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First published online 21 September 2004
doi: 10.1242/jcs.01366

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A novel partner for Dictyostelium filamin is an -helical developmentally regulated protein

Monika Knuth^1,*, Nandkumar Khaire^1,*, Adam Kuspa², Si Jie Lu², Michael Schleicher³ and Angelika A. Noegel,^1,

¹ Zentrum Biochemie, Institut für Biochemie I, Medizinische Fakultät, Universität zu Köln, 50931 Köln, Germany
² Department of Biochemistry, Baylor College of Medicine, Houston, TX 77030, USA
³ Institut für Zellbiologie, Ludwig-Maximilians-Universität München, Schillerstrasse 42, 80336 München, Germany

View larger version (59K): [in a new window]   Fig. 1. The FIP cDNA codes for a 230 kDa protein. (A) The sequence predicts a protein of 229.8 kDa. The predicted coiled coil domains are underlined. The two leucine-zipper motives are boxed and the filamin-binding region is double boxed. These sequence data are available from GenBank/EMBL/DDJB under accession number AF356600. (B) mAb K12-454-2 recognizes a 230 kDa protein in protein extracts from developing AX2 cells (t6).

View larger version (45K): [in a new window]   Fig. 2. The presence of ddFIP mRNA and protein during development. Dictyostelium amoebae of strain AX2 were starved on phosphate agar. At the indicated time points, samples were taken for RNA extraction or preparation of total cell homogenates. (A) RNA blots containing 30 µg of total RNA per lane were probed with a 2.7 kb fragment encompassing the 5'-end of the FIP DNA. Transcript levels are rising during the early developmental stages and are maintained throughout culmination. During maturation of the fruiting body, the transcript level decreases. (B) Total cell homogenates of 1x106 cells per lane were resolved in 8% polyacrylamide gels and blotted onto nitrocellulose membranes. Blots were incubated with mAb K12-454-2. The antibody recognizes a 230 kDa band throughout all stages of development.

View larger version (72K): [in a new window]   Fig. 3. Colocalization of FIP with filamin in aggregating AX2 cells as analyzed by confocal microscopy. Cells were starved for 10 hours in shaking suspension, fixed with methanol and stained with FIP-specific mAb K12-362-8 (A) or K12-454-2 (D) and a polyclonal filamin serum (B,E). C and F show the overlays. Detection was with Alexa488-conjugated goat anti-mouse IgG and Alexa568-labeled goat anti-rabbit IgG. The arrows point to areas of colocalization. Bar, 10 µm.

View larger version (38K): [in a new window]   Fig. 4. FIP distribution in cell fractionation experiments and coimmunoprecipitation of filamin and FIP. (A) 2x108 AX2 cells were developed for 15 hours on phosphate agar, opened by freezing and thawing (Lys), and cytoplasm and membrane fractions were separated by centrifugation at 10,000 g into supernatant (S10) and pellet (P10). The supernatant (S10) was further fractionated by centrifugation at 100,000 g into supernatant (S100) and pellet (P100). The fractions were resolved in 8% polyacrylamide SDS gels and blotted onto nitrocellulose membranes. Blots were incubated with mAb K12-454-2 for FIP detection and filamin-specific mAb 82-454-12. Both proteins are mostly present in the cytosolic fraction and to a lesser extent in the membrane sediment. (B) FIP (a) and filamin (b) can be coimmunoprecipitated from AX2 cell homogenates. In (a), immunoprecipitation was performed with filamin-specific mAb 82-454-12, and the immunoblot containing the immunocomplexes was probed for the presence of FIP with mAb K12-454-2. In (b), immunoprecipitation was performed with FIP-specific mAb K12-454-2, and filamin was detected in the immunoprecipitate using mAb 82-454-12. The immunocomplexes were resolved by SDS-PAGE (10% acrylamide). The bands observed at approximately 170 kDa in (a) are breakdown products of FIP. Likewise, the band below 94 kDa in (b) is a breakdown product of filamin. Both proteins are highly susceptible to proteolysis. The bands at about 60 kDa (star) are the immunoglobulin heavy chains. The homogenates were obtained from cells that were allowed to develop on phosphate agar plates for 12 hours.

View larger version (27K): [in a new window]   Fig. 5. Verification of the filamin-FIP interaction. (A) 20 µg/ml each of filamin rod polypeptides rod 3, rod 4, rod 5-6, rod 2-4, rod 4-6 and an unrelated polypeptide (gst-rez 1) were immobilized on plastic and increasing amounts of FIP polypeptide (rec c-FIP; residues 1612-1907) were added. The ability of the rod proteins to bind the recombinant FIP polypeptide was measured by ELISA using mAb K12-454-2. Measurements were performed in duplicate. Data from a representative experiment are shown. (B) Direct interaction of FIP polypeptide and rod 2-4. Both polypeptides (30 µg each) were mixed and immunoprecipitation was performed using mAb K12-454-2. The immunoprecipitate was resolved by SDS-PAGE (12% acrylamide) and stained with Coomassie Blue (a). The control in c shows that rod 2-4 polypeptide (see b) is not precipitated by mAb K12-454-2 in the absence of the FIP polypeptide. The proteins were separated by SDS-PAGE (10% acrylamide). The location of the molecular weight markers as well as of the immunoglobulin light (lc) and heavy chain (hc) is indicated by arrows. The band at approximately 70 kDa also represents the antibody.

View larger version (108K): [in a new window]   Fig. 6. Localization of the GFP-cFIP fusion protein during phagocytosis and exocytosis. Single confocal sections through living AX2 cells expressing the GFP-cFIP fusion protein. (A) Uptake of TRITC-labeled yeast cells (indicated by a star). GFP-cFIP is located at the plasma membrane during the formation (upper panel) and the engulfment of the phagosome (lower panel). GFP-cFIP is released from the phagosomal membrane after complete engulfment of the yeast particle. (B) GFP-cFIP reassembles at the membrane during exocytosis of TRITC-labeled yeast cells. Bar, 5 µm.

View larger version (70K): [in a new window]   Fig. 7. Cytoskeletal association of the GFP-cFIP fusion protein during cytoskeletal rearrangements in response to cytochalasin A. Dictyostelium AX2 cells expressing the GFP-cFIP fusion protein were attached to coverslips and treated with 20 µM cytochalasin A in phosphate buffer for 1 hour prior to fixation with methanol. In the upper panel, a cell labeled with the filamin-specific mAb 82-382-8 and, in the lower panel, a cell stained with the actin-specific mAb Act 1-7 is shown (A,B, GFP fluorescence; A',B', antibody staining; A",B", overlay). The merged pictures (A",B") reveal a cytoskeletal association of the GFP-cFIP fusion protein. It colocalizes with the cytoskeletal elements in patches at the plasma membrane. Bar, 5 µm.

View larger version (77K): [in a new window]   Fig. 8. Expression of GFP-cFIP in AX2 affects development and phototaxis. (A) 1x108 cells were deposited onto a phosphate agar plate, allowed to developed at 21°C and documented at the indicated times. (A) Microscopic images of the developmental stages of GFP-cFIP-expressing cells and AX2 cells at the indicated time points. GFP-cFIP-expressing cells have completed their developmental program after 16 hours of starvation, whereas AX2 cells need 24 hours to form fruiting bodies. The figures also reveal that the GFP-cFIP-expressing cell line forms smaller aggregates that develop into shorter fruiting bodies. Bar, 1 mm. (B) Differences in the size of the aggregates from GFP-cFIP-expressing and AX2 cells measured after 8 hours of starvation (left). The diameter of the GFP-cFIP aggregates is reduced by about 30% compared with the wild-type aggregates. Likewise, the length of the fruiting bodies was measured after 24 hours of development (right). The GFP-cFIP-expressing cells form fruiting bodies that are reduced in size by 30% compared with the AX2 cells. The columns represent mean±s.e. value [t8: n=268 (GFP-cFIP), 270 (AX2); t24: n=96 (GFP-cFIP), 41 (AX2)]. The experiments were performed three times. (C) AX2 cells expressing GFP-cFIP have a phototaxis defect. Slugs do not move straight towards the light but at an angle and travel shorter distances. (a) Behavior of AX2 wild-type slugs, (b), behavior of GFP-cFIP-expressing AX2 slugs. Slug migration was for 48 hours. The location of the light source is indicated by an arrowhead.

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