Single-cell green algae (C. Reinhardtii) is a key model organism to study ciliogenesis. Cilia have important roles in sensory signaling pathways and in clearing the airways of mucus and dirt in multiple systems of the human body. As cilia are found on most eukaryotic cells, defects in ciliogenesis result in many symptoms and disorders. We are testing the hypothesis that when a flagellum is removed, the long flagellum shrinks because it is competing with the shorter regrowing flagellum. We used a 780-nm 200-fs laser to perform laser ablation to amputate one of two flagella on wildtype and mutant algae. Fla3 and Fla10 mutants were altered to inhibit the KAP kinesin motor that drives the intraflagellar transport (IFT) pathway. Impaired IFT pathways would demonstrate a lag in response to flagellar length equalization and a reduced disassembly rate. Quantified images following the long flagellum for 20 min post-ablation demonstrate a delayed disassembly rate in the Fla3 mutant compared to wildtype; Fla10 was inconclusive. Therefore, it was concluded that the proper function of KAP motor protein serves a significant role in length control of cilia. In the future, we will compare the assembly rates of flagellar regrowth for the wildtype and mutants.
We describe an approach for using the flagella axoneme as the basis for biological self-assembling protein nanoarrays. The axoneme is the insoluble protein core of the eukaryotic flagellum or cilium. By attaching a protein of interest to particular axonemal proteins, it is possible to exploit the intraflagellar transport system to incorporate those proteins into the axoneme as it assembles. Using the axoneme as a protein array confers several advantages, such as high protein loading capacity compared to other bioparticle systems; genetically programmed self-assembly without the need for any linking steps; single-step purification of particles without the need for cell lysis, allowing retention and re-use of biomass; and choice of isolating the particle as a membrane enclosed vesicle or as an exposed protein array. Here we test several potential axonemal proteins as adaptor proteins, using green fluorescent protein as a test case. We find that FAP20 is an ideal scaffold protein for this purpose in that it shows high incorporation and uniform localization. We verify that FAP20-GFP constructs are stably associated with the axoneme during purification and storage, that the GFP moiety can be released by protease cleavage, and that the flagellar array can be easily encapsulated in aqueous-oil emulsion droplets for use in microfluidic assays.
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