Figure 1.
Structure of the cytoskeleton and ventral disc in Giardia.
Panels A (DIC) and B (anti-alpha-tubulin immunostaining) illustrate the primary microtubule structures of interphase trophozoites including the ventral disc (vd), and the four pair of flagella: anterior (afl), ventral (vfl), posteriolateral (pfl) and caudal (cfl). Scale = 5 µm. The structures of the ventral disc and flagellar basal bodies are illustrated in the schematic in panel C, including the ventral disc microtubule (MT) array (vdMTs) and the lateral crest (lc), the overlap zone of the ventral disc MT spiral (oz), the supernumerary MT array (snMTs), and the four pair of flagella and eight basal bodies (bb). Other areas of the ventral side of the giardial cell, including the lateral shield (ls) and the marginal groove (mg) are also shown. The detergent-extracted disc preparation used for proteomic analysis is shown in panel D (phase contrast), and a similarly extracted cytoskeletal preparation is shown in panel E. The ventral disc (vd), ventrolateral flange (vlf), lateral crest (lc) and overlap zone (oz) are shown, as well as the bare area (ba) and underlying flagellar basal bodies (SEM courtesy of Joel Mancuso). Scale = 2 µm.
Figure 2.
GFP tagging of known disc-associated proteins does not affect subcellular localization, disc conformation, or attachment.
Panels A–H show the ventral disc localization of C-terminal GFP-tagged, previously described disc-associated proteins beta-giardin (A,B), delta-giardin (C,D), gamma-giardin (E,F) and SALP-1 (G,H) (also see Table 1). The dome-shaped structure of the ventral disc is visible using 3D live imaging in a GFP-tagged beta-giardin strain (with the plasma membrane counterstained in red using CellMask (Invitrogen)) (I). Note the curvature of the ventral disc as visualized by live imaging in attached beta-giardin::GFP trophozoites. Scale = 5 µm. A schematic of the structural elements of the ventral disc (J) shows the curved ventral disc microtubules with associated trilaminar microribbons that protrude into the cytoplasm. The crossbridges connecting the microribbons are also shown.
Table 1.
Known and novel disc-associated proteins.
Figure 3.
Novel disc-associated proteins associated with the entire disc.
Six new DAPs localize to the entire ventral disc spiral in a manner similar to the previously described microribbon-associated proteins (see Figure 2 and Figure S3) as visualized by C-terminal GFP tagging (grey or green) and either anti-alpha-tubulin immunostaining of the MT cytoskeleton (red), or anti-beta-giardin immunostaining of the ventral disc microribbons (red). The two nuclei are visible with DAPI staining (blue). One putatively microtubule-associated DAP possesses a CAP-Gly motif (A,B,C). Another disc-localizing DAP is the possibly mis-named median body protein (MBP, H,I,J). Three ventral disc-localizing DAPs have ankyrin repeat domains (D–F, K–M, and Q–S). Note the absence of GFP localization in the ventrolateral flange (vlf) area in DAP17053 (Q–S). Finally, one disc-localizing DAP is a Nek kinase that has a greater localization to the posterior region of the ventral disc (N–P). Scale = 5 µm. Schematic shows areas of GFP localization in red.
Figure 4.
A diversity of disc-associated proteins localize to the lateral crest.
Ten new DAPs localize to the lateral crest (lc) structure that surrounds the ventral disc (see red, schematic) and comes in close contact with the intestinal epithelium during attachment [12]. In all immunostained images except (D), DAPs are visualized using C-terminal GFP tagging (grey or green) in trophozoites counterstained with anti-alpha-tubulin immunostaining of the MT cytoskeleton (red) and DAPI staining of the two nuclei (blue). DAP13981 (A,B) is an ankyrin repeat-containing Nek kinase that localizes clearly to the lateral crest when imaged using anti-GFP immunogold labeling and negative staining (C); scale = 200 nm. In (D), DAP17096::GFP localization (green) is shown in a trophozoite immunostained with anti-beta-giardin (red) to highlight the ventral disc microribbons, emphasizing the lack of colocalization of the lateral crest proteins with the main structure of the ventral disc. In some cases, lateral crest-localizing DAPs associate with other structures, such as the basal bodies (bb) (E-H,U,V and see Figure 6) and the inner region of the ventral disc surrounding the bare area (ba) (DAP103810; Q,R). All of the lateral crest localizing-DAPs contain ankyrin repeats except DAP16424 (U,V), a novel protein that has a punctate localization to the lateral crest and the cytoplasmic region of the anterior axonemes (afl) and basal bodies (bb). Scale for immunostained images = 5 µm. Schematic shows areas of GFP localization in red.
Figure 5.
DAP17090::GFP and DAP16263::GFP localize to the supernumerary microtubules and axonemes.
Both DAP17090 (A–C), which contains a SAM protein-protein interaction motif, and DAP16263 (D–F), a DIP13 microtubule binding protein homolog, localize to the supernumerary MTs that are slightly dorsal or ventral to the main ventral disc microtubules (for 3D stacks, see Video S1 and Video S2). These DAPs also have a transient (see Figure 6) localization to the basal bodies (bb) and the caudal (cfl) and ventral (vfl) flagella. DAPs are again visualized using C-terminal GFP tagging (grey or green) in cells counterstained with anti-alpha-tubulin immunostaining of the MT cytoskeleton (red) and DAPI staining of the two nuclei (blue). Scale = 5 µm. Schematic shows areas of GFP localization in red.
Figure 6.
DAPs recover only at non-disc, axoneme or basal body regions.
Cells stably transformed with expression plasmids encoding full-length DAP::GFPs were subjected to quantitative FRAP analysis. Panel A shows representative images of DAP4410::GFP, from the group of proteins that localize to the entire disc spiral. No recovery is observed for DAP4410::GFP 10 minutes post-bleaching (B), and no additional non-disc localization was observed throughout the cell cycle. Panel C shows DAP13981::GFP, a representative of the lateral crest-localizing DAPs. DAP13981::GFP does not recover after 10 minutes post-bleach when localized to the lateral crest of the ventral disc (black line, D), but exhibits significant recovery when localized to the basal bodies or axonemes (gray line, D). DAP17090::GFP (E) is a representative supernumerary MT-associated DAP. No recovery of DAP17090 is observed at the supernumerary MTs at 10 minutes post-bleach (black line, F). Panel (G) shows a DAP17090::GFP cell with basal body and axoneme localization of the protein, which partially recovers within 3 minutes post-bleach (gray line, F). Propidium iodide was used to monitor cell viability (G). Relative fluorescence intensity was corrected for overall loss of fluorescence due to imaging and normalized for background. Black lines indicate a disc-associated region of interest (N = 10), whereas gray lines indicate a BB/axoneme recovery region (N = 10).