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First published online 11 May 2004
doi: 10.1242/jcs.01113

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EBV attachment stimulates FHOS/FHOD1 redistribution and co-aggregation with CD21: formin interactions with the cytoplasmic domain of human CD21

Michael B. Gill^1,2,5,*, Jennifer Roecklein-Canfield^1,*, David R. Sage^1,2, Maria Zambela-Soediono¹, Nina Longtine^4,5, Marc Uknis³ and Joyce D. Fingeroth^1,2,5,

¹ Division of Infectious Diseases, Beth Israel Deaconess Medical Center, Boston, MA 02115, USA
² Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Boston, MA 02115, USA
³ Division of Surgery, Beth Israel Deaconess Medical Center, Boston, MA 02115, USA
⁴ Department of Pathology, Brigham and Women's Hospital, Boston, MA 02115, USA
⁵ Harvard Medical School, Boston, MA 02115, USA

View larger version (55K): [in a new window]   Fig. 1. The intracellular domain of CD21 interacts with FHOS. (a) in yeast. A GAL4 activation domain plasmid encoding the C-terminal amino acids of FHOS was serially diluted (10–1-10–4; UD, undiluted) and co-transformed into a yeast reporter strain with plasmids encoding the GAL4 DNA binding domain fused to one of the following: the 34 aa residues of the CD21 cytoplasmic tail (CD21-CT), the 34 aa residues of the CD21 cytoplasmic domain plus nine residues of the transmembrane domain (CD21-TMCT), lamin, the 105 aa residues of the CAR cytoplasmic domain (CAR-CT), or bacteriophage T7 gene 2 (T7/gp2; see Materials and Methods). Co-transformants were selected on minimal medium. Growth (i.e. activation of the HIS3 reporter) indicates a positive interaction. +C (positive control) represents co-transformation of plasmids encoding SNF1 and SNF4, proteins that interact in the two-hybrid assay. (b) In vitro. Purified fusion proteins expressing the C terminus of FHOS, GST-FHOS, GST alone or GST-TK (irrelevant protein) were incubated with cell lysates prepared from the B-cell lines Raji (CD21+; lanes 2-4) and Nalm6 (CD21–; lanes 5 and 6). Proteins interacting with FHOS were co-purified on glutathione beads, separated by SDS-PAGE and detected by immunoblot using a monospecific rabbit anti-CD21 antiserum. A total protein lysate from Raji cells (lane 1) was analyzed as a CD21 positive control. (c) In vivo. 293T cells were transfected with pG5CAT-EGFP (reporter) plus the desired combination of bait (pM-X) and target (pVP16-X) vectors as indicated. X denotes the fusion protein of interest. Twenty-four hours after transfection, cells were identified with Hoechst (nuclear stain), fixed, and analyzed by fluorescence microscopy. Fluorescent EGFP+ cells indicates a positive interaction between bait and target fusion proteins. Top panel (method verification): EGFP induction was assessed using positive control vectors (pM53-VP16 and pM-53 + pVP16-T) compared with negative control vectors (pM-53 + pVP16-CP, control protein; Clontech). Middle panel: EGFP induction following co-transfection of recombinant vectors encoding the cytoplasmic domain of CD21, CD21.CT together with the C terminus of FHOS, FHOS.CT. Right and left panels show reciprocal experiments. Bottom panel (control), EGFP induction following transfection of CD21.CT or of FHOS.CT together with cognate two-hybrid vectors encoding irrelevant proteins (T antigen or p53).

View larger version (76K): [in a new window]   Fig. 2. FHOS associates with the actin cytoskeletion. (a) FHOS co-localizes with actin in the cytoplasm and in protrusions. Transfected 293T cells expressing full length FHOS fused to EGFP, FHOS (green, panels 1-3, 6) were co-stained with either TRITC-phalloidin (panel 4; red) to detect actin or with anti -tubulin/goat F(ab')2 anti-mouse Ig R-PE (panel 7; red). Merged images (panel 3 with 4 and panel 6 with 7) are shown in panels 5 and 8, respectively. Hoechst 33342 (blue) was used to detect nuclei in panels 5 and 8. (b) FHOS accumulates with actin at the border between cells and without actin in a perinuclear patch. Transfected 293T cells expressing EGFP-FHOS (green; panels 1,4-5) were co-stained with TRITC-phalloidin (panels 2 and 6; red) and merged images (panel 1 with 2 and panel 5 with 6) are shown in panels 3 and 7, respectively. Nuclei are stained blue as in a. All cells were fixed, stained and imaged using an Eclipse E600 fluorescence microscope and processed using Spot Advance technology.

View larger version (62K): [in a new window]   Fig. 3. FHOS co-localizes, aggregates and caps with CD21 in response to cell surface EBV binding. (a) FHOS co-localizes with aggregated CD21. Transfected 293T cells expressing EGFP-FHOS (green) and CD21 were co-stained with an isotype control mAb (UPC10; panel 2) or with anti-CD21 (mAb HB-5; panels 5 and 8) followed by goat F(ab')2 anti-mouse IgG-R-PE (red). Cells challenged with concentrated EBV are shown in panels 4-9. Merged imaged (panel 4 with 5 and panel 7 with 8) are shown in panels 6 and 9 respectively. (b) The C terminus of FHOS is required for efficient aggregation and co-localization. Transfected HeLa cells expressing either FHOS (EGFP-FHOS, green; panels 1-9) or FHOSCT (EGFP-FHOSCT, green; panels 10-15) and CD21 (red; stained as in a) were incubated with EBV (panels 4-9 and 13-15) or without EBV (panels 1-3 and 10-12). Merged images of FHOS with CD21 are shown in panels 3 (panel 1+2), 6 (panel 4+5) and 9 (panel 7+8). Merged images for FHOSCT with CD21 are shown in panels 12 (panel 10+11) and 15 (panel 13+14). All cells were fixed, stained and imaged as stated in Fig. 2.

View larger version (33K): [in a new window]   Fig. 4. FHOS is abundantly expressed in splenic littoral cells. (a) FHOS expression in primary hematopoietic cells. RT-PCR of total RNA was performed using internal FHOS-specific primers (Materials and Methods) and demonstrates the ubiquitous expression of FHOS RNA in distinct primary hematopoietic cells (B cells, T cells, monocytes, dendritic cells, FDCs) and certain cell lines (HeLa, JY). Negative control, murine 3T3 cells. (b) FHOS expression in spleen. Normal frozen human spleen was sectioned and analyzed by in situ hybridization. The antisense panel demonstrates high expression of FHOS mRNA in splenic littoral cells, whereas no signal can be detected in the sense panel (control).

View larger version (21K): [in a new window]   Fig. 5. Schematic representation of proposed EBV-CD21-FHOS interactions. (Left) In the unstimulated cell FHOS is in an auto-inhibited conformation in the cytoplasm. (Middle) When CD21 is engaged by one of its ligands (e.g. EBV) an intracellular signal is generated (? Rac1 activation/mechanical force) that results in release of nearby FHOS from intramolecular inhibition. FHOS unfolds and interacts with the cytoplasmic domain of CD21 via its unique C-terminal domain (CT). (Right) The exposed FH domains engage cytoskeleton associated-proteins and the FH2 domain nucleates actin leading to localized formation of actin cables. Actin clustering enables the physical relocalization of CD21 into membrane aggregates, provides an anchor for ligand bound CD21 and may regulate endocytosis.