Silencing of Pnut in Drosophila L2 cells by dsRNA causes multinucleation and Orc6 mislocalization. (A) Immunoblotting with anti-Pnut antibody of whole cell extract Drosophila L2 cells transfected with either Orc6 dsRNA or Pnut dsRNA for 72 h, or nontransfected (control). Tubulin was used as a loading control. (B) Drosophila L2 cells, either control or transfected with Pnut dsRNA were fixed 72 h after transfection, and stained as described in Materials and Methods. For the Pnut dsRNA-treated cells, the differential interference contrast (DIC) is also shown.

Purification of native and recombinant Drosophila septin complexes. (A) SDS-PAGE gel stained with silver of glycerol gradient fractions. In this last step in the purification protocol of septin complex from Drosophila embryos, samples enriched for septin proteins were fractionated by glycerol gradient centrifugation. Fractions of the gradient were analyzed by SDS-PAGE followed by silver stain for the presence of septin proteins as depicted for a typical purification. (B) SDS-PAGE gel stained with colloidal Coomassie depicts purified reconstituted septin complexes next to purified native complex for comparison. His-tagged Pnut runs slightly higher than native Pnut. The E. coli-derived complex preparation contains some degraded Pnut protein. (C) Phosphorimaging picture of TLC plate containing separated nucleotides eluted from native and recombinant septin complex incubated with GTP at 21°C for the time points indicated. The conversion of GTP to GDP was traced with [α-³²P]GTP. (D) GTPase activity of purified septin complex. Graph depicts the amount of GDP generated as a fraction of the total of GTP and GDP measured on the TLC plate. The origin of the septin complex is as follows: black bars, native; white bars, E. coli; and gray bars, baculovirus.

Orc6 increases GTP hydrolysis of septin complex. Phosphorimaging picture of TLC plate containing separated nucleotides eluted from either native or recombinant baculovirus septin complex incubated with GTP at 21°C in the presence of BSA, wild-type Orc6, and the Orc6-200 mutant for the time points indicated. BV(wt), baculovirus-derived recombinant septin complex containing wild-type Pnut. BV(3L1), baculovirus derived recombinant septin complex containing 3L1 triple leucine mutant of Pnut.

Model for the interaction of Orc6 with the septin complex. Pnut subunits within the linear complex are marked. Sep1 is marked as S1, Sep2 is marked as S2, and Orc6 is marked as O6. For details, see text.

Interaction of Orc6 with Pnut and septin complex. (A and B) The coiled-coil domain of Pnut is essential for its interaction with Orc6. (A) Results from the Cytotrap yeast two-hybrid system are presented. Individual colonies expressing both myristylated Orc6 and hSos-Pnut fusion proteins were picked and resuspended in 35 μl of TE. Then, 2.5 μl of the suspension was applied on gal/raff plates, which were incubated at either the permissive (25°C) or restricted (37°C) temperature. hSos-ColI is a fusion protein that serves as a negative control. A schematic representation is given for each mutant of Pnut tested. The lines for the representations of Pnut(3L1) and Pnut(3L2) represent the relative locations of the mutated leucines, which are Pnut(L463A,L468A,L470A) and Pnut(L481A,L483A,L488A), respectively. (B) Direct interaction studies of in vitro-synthesized Pnut and Orc6 proteins. Proteins were synthesized individually in an in vitro transcription translation reaction containing [³⁵S]methionine. Reactions containing wild-type or mutant Pnut protein were mixed with Orc6. Proteins were immunoprecipitated with either anti-Pnut antibody or anti-Orc6 antibody and further analyzed by SDS-PAGE followed by autoradiography. The GST-Pnut(427-539) synthesis reaction contained a degradation product of slightly bigger size than Orc6 that was pulled down with anti-Pnut antibody only. (C) The coiled-coil domain of Pnut is essential for septin complex formation. Sep1, Sep2, and wild-type or mutant Pnut protein were simultaneously synthesized in an in vitro transcription translation reaction containing [³⁵S]methionine. Proteins were immunoprecipitated with anti-Pnut antibody and further analyzed by SDS-PAGE followed by autoradiography. Wild-type (Pnut1-539) and deletion mutants (Pnut1-460, Pnut 1-427) as well as the triple leucine mutants within the coiled coil domain Pnut(3L1) and Pnut(3L2) are shown. (D) Orc6 interacts with the septin complex via Pnut. Sep1, Sep2, and wild-type or mutant Pnut protein were simultaneously synthesized in an in vitro transcription translation reaction containing [³⁵S]methionine, whereas Orc6 was synthesized individually. Reactions were mixed and proteins immunoprecipitated with either anti-Pnut antibody or anti-Orc6 antibody. Immunoprecipitates were analyzed by SDS-PAGE followed by autoradiography. (E) Expression of Pnut mutants under control of native promoter in Drosophila tissue culture cells. Drosophila L2 cells were transfected with pCasper constructs expressing FLAG-tagged Pnut under control of the Pnut promoter. Cells were fixed 72 h after transfection, stained with anti-FLAG antibody, and counterstained with DAPI. Columns of three representative images for wild-type Pnut and Pnut mutants are shown.

Negative-stain electron microscopy of filaments of native (A–D) and recombinant septin complex (E and F). Native complex shows smaller filaments due to the lower concentration of protein (A and B). The addition of 1.5% PEG 8000 (C) or concentrating the sample (D) induces larger cable-like aggregates. Cable-like aggregates are also present in the concentrated recombinant protein complex (E and F). Images taken at different magnifications. Bars, 100 nm.

Orc6 enhances septin filament assembly. Recombinant baculovirus derived wild-type septin complex was diluted in GTPase buffer to 80 ng Pnut/μl, and incubated with 80 ng/μl Orc6 protein and 2 μM nucleotide where indicated. Samples were incubated at room temperature for 2 h after which they were prepared for EM. Proteins incubated are recombinant septin complex (A), recombinant septin complex and Orc6 (B), recombinant septin complex and Orc6-200 (C), recombinant septin complex and Orc6 in the presence of GDP (D), recombinant septin complex and Orc6 in the presence of GTP (E), and recombinant septin complex and Orc6 in the presence of GTPγS (F). (G) Recombinant baculovirus derived wild-type septin complex was diluted in GTPase buffer to 80 ng Pnut/μl and incubated with 80 ng/μl wild-type Orc6 at room temperature for 2 h. At this point, half the sample was used for EM analysis, confirming filament formation. Subsequently, 2 μM GTP was added to the remaining sample after which incubation at room temperature was continued for an additional 2 h. Sample was processed and analyzed with EM. Bars, 100 nm.