure 1

Domain organization of Mayven. Amino acid positions demarcating the individual domains are numbered and the sequence of the SH3 motif is highlighted.

Figure 2

Subcellular distribution pattern of exogenous Mayven following expression of a panel of pMayven-EGFP mutants. Group (I) Amino mutants, Group (II) Carboxyl mutants and Group (III) SH3 motif mutants. OECs were transfected with three series of mutant cDNAs: amino terminus deletion constructs (A-D), carboxyl terminus deletion constructs (E-G), SH3 motif mutants (H-J) and actin stained. Only pDK transfectants (J) gave a wild type distribution pattern.

Figure 3

Subcellular distribution pattern of exogenous Mayven in neural and non-neural cells. (A) pMayven-EGFP/OECs, (B) (pMayven-HA/pEGFP-βActin)/OECs, (C) (pMayven-HA/pLasp-EGFP)/OECs. (D) Schwann cells, (E) astrocytes, (F) Oli-Neu cells, (G) B104 cells, (H) Cos7 cells and BHK cells (I) were transiently transfected with pMayven-EGFP and actin stained. Mayven is transported to the tips and overlaps in part with actin in all neural cell types (A-I) in contrast to the distribution pattern in Cos7 and BHK transfectants (H,I).

Figure 4

RT.PCR depicting endogenous Mayven message levels in neural and non-neural cells. Mayven (Product length 1323 bps) and Cyclophilin (Product length 300 bps) were amplified from cDNAs prepared from O2A c-myc (OPC), (OECs), astrocytes (Ast), Schwann cells (Sc), (B104), (Cos7) and (BHK). Mayven mRNA abundance is much higher in neural cell types.

Figure 5

Cofocal analysis depicting the relationship between the subcellular distribution of Mayven and actin in OEC transfectants. OECs were transiently transfected with pMayven-EGFP and actin stained. Mayven is transported towards the lamellipodia in both flat (A) and (B) spindle cells and overlaps in part with actin in both cell types as highlighted in the blow ups.

Figure 6

Subcellular distribution pattern of exogenous Mayven following drug induced disruption of the OEC cytoskeleton with Cytochalasin B (A-D), Wiskostatin (E-H) and Nocodazole (I-L). Non-transfected (A,B) pMayven-EGFP (C) and pEGFP-N1 (D) transfected cells were exposed to 0.5 μM Cytochalasin B for 36 hrs (B-D) and stained for actin (A-D). Cytochalasin B treatment resulted is a redistribution of Mayven (C) compared to EGFP (D). Transfected pMayven-EGFP cells were administered 2, 4 and 8 μM Wiskostatin for 2 hrs (E-G) and pEGFP-N1 cells were treated for the same duration with 8 μM (H). In the absence of cell death there was a relocation of Mayven (F) while cell death did not accompany any change in the distribution pattern in pEGFP-N1 transfectants (H). Non-transfected cells (I,J) and pMayven-EGFP transfected cells (K,L) were treated with 30 μM Nocodazole for up to 3 hrs (J,L) and immunostained for β-tubulin (I-L). Mayven loclaization remains unaffected despite a breakdown in the microtubular network (K,L).

Figure 7

Relative process length between non-transfected OECs compared to pMayven, pDK, and a pool of mutant transfectants. Compared to non-transfected cells there is a significant reduction in process length in the pool of mutant transfectants.

Figure 8

Subcellular distribution pattern of exogenous Mayven following co-expression of a series of pMayven-EGFP amino deletion mutants with pMayven-HA and RT.PCR comparing endogenous and exogenous Mayven message levels in non-transfected and transfected OECs. OECs were co-transfected with pMayven-HA and mutant constructs pΔSH3 (A), pΔFYN (B), pΔBTB/POZ (C) and pΔBACK and immunostained for HA. Homomeric interaction (A,B) between the wild HA tagged fusion protein and EGFP tagged mutant form was dependent on the presence of a BTB/POZ domain. (E) RT. PCR Mayven (Product length 1323 bps) Cyclophilin (Product length 300 bps), β actin (146 bps) were amplified from cDNAs prepared from non-transfected OECs (lane 1), pΔBTB/POZ transfectants (lane 2) and pΔFYN transfectants (lane 3) confirming an increase in Mayven mRNA levels in transfected cells.

Figure 9

Schematic depicting proposed individual domain function in Mayven multi-protein complex formation