Restriction of 480/270-kD ankyrin G to axon proximal segments requires multiple ankyrin G-specific domains - PubMed (original) (raw)

Restriction of 480/270-kD ankyrin G to axon proximal segments requires multiple ankyrin G-specific domains

X Zhang et al. J Cell Biol. 1998.

Abstract

AnkyrinG (-/-) neurons fail to concentrate voltage-sensitive sodium channels and neurofascin at their axon proximal segments, suggesting that ankyrinG is a key component of a structural pathway involved in assembly of specialized membrane domains at axon proximal segments and possibly nodes of Ranvier (Zhou, D., S. Lambert, D.L. Malen, S. Carpenter, L. Boland, and V. Bennett, manuscript submitted for publication). This paper addresses the mechanism for restriction of 270-kD ankyrinG to axon proximal segments by evaluation of localization of GFP-tagged ankyrinG constructs transfected into cultured dorsal root ganglion neurons, as well as measurements of fluorescence recovery after photobleaching of neurofascin- GFP-tagged ankyrinG complexes in nonneuronal cells. A conclusion is that multiple ankyrinG-specific domains, in addition to the conserved membrane-binding domain, contribute to restriction of ankyrinG to the axonal plasma membrane in dorsal root ganglion neurons. The ankyrinG-specific spectrin-binding and tail domains are capable of binding directly to sites on the plasma membrane of neuronal cell bodies and axon proximal segments, and presumably have yet to be identified docking sites. The serine-rich domain, which is present only in 480- and 270-kD ankyrinG polypeptides, contributes to restriction of ankyrinG to axon proximal segments as well as limiting lateral diffusion of ankyrinG-neurofascin complexes. The membrane-binding, spectrin-binding, and tail domains of ankyrinG also contribute to limiting the lateral mobility of ankyrinG-neurofascin complexes. AnkyrinG thus functions as an integrated mechanism involving cooperation among multiple domains heretofore regarded as modular units. This complex behavior explains ability of ankyrinB and ankyrinG to sort to distinct sites in neurons and the fact that these ankyrins do not compensate for each other in ankyrin gene knockouts in mice.

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Figures

Figure 2

Schematic diagram of cDNA constructs used in transfection experiments. Preparation of cDNA constructs of ankyrin and neurofascin is described in Materials and Methods. Ank270–GFP represents the full-length 270-kD ankyrinG with GFP tag at its COOH terminus. Construct Ank270(ΔSR)–GFP has a deleted serine-rich domain and Ank270(ΔM1,2)–GFP lacks the first half of the membrane-binding domain. Construct Ank270(ΔSR,T)– GFP is a 190-kD natural isoform of ankyrinG without the serine-rich and tail domains. Deletion of the COOH-terminal domain from construct Ank270(ΔSR,T)–GFP results in construct Ank270(ΔSR,T,Ct)–GFP. M-GFP, SB-GFP, SR-GFP, T-GFP, and Ct-GFP represent the membrane-binding domain, the spectrin-binding domain, the serine-rich domain, the tail domain, and the COOH-terminal domain, respectively. In the neurofascin construct family, construct HA–NF contains the full-length of neurofascin with HA-epitope at the NH2 terminus. Constructs HA– NF(ΔEC) and HA–NF(ΔCD) have the deletion of the extracellular domain and the cytoplasmic domain respectively. HA–NF–GFP contains a GFP tag at the COOH terminus of the full-length neurofascin.

Figure 1

Distribution of 270/ 480-kD native ankyrinG, ankyrinB, spectrin, and neurofascin in cultured DRG neurons. 270/480-kD ankyrinG (A) and ankyrinB (B) are double labeled in 9-d-old embryonic DRG culture. (C) Composite image of A and B. AnkyrinG is concentrated at the proximal segment (A, arrows), whereas ankyrinB is more evenly distributed in axons (B). Spectrin (E), compared with double-labeled ankyrinG (D, arrow, proximal segment), also lacks polarized distribution at the proximal segment. (F) Composite image of D and E. Neurofascin (H), a member of L1 CAM family of cell adhesion molecules, is concentrated and colocalized with ankyrinG at the proximal segment (G, arrow). (I) Composite image of G and H. The green channel (FITC) corresponds to ankyrinG stain whereas the red channel (TRITC) represents other proteins. Bar, 25 μm.

Figure 3

The unique serine-rich and tail domain of ankyrinG contribute to restriction of 270-kD ankyrinG at the axon proximal segment. The cDNAs of 270-kD ankyrinG and its variants lacking the serine-rich and/or tail domains are transfected into 8-d-old DRG culture using Helios™ Gene Gun system. Expression of transfected proteins is visualized by immunostaining of the GFP tag. The transfected 270-kD ankyrinG (Ank270-GFP) is highly restricted to the proximal segment (A). High zoom recording reveals plasma membrane stain of the transfected 270-kD ankyrinG at the proximal segment (B) and the cell body (C). The restricted location of transfected Ank270–GFP (D) is displayed against the full length of the transfected axon which is revealed by double labeling of the cotransfected cytoplasmic domain-deleted neurofascin (E). The transfected 190-kD ankyrin (Ank270[ΔSR,T]–GFP) is also localized at the plasma membrane of the proximal segment (G) and the cell body (H). However, a significant amount of the transfected Ank270(ΔSR,T)–GFP is distributed beyond the proximal segment into the axon (F). Insertion of the tail domain into Ank270(ΔSR,T)– GFP (Ank270[ΔSR]–GFP) improves but does not totally restore restriction of the transfected ankyrin to the proximal part of the axon (I). J and K show membrane localization of Ank270(ΔSR)–GFP at the proximal segment and the cell body respectively. Bar, 25 μm.

Figure 4

The immunofluorescence intensity profile of transfected ankyrins along the length of axons. The immunofluorescence intensity of the GFP tag of transfected ankyrins is measured using Carl Zeiss LSM measurement program. The distance is calculated from the starting region of the proximal segment. The fluorescence intensity is normalized to the intensity at the plasma membrane of the cell body. (A) Measurement of the intensity drop along the length of the axon. (B) Two-thirds of the intensity-drop distance (the distance for the intensity of immunofluorescence to drop to one-third of the intensity at the starting region of the proximal segment). Data in B represent the mean ± SEM for five transfected neurons.

Figure 5

The spectrin-binding domain and the tail domain of ankyrinG contain binding sites to the axolemma of axon proximal segments. Different domains of 270-kD ankyrinG tagged with GFP at their COOH termini were individually transfected into DRG neurons. Expression of transfected proteins is examined by immunostaining of the GFP tag. The spectrin-binding domain SB-GFP (A and B) and the tail domain T-GFP (K and L) are targeted to the plasma membrane of the proximal segment and the cell body. The membrane-binding domain M-GFP (E and F), COOH-terminal domain Ct-GFP (I and J), and the serine-rich domain SR-GFP (G and H), however, are predominantly localized in the cytoplasm of the axon and the cell body. The spectrin-binding domain of ankyrinB (SB-GFP [ankyrinB]) is not targeted to the axolemma of the proximal segment (C), although a small fraction is distributed at the membrane of the cell body (D). Bar, 25 μm.

Figure 6

The immunofluorescence intensity profile of transfected spectrin-binding and tail domains of ankyrinG in axons. (A) Immunofluorescence profile. (B) the two-thirds intensity-drop distance of transfected spectrin-binding and tail domains. Data represent the mean ± SEM for five transfected neurons.

Figure 7

Association of 270-kD ankyrinG to the plasma membrane of human kidney 293 cells requires the membrane-binding domain and cotransfected neurofascin. Transfected 270-kD ankyrinG (Ank270-GFP) alone visualized by the GFP fluorescence distributes throughout the cytoplasm of 293 cells (A). Cotransfection with neurofascin (HA–NF) recruits Ank270–GFP to the plasma membrane (C). (B) Double labeling of cotransfected neurofascin by immunostaining of the HA epitope. The membrane-binding domain of ankyrin (M-GFP) is sufficient to be recruited to the plasma membrane (E) by cotransfected neurofascin (D). Deletion of the first half of the membrane-binding domain (Ank270(ΔM1,2)–GFP) abolishes the membrane-recruitment by cotransfected neurofascin (F and G). Bar, 3 μm.

Figure 8

Multiple domains of 270-kD ankyrinG contribute to immobilization of ankyrinG at the plasma membrane of human kidney 293 cells. (A) FRAP plasma membrane-localized 270-kD ankyrinG and its variants with deleted domains. Ankyrins with the membrane-binding domain are capable of being recruited to the plasma membrane by cotransfected neurofascin HA–NF or the extracellular domain-truncated neurofascin HA–NF(ΔEC) (Zhang et al., 1998). Dynamics of membrane-associated ankyrins are evaluated by fluorescence recovery measurement after photobleaching the GFP signals. Also included are the FRAP of cytoplasm-located 270-kD ankyrinG and membrane-associated GFP-neurofascin (HA–NF–GFP). (B) Comparison of recovery rate (the time taken to achieve half of the 50-s recovery) and recovered fraction (the fraction recovered after 50 s) between the cytoplasm-localized ankyrin and the membrane-associated ankyrin. (C) Recovery rate and recovered fraction of membrane-associated ankyrins. HA–NF–GFP is measured as a reference for freely diffusing membrane proteins. Data in B and C represent the mean ± SEM for six experiments.

Figure 8

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Restriction of 480/270-kD ankyrin G to axon proximal segments requires multiple ankyrin G-specific domains - PubMed (original) (raw)