Fig 1.
Depletion of GlActin affects attachment and ventral disc morphology.
A) Left: Diagram of a Giardia trophozoite showing GlActin (magenta), disc marker delta-giardin (cyan), and DAPI/nuclei (blue). Anterior (Af), posteriolateral (Plf), ventral (Vf), and caudal flagella (Cf) are shown. Right: Enlarged diagram of ventral disc and areas: overlap zone (OZ), ventral groove (VG), and bare area (BA). Arrows indicate the origin of the microtubule sheet and orientation of the microtubule plus ends. B) Quantification of non-challenged attachment assays. Attached and unattached cells were counted 24 hours after treatment with either a standard control or anti-GlActin morpholino. Graph is representative of an average of three replicates, mean ± sem. For control, 74% ± 6 were attached and 25% ± 6 were detached. For GlActin-depleted cells 23% ± 2 were attached and 77% ± 2 detached. Raw numbers and detailed statistics reported in S1 Table. Each replicate was analyzed separately with a two-sided chi-square test with Yates’ correction, P<0.0001 for each. C) Unattached cells from B were stained with propidium iodide and fluorescein diacetate to distinguish dead from living cells respectively. N = 198 control cells and 222 GlActin-depleted cells. There was no significant difference between the two groups based on a two-sided Fisher’s exact test P = 0.7307. D) Both attached and unattached GlActin-depleted and control cells were fixed and immunostained for delta-giardin to assess ventral disc morphology using fluorescence microscopy. N = 126 cells for each condition. There was a significant difference in unattached cells based on a two-sided chi-square test with Yates’ correlation, P<0.0001. No significant difference was detected in attached cells based on the same test, P<0.0903 E) Representative images of cells from D, with examples of normal discs (teal), mild defects (yellow), and severe defects (pink). Scale bar denotes 5 μm.
Fig 2.
DAAP1 is a GlActin interactor and a ventral disc-associated protein.
A) Immunoprecipitation from extracts of cells expressing DAAP1-3xHA and wild type cells, followed by western blots probed with anti-GlActin and anti-HA antibodies, revealed interaction between the two proteins. B) Image of DAAP1-mNeonGreen in a live cell. The fusion protein localizes to the disc and is enriched in the ventral groove (asterisk) and overlap zone (arrow). Scale bar denotes 5 μm. C) Immunofluorescence of GlActin (magenta), tubulin (cyan), DAAP1-mNeonGreen (green), and DAPI/nuclei (blue) during the cell cycle. DAAP1-mNeonGreen remains on the disc into telophase, is incorporated into the nascent discs by mid cytokinesis (see arrows), and localization is fully restored in newly divided cells. Pearson’s correlation coefficients using Costes randomization were calculated for colocalization between DAAP1 and GlActin in each image, shown in the lower righthand corner. The Costes P-value was 1 for all images. Scale bar denotes 5 μm.
Fig 3.
DAAP1 loading onto the ventral disc requires GlActin.
A) Representative images of attached cells 24 hours after treatment with either an anti-GlActin or control morpholino. Fluorescence intensity of DAAP1 at the ventral groove (see green circle) was measured at the brightest section in each stack. GlActin shown in magenta, mNeonGreen-DAAP1 in green, DAPI in blue. Scale bar denotes 5 μm. B) A two-tailed Mann-Whitney test indicated that this difference was not statistically significant, (NControl = 57, NGlActin-depletd = 55), P = 0.227. Median and 95% confidence interval shown. Median of control = 3131445. Median of GlActin-depleted = 3298353. Two and four outliers were detected by Tukey Fence, k = 1.5 in the control and GlActin-depleted condition respectively and were not included in the analysis. C) Representative images of unattached cells 24 hours after treatment with either an anti-GlActin or control morpholino. Fluorescence intensity of DAAP1 at the ventral groove (see green circle) was measured at the brightest optical section in each stack. D) A two-tailed Mann-Whitney test indicated that this difference was statistically significant, (NControl = 55, NGlActin-depletd = 54), P<0.0001. Median and 95% confidence interval shown. Median of control = 3508064. Median of GlActin-depleted = 1609904. Four outliers were detected by Tukey Fence, k = 1.5 in the GlActin-depleted condition and were not included in the analysis.
Fig 4.
DAAP1 is stably associated with the ventral disc.
Fluorescence recovery after-photobleaching (FRAP) in DAAP1-mNeonGreen-cells showed no recovery after 12 minutes for bleached areas in both the ventral groove (A) and overlap zone (B). Three points of reference were taken, including bleached (ROI1, blue), unbleached (RO2, red), and background (green), and normalized accordingly. Scale bar denotes 5 μm. N = 10 for each ROI in both A and B.
Fig 5.
DAAP1-depleted discs do not have defects detectable by light microscopy.
A) Fluorescence of DAAP1-mNeonGreen cells transfected with a dead Cas9 vector containing a HALO-tagged delta-giardin and either a non-specific small guide RNA (dCas9-CTRL) or a small guide RNA targeting DAAP1 (dCas9+sgRNA392-411). Normalized to the control, the DAAP1-depleted cells demonstrated a population-average knockdown of 40% ± 3 (three independent transformations). B) Attached and unattached control and DAAP1-depleted cells were fixed and stained separately before assessing their disc morphology. Based on a chi-square test, there was no significant difference in disc defects observed between either attached or unattached groups. (Control attached N = 129 cells, control unattached N = 126, DAAP1-depleted attached N = 123, DAAP1-depleted unattached N = 128.) Attached cells, P = 0.3483. Unattached cells:, P = 0.1288. C) Representative images from B: GlActin (magenta), delta-giardin-HALO (cyan), DAAP1-mNeonGreen (green), and DAPI/nuclei (blue). Scale bar denotes 5 μm.
Fig 6.
DAAP1 depletion results in reduced attachment.
A) DAAP1 fluorescence levels from control (dCas9-CTRL) and DAAP1-depleted (dCas9+sgRNA392-411). Unattached cells had a greater decrease in DAAP1 expression (38% ± 6) than attached cells (23% ± 2). Average of two independent transformations with three technical replicates for each is shown. B) In this flow chamber assay, attached cells were challenged with a flow rate of 100 μL/min. Cells were tracked for 10 seconds using TrackMate software in ImageJ, colored lines indicating resulting paths. C) Distribution of mean flow-induced speed. D) Mean flow-induced speed of each cell for 10 seconds after challenge, with median and 95% confidence intervals. A two-tailed Mann-Whitney test indicated that this difference was statistically significant, (NdCas9-CTRL = 124, NdCas9+sgRNA392-411 = 111), P = 0.001. Median of control = 10.56, upper 95% CI = 13.33, lower 95% CI = 10.15. Median of DAAP1-depleted is 14.62, upper 95% CI = 18.62, lower 95% CI = 14.41. E) Kaplan-Meier curve of cells displaced from the field of view over time, P = 0.0077 N = 124 for control, N = 111 for DAAP1-depleted cells. Median and standard error shown.
Fig 7.
DAAP1 has a role in ventral disc seal formation.
A) Fluorescent microspheres were added to live attached control (dCas9-CTRL) and DAAP1-depleted (dCas9+sgRNA392-411) and imaged. Left: significantly more cells with microspheres under the disc were observed in DAAP1-depleted cells based on a chi-square test exact test, P<0.001. N = 175 for control, 180 for DAAP1-depleted cells. Right, top: control cell. Right, bottom: DAAP1-depleted cell with microsphere under disc (yellow arrow). Merged images shown with DIC, microspheres (magenta), delta-giardin (cyan). Two-sided Fisher’s exact test, P = 0.0156 was used for analysis. B) TIRF microscopy assay to monitor the quality of attachment. Control and DAAP1-depleted cells were stained with the plasma membrane stain CellMask Orange and imaged with TIRF microscopy. Left: significantly more DAAP1-depleted cells were partially attached than control cells based on a chi-square test, N = 317 for each condition, P<0.005. Right: images of cells in initial attachment (stage 2) with lateral crest (LC) contact, partial attachment (stage 3), with lateral crest and lateral shield (LS) contact, or fully attached (stage 4), with lateral crest, lateral shield, and bare area (BA) contact.