Intraflagellar transport proteins are involved in thrombocyte filopodia formation and secretion

Abstract

Intraflagellar transport (IFT) proteins are vital for the genesis and maintenance of cilia. Our identification of ift122 transcripts in zebrafish thrombocytes that lack primary cilia was unexpected. IFT proteins serve transport in cilia, whose narrow dimensions may have necessitated the evolution of IFT from vesicular transport in ancestral eukaryotes. We hypothesized that IFTs might also facilitate transport within the filopodia that form when thrombocytes are activated. To test this possibility, we knocked down ift122 expression by injecting antisense Morpholino oligonucleotides (MOs) into zebrafish embryos. Laser-induced arterial thrombosis showed prolonged time to occlusion (TTO) of the vessel, as would be expected with defective thrombocyte function. Acute effects in adult zebrafish were evaluated by Vivo-Morpholino (Vivo-MO) knockdown of ift122. Vivo-MO morphants showed a prolonged time to thrombocyte aggregation (TTA) in the plate tilt assay after thrombocyte activation by the following agonists: ADP, collagen, PAR1 peptide, and epinephrine. A luminescence assay for ATP revealed that ATP secretion by thrombocytes was reduced in collagen-activated blood of Vivo-MO ift122 morphants. Moreover, DiI-C18 labeled morphant thrombocytes exposed to collagen showed reductions in filopodia number and length. Analysis of ift mutants, in which cilia defects have been noted, also showed prolongation of TTO in our arterial laser thrombosis assay. Additionally, collagen activation of wild-type thrombocytes led to a concentration of IFT122 both within and at the base of filopodia. Taken together these results, suggest that IFT proteins are involved in both the extension of filopodia and secretion of ATP, which are critical in thrombocyte function.

Keywords: IFT122; Intraflagellar transport; Thrombocytes; Zebrafish.

Figure 1.

Knockdown of ift122. A. Left panel: primer (red line), exon sites, and alternatively spliced product. Right panel: RT-PCR products resolved by agarose gel electrophoresis. Arrows point to bands corresponding to unspliced (431 bp) and alternatively spliced (270 bp) products in control and ift122 knockdown embryos. Marker lane shows DNA size-markers. B. Immunostaining of zebrafish embryos. Upper and lower panels are control MO (Control) and IFT122 MO-injected (IFT122 KD) embryos. Left, middle and right panels, show brightfield, green fluorescence (IFT122), and red fluorescence (β-tubulin IV) respectively. Embryos were labeled with IFT122 antibody (green) and β-tubulin IV antibody (red). Arrow head points to the notochord. C. Overlay histogram of the mean fluorescence intensity of blood cells immunolabeled with IFT122 antibody. The fluorescence intensities of 10,000 cells from ift122 Vivo-MO injected (red line), and control Vivo-MO injected (black line) adult zebrafish were analyzed by BD Accuri C6 flow cytometer. The overlay histograms show cell number (Count) on the y-axis, and the decrease in overall fluorescence of ift122 Vivo-MO treated blood cells as left shift that reflects the reduced IFT122 proteins levels on the x-axis (FL1-A) relative to that observed in the control blood cells.

Figure 2.

Effects of ift122 knockdown on arterial thrombosis in zebrafish larvae. A. TTO (time to occlusion) of 5 dpf knockdown larvae derived from ift122 MO injected embryos (labeled as IFT122-MO) increased twofold over control MO injected embryos (labeled as Control). Knockdown and control groups were compared by one way ANOVA; error bars represent standard deviation; n=55 zebrafish per group; *p < 0.001. B. Rescue of IFT122 morphant. TTO of 5 dpf larvae obtained by coinjection of wild-type sense ift122 RNA and ift122 MO labeled as IFT122-MO (S mRNA) was shorter than the TTO of larvae obtained by coinjection of antisense ift122 RNA and ift122 MO labeled as IFT122-MO (AS mRNA). The above two groups were compared by one way ANOVA; error bars represent standard deviation; n=38 zebrafish per group; *p < 0.001.

Figure 3.

Effect of ift122 Vivo-MO knockdown on thrombocyte aggregation time in response to agonists. Panels A, B, C, and D show the results of plate-tilt assays in TTA (time to aggregation) for blood samples from adult fish injected with ift122 Vivo-MO (labeled as IFT122) and from control fish injected with control Vivo-MO (labeled as Control) in the presence of the agonists ADP, collagen, PAR1 peptide, and epinephrine. Each panel shows significant differences between ift122 knockdown and its corresponding control. For each panel, knockdown and control groups were compared by one way ANOVA; error bars represent standard deviation; n=12 zebrafish per group; *p < 0.001.

Figure 4.

Laser-induced arterial thrombosis of ift122 Vivo-MO knockdown larvae. 4 dpf larvae were injected with either ift122 Vivo-MO or control Vivo-MO and after 24 hrs laser thrombosis assay was performed on these larvae. TTO (time taken to occlusion) of 5 dpf ift122 Vivo-MO injected larvae (labeled as IFT122) showed a twofold increase compared to control Vivo-MO injected larvae (labeled as control). Knockdown and control groups were compared by one way ANOVA; error bars represent standard deviation; n=42 larvae per group; *p < 0.001.

Figure 5.

Reduction of ATP release in thrombocytes subjected to ift122 Vivo-MO knockdown. ATP luminescence (relative luminescence units; RLU) showed a 1.5-fold decrease in ATP luminescence from ift122 knockdown thrombocytes (IFT122) compared to controls, indicating a decrease in ATP release. Knockdown and control groups were compared by one way ANOVA; error bars represent standard deviation; n=12 zebrafish per group; *p < 0.001.

Figure 6.

Qualitative assessment of filopodia reduction in ift122 Vivo-MO knockdown thrombocytes. Representative images of ift122 knockdown (labeled as IFT122) and control samples derived from control Vivo-MO injected zebrafish, showing filopodia formed upon collagen activation of DiI-C18 labeled thrombocytes. Seventy-five thrombocytes in each of the control and experimental samples were analyzed. Arrows show the filopodia. Note ift122 Vivo-MO knockdown thrombocytes have fewer filopodia compared to thrombocytes from control Vivo-MO injected zebrafish.

Figure 7.

Localization of IFT and β-tubulin IV in collagen-activated thrombocytes. A. Immunofluorescence images wild-type thrombocytes. Panels a, b, c, and d show brightfield, red fluorescence, green fluorescence, and merge of red and green fluorescence images, respectively. Thrombocytes from wild-type zebrafish were labeled with β-tubulin IV antibody (red, β-tubulin) and IFT122 antibody (green, IFT122). Note the colocalization of IFT122 and β-tubulin IV in both wild-type control and collagen-treated cells. In collagen-treated cells, IFT122 and β-tubulin IV are concentrated at the base of and within filopodia, while the control cells show an even, ring-like distribution. Scale bar (lower right panel) applies to all panels. B. Quantification of fluorescence intensities at the base of filopodia compared to the other areas in the peripheral regions of thrombocytes. From collagen-activated thrombocytes mean fluorescent intensities of the base of the filopodia and another equal area were measured by Image J software and their ratio was calculated, and these ratios were compared with the similar ratio from the wild-type control cells. Collagen-activated and control groups were compared by one way ANOVA; error bars represent standard deviation; n=30 thrombocytes per group; *p < 0.001. C. Immunofluorescence images of ift122 knockdown thrombocytes. Panels a, b, c, and d show brightfield, red fluorescence, green fluorescence, and merge of red and green fluorescence images, respectively. Thrombocytes from ift122 knockdown zebrafish were labeled with β-tubulin IV antibody (red, β-tubulin) and IFT122 antibody (green, IFT122). Note the IFT122 and β-tubulin IV are not concentrated but are diffuse although colocalization of IFT122 and β-tubulin IV was noted in both ift122 knockdown control and collagen treated cells. Scale bar (lower right panel) applies to all panels.

Figure 7.

Localization of IFT and β-tubulin IV in collagen-activated thrombocytes. A. Immunofluorescence images wild-type thrombocytes. Panels a, b, c, and d show brightfield, red fluorescence, green fluorescence, and merge of red and green fluorescence images, respectively. Thrombocytes from wild-type zebrafish were labeled with β-tubulin IV antibody (red, β-tubulin) and IFT122 antibody (green, IFT122). Note the colocalization of IFT122 and β-tubulin IV in both wild-type control and collagen-treated cells. In collagen-treated cells, IFT122 and β-tubulin IV are concentrated at the base of and within filopodia, while the control cells show an even, ring-like distribution. Scale bar (lower right panel) applies to all panels. B. Quantification of fluorescence intensities at the base of filopodia compared to the other areas in the peripheral regions of thrombocytes. From collagen-activated thrombocytes mean fluorescent intensities of the base of the filopodia and another equal area were measured by Image J software and their ratio was calculated, and these ratios were compared with the similar ratio from the wild-type control cells. Collagen-activated and control groups were compared by one way ANOVA; error bars represent standard deviation; n=30 thrombocytes per group; *p < 0.001. C. Immunofluorescence images of ift122 knockdown thrombocytes. Panels a, b, c, and d show brightfield, red fluorescence, green fluorescence, and merge of red and green fluorescence images, respectively. Thrombocytes from ift122 knockdown zebrafish were labeled with β-tubulin IV antibody (red, β-tubulin) and IFT122 antibody (green, IFT122). Note the IFT122 and β-tubulin IV are not concentrated but are diffuse although colocalization of IFT122 and β-tubulin IV was noted in both ift122 knockdown control and collagen treated cells. Scale bar (lower right panel) applies to all panels.

Figure 8.

Analyses of homozygous ift172 and ift88 mutant zebrafish larvae. A. Photograph of agarose gel shows RT-PCR products of ift172 and ift88 thrombocyte mRNAs. Arrows point to the bands corresponding to the ift172 (255 bp) and ift88 (188 bp) transcripts and marker lane shows DNA size-markers. B. Top panels: Representative homozygous mutant zebrafish larvae at 3 days post-fertilization. Note the characteristic tail curvature associated with IFT deficiency in both ift172 and ift88 mutant larvae, while control 3 dpf larva lacks curvature. Bottom panels: TTO (time to occlusion) of 4 dpf ift88 and ift172 mutant larvae increased more than 2.5-fold compared to controls larvae from the same progeny. Mutant and control groups were compared by one way ANOVA; error bars represent standard deviation. For the group of ift88 and the corresponding control n=30 and the group of ift172 its control n=32.*p < 0.001 for both groups.