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First published online 14 April 2008
doi: 10.1242/jcs.027466

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ABBA regulates plasma-membrane and actin dynamics to promote radial glia extension

¹ Program in Cellular Biotechnology, Institute of Biotechnology, PO Box 56, 00014 University of Helsinki, Finland
² Program in Developmental Biology, Institute of Biotechnology, PO Box 56, 00014 University of Helsinki, Finland
³ Division of Biochemistry, Department of Basic Veterinary Sciences, PO Box 66, 00014 University of Helsinki, Finland
⁴ W. M. Keck Center for Collaborative Neuroscience, Rutgers, State University of New Jersey, NJ, USA
⁵ Neuroscience Center, PO Box 56, 00014 University of Helsinki, Finland

View larger version (100K): [in this window] [in a new window]   Fig. 1. Domain structure and expression of ABBA. (A) ABBA is composed of an N-terminal IM domain, a serine-rich region, three proline-rich motifs and a C-terminal WH2 domain. (B) Northern blot analysis of ABBA expression in adult mouse tissues shows a single band that was predominant in brain. Each lane contains 2 µg total RNA. RNA in situ hybridisation analysis on sections from mouse embryos at E12.5 (C-F), E14.5 (G-I) and E18.5 (J) and adult cerebellum (K). Probes are indicated in the images. At E12.5 ABBA is strongly expressed in the floorplate of the spinal cord (C) and hindbrain (F), whereas weaker expression is detected in parenchyma and in the outer border of the marginal zone (arrowheads) where radial glia endplates locate. (D) MIM is expressed in spinal cord neurons and its expression is distinct from that of ABBA. (E) No specific signal was obtained with the ABBA sense probe. (G) Sagittal section from E14.5 mouse head where ABBA mRNA was detected in the pial surface surrounding the brain and in the midline glial raphe. (H) ABBA mRNA was detected in the developing cortex at E14.5 and especially in the outer border of the marginal zone (arrowhead). Cross section at E14.5 (I) and sagittal section at E18.5 (J) show ABBA expression in the brainstem raphe. (K) In the adult brain, the strongest ABBA expression is confined to the molecular layer of the cerebellum. cp, cortical plate; fp, floorplate; hb, hindbrain; mz, marginal zone; ra, glial raphe; spc, spinal cord; vz, ventricular zone. Scale bars: 0.1 mm in C-F, H-K and 0.2 mm in G.

View larger version (112K): [in this window] [in a new window]   Fig. 2. ABBA is enriched in radial glia. (A) Anti-ABBA antibody recognises a single band from NIH3T3 cell lysate corresponding to the GFP-tagged ABBA, and a lower molecular weight protein from mouse whole brain lysate corresponding to endogenous ABBA. Immunohistochemical detection of ABBA protein (red) on sections from E12.5 (B,E) and E14.5 (I,M,Q) mouse embryonic tissue. Double labelling was performed either with RC2 (C,J,R) or Tuj1 (F,N) (green) and colocalisation with ABBA appears yellow in merged images (D,G,K,O,S) and in corresponding higher magnifications (H,L,P,T). At E12.5, the strongest ABBA expression was detected in the hindbrain floorplate where it colocalised with RC2. ABBA was detected in the radial glia fibers throughout the hindbrain region (arrowhead) and was enriched in the marginal zone where the radial glia end feet are located (arrow) (B-D). No colocalisation was observed with Tuj1, which labelled interneuron axons crossing the ABBA-positive spinal cord floorplate (E-H). At E14.5, ABBA colocalises in glial raphe (arrow) with RC2 (I-L) but not with Tuj1 (M-P). ABBA was detected throughout the developing cortex, where it colocalises in radial glia with RC2 (Q-T). cp, cortical plate; fp, floorplate; hb, hindbrain; mz, marginal zone; ra, glial raphe; gf, glial fiber; vz, ventricular zone. Scale bars: 20 µm in B-G, I-K, M-O, Q-S and 10 µm in H,L,P,T.

View larger version (66K): [in this window] [in a new window]   Fig. 3. ABBA is abundantly expressed in glial cell lines where it localises to the interface between plasma membrane and cortical actin cytoskeleton. (A) Western blot analysis demonstrating abundant expression of ABBA in primary glia, whereas cortical neurons derived from E16.5 mouse embryos are negative for ABBA. Anti-actin was used a loading control. (B) ABBA is expressed in glial cell lines (C6 and C6-R), but was not detected in neuronal cell lines (N18, Neuro2A, or Shep). As a loading control, anti-actin antibody was used. (C-E) Immunofluorescence microscopy images from C6-R cells revealed that endogenous ABBA localises in lamellipodia at the leading edge of the cortical actin cytoskeleton (white arrowhead). (F-H) Co-labelling of C6-R cells with ABBA antibody and membrane marker (CM-Dil) demonstrates that ABBA localises to the plasma membrane at the leading edge. (H) Magnified areas show individual channels from the boxed region of the image. (I-K) Three-dimensional confocal microscopy analysis from C6-R cells confirmed the localisation of ABBA to the interface between plasma membrane and the actin cytoskeleton (white arrowheads). ABBA is in green whereas F-actin (panels D,E,I,J,K) and plasma membrane (panels G,H) are in red. Scale bars: 20 µm in C-H; 5 µm in I; 2 µm in J,K.

View larger version (28K): [in this window] [in a new window]   Fig. 4. ABBA binds ATP-G-actin with a high affinity through its C-terminal WH2 domain but does not bundle actin filaments. (A-D) A change in fluorescence of NBD-labelled ATP- and ADP-G-actin was measured over a range of concentrations of ABBA274-715. Symbols represent mean data from three experiments and solid lines indicate fitted binding curves with a 1:1 stoichiometry. ABBA interacts with ATP-actin monomers with four times higher affinity than ADP-actin monomers. ABBA-mutWH2 did not display detectable binding to G-actin. (E) Low-speed cosedimentation analysis measuring actin-filament-bundling activity of ABBA IM domain (ABBA-IMD). In the presence of -actinin, majority of actin filaments were in the pellet fraction `P', demonstrating the actin-filament-bundling or crosslinking activity of the protein. By contrast, under identical conditions, ABBA IMD did not induce detectable actin-filament bundling or crosslinking, and the majority of actin was in the supernatant `S'. (F) Quantification of bundling activities of ABBA-IMD and -actinin from two independent experiments. Data are means ± s.e.m. (G) A three-dimensional confocal microscopy analysis from C6-R cells expressing GFP-tagged ABBA-IMD revealed that ABBA-IMD does not localise to peripheral actin bundles (white arrowhead), but instead localises to the plasma membrane surrounding the protruding bundles (black arrowhead). F-actin is red and ABBA-IMD green. Scale bars: 10 µm (left) and 2 µm (magnified region on right).

View larger version (83K): [in this window] [in a new window]   Fig. 5. The ABBA IM domain interacts with PtdIns(4,5)P2-rich membranes and deforms them into tubular structures. (A) ABBA IM domain (ABBA-IMD) or ABBA-IMDmut were incubated in the absence or presence of vesicles. After centrifugation, equal amounts of supernatant `S' and pellet `P' were analysed on SDS-PAGE. ABBA-IMD bound PtdIns(4,5)P2-containing vesicles with high affinity, whereas ABBA-mutIMD displayed a severe defect in PtdIns(4,5)P2 binding. (B) Quantification of wild-type ABBA-IMD and ABBA-mutIMD binding to lipid vesicles from three individual experiments. Data are represented as mean ± s.e.m. (C) Electron micrographs of vesicles containing 30% PtdIns(4,5)P2 mixed with buffer, ABBA-IMD or ABBA-IMDmut. Only wild-type ABBA-IMD induced clustering of vesicles and membrane tubulation. (D) Micrographs of different magnifications showing that ABBA-IMD induced membrane tubules of vesicles containing 5% PtdIns(4,5)P2. Scale bars: 1 µm in C, upper row; 0.2 µm in C, bottom row; 0.5 µm (left) and 0.2 µm (middle and right) in D.

View larger version (45K): [in this window] [in a new window]   Fig. 6. Depletion of ABBA by siRNA results in glial process extension defects. (A) Time-lapse images from C6-R cells expressing GFP-tagged ABBA IM domain and GFP-tagged full-length protein. Cells expressing the IM domain typically displayed multiple protruding microspikes (black arrowhead), whereas the full-length protein localised to the plasma membrane but did not induce dramatic microspike formation. (B) Western blot analysis demonstrating ABBA protein levels before and after transfection with siRNA oligonucleotides. Anti-actin antibody was used as a loading control. (C) Representative images derived from CellIQ cell imaging platform at 2 and 12 hour time points after replating control siRNA oligonucleotide duplex or with siRNA specific to ABBA-transfected C6-R cells. Scale bar: 5 µm. (D) Graph of mean lengths of C6-R cell processes from six individual wells per group. More than 50 cells were counted in each well. Error bars represent s.e.m. (E) Western blot analysis demonstrating the resistance of ABBA-GFP rescue construct to ABBA siRNA. (F) Quantification of cell lengths from control siRNA or ABBA siRNA-transfected cells that were cotransfected with GFP or siRNA-resistant GFP-ABBA constructs 1.5 hours after replating. Data are represented as mean ± s.e.m. (n=49). Statistical significance was established by one-way ANOVA using Tukey test for comparison.

View larger version (73K): [in this window] [in a new window]   Fig. 7. Depletion of ABBA alters lamellipodial dynamics. (A) Time-lapse movies of C6-R cells transfected with control or ABBA siRNA oligonucleotides were analysed by drawing a one-pixel-wide line across lamellipodium in the direction of protrusion. The kymograph was constructed by copying the image from this line from 200 frames of the movie and pasting along the x-axis. Steeper slopes in the kymograph correspond to higher velocity rates in the lamellipodial protrusion. (B) Velocity of individual protrusions was calculated and plotted in the graph. Data were collected from 98 ABBA-knockdown and 105 control protrusion events, error bars represent s.e.m. (C) Individual ruffling events (>0.5 µm) were calculated from kymographs of 20 cells. Data are represented as mean ± s.e.m. **P<0.02; *P<0.05; Student's t-test. Scale bar: 5 µm.

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