Intraflagellar transport-20 mediates the ciliary membrane trafficking of channelrhodopsin in Chlamydomonas reinhardtii
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Abstract
The primary cilium is a microtubule-based organelle essential for cellular signaling, whose assembly depends on intraflagellar transport (IFT). IFT20, a unique IFT-B component, localizes to both Golgi and cilium/flagellum, and plays a role in ciliary membrane protein trafficking. Here, we analyzed IFT20-mediated ciliary membrane trafficking of channelrhodopsin-1 (ChR1) in Chlamydomonas reinhardtii . IFT20 and ChR1 was found to be co-localized throughout the cilia of C. reinhardtii wild-type strain. In bbs1 mutants, IFT20 exhibited distribution along the flagellar length and basal body region. In contrast, ChR1 was found to be accumulating in the distal region of the cilia. Further, protein interaction network analysis reveals that IFT20 serves as a central adaptor, interfacing ancillary ciliary trafficking components, including Arf, the IFT complex, and BBSome subunits. The Arf co-localized with IFT20 in the cilia of the wild-type strain, suggesting a potential interaction. The fluorescence spectroscopic analysis shows that upon GTP binding, IFT20 undergoes concentration-dependent fluorescence quenching. From the far-UV CD spectroscopic analysis, it was observed that recombinant IFT20 has a predominantly helical structure, which does not change upon GTP binding. These findings extend the mechanistic understanding of ciliary membrane protein delivery in C. reinhardtii and reveal a conserved role of IFT20 in photoreceptor trafficking.
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demonstrated significant spatial overlap
For this and subsequent figures, some quantification or multiple cells rather than images of a single cell would be more convincing (even just a line scan of fluorescence intensity along the length of the flagella in each channel, for spatial coincidence). Single channel grayscale would also make it visually more clear. It’s a bit difficult to discern as shown.
There is also an IFT20-mCherry strain available in the chlamy center. It’s expressed in the ift20 mutant background. Curious also what the chr1 localization is in ift20 mutants.
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The same blot probed with pre-immune serum at the same dilution failed to detect any corresponding band, indicating a lack of non-specific binding and confirming the high specificity of the antibody for CrIFT20
Since you have pre-immune serum, I’m guessing you generated this chlamy-specific ift-20 antibody yourself? Wild type and IFT-20 mutant (either ift20-1 or a CLiP mutant) lysate by side on the same blot would be a convincing demonstration of antibody specificity.
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CrIFT20 (1:250) and ChR1-Ct
What is the source of these antibodies?
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