Intraflagellar transport (IFT) is essential for construction and maintenance of cilia. IFT proteins concentrate at the basal body, where they are thought to assemble into trains and bind cargoes for transport. To study the mechanisms of IFT recruitment to this peri-basal body pool, we quantified protein dynamics of eight IFT proteins, as well as five other basal body localizing proteins, using fluorescence recovery after photobleaching in vertebrate multiciliated cells. We found that members of the IFT-A and IFT-B protein complexes show distinct turnover kinetics from other basal body components. Additionally, known IFT sub-complexes displayed shared dynamics, suggesting shared basal body recruitment and/or retention mechanisms. Finally, we evaluated the mechanisms of basal body recruitment by depolymerizing cytosolic MTs, which suggested that IFT proteins are recruited to basal bodies through a diffusion-to-capture mechanism. Our survey of IFT protein dynamics provides new insights into IFT recruitment to basal bodies, a crucial step in ciliogenesis and ciliary signaling.

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BackgroundClustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided adaptive immune systems are found in prokaryotes to defend cells from foreign DNA. CRISPR Cas9 systems have been modified and employed as genome editing tools in wide ranging organisms. Here, we provide a detailed protocol to truncate genes in mammalian cells using CRISPR Cas9 editing. We describe custom donor vector construction using Gibson assembly with the commonly utilized pcDNA3 vector as the backbone.ResultsWe describe a step-by-step method to truncate genes of interest in mammalian cell lines using custom-made donor vectors. Our method employs 2 guide RNAs, mutant Cas9D10A nickase (Cas9 = CRISPR associated sequence 9), and a custom-made donor vector for homologous recombination to precisely truncate a gene of interest with a selectable neomycin resistance cassette (NPTII: Neomycin Phosphotransferase II). We provide a detailed protocol on how to design and construct a custom donor vector using Gibson assembly (and the commonly utilized pcDNA3 vector as the backbone) allowing researchers to obtain specific gene modifications of interest (gene truncation, gene deletion, epitope tagging or knock-in mutation). Selection of mutants in mammalian cell lines with G418 (Geneticin) combined with several screening methods: western blot analysis, polymerase chain reaction, and Sanger sequencing resulted in streamlined mutant isolation. Proof of principle experiments were done in several mammalian cell lines.ConclusionsHere we describe a detailed protocol to employ CRISPR Cas9 genome editing to truncate genes of interest using the commonly employed expression vector pcDNA3 as the backbone for the donor vector. Providing a detailed protocol for custom donor vector design and construction will enable researchers to develop unique genome editing tools. To date, detailed protocols for CRISPR Cas9 custom donor vector construction are limited (Lee et al. in Sci Rep 5:8572, 2015; Ma et al. in Sci Rep 4:4489, 2014). Custom donor vectors are commercially available, but can be expensive. Our goal is to share this protocol to aid researchers in performing genetic investigations that require custom donor vectors for specialized applications (specific gene truncations, knock-in mutations, and epitope tagging applications).Electronic supplementary materialThe online version of this article (10.1186/s12867-018-0105-8) contains supplementary material, which is available to authorized users.

Intraflagellar transport (IFT) is essential for construction and maintenance of cilia. IFT proteins concentrate at the basal body, where they are thought to assemble into trains and bind cargoes for transport. To study the mechanisms of IFT recruitment to this peri-basal body pool, we quantified protein dynamics of eight IFT proteins, as well as five other basal body localizing proteins, using fluorescence recovery after photobleaching in vertebrate multiciliated cells. We found that members of the IFT-A and IFT-B protein complexes show distinct turnover kinetics from other basal body components. Additionally, known IFT sub-complexes displayed shared dynamics, and these dynamics were not altered during cilia regeneration as compared to homeostasis. Finally, we evaluated the mechanisms of basal body recruitment by depolymerizing cytosolic MTs, which suggested that IFT proteins are recruited to basal bodies through a diffusion-to-capture mechanism. Our survey of IFT protein dynamics provides new insights into IFT recruitment to basal bodies, a crucial step in ciliogenesis.

Cilia are multifunctional organelles that originated with the last eukaryotic common ancestor and play central roles in the life cycles of diverse organisms. The motile flagella that move single cells like sperm or unicellular organisms, the motile cilia on animal multiciliated cells that generate fluid flow in organs, and the immotile primary cilia that decorate nearly all cells in animals share many protein components in common, yet each also requires specialized proteins to perform their specialized functions. Despite a now-advanced understanding of how such proteins are transported within cilia, we still know very little about how they are transported from their sites of synthesis through the cytoplasm to the ciliary base. Here, we review the literature concerning this underappreciated topic in ciliary cell biology. We discuss both general mechanisms, as well as specific examples of motor-driven active transport and passive transport via diffusion-and-capture. We then provide deeper discussion of specific, illustrative examples, such as the diverse array of protein subunits that together comprise the intraflagellar transport (IFT) system and the multi-protein axonemal dynein motors that drive beating of motile cilia. We hope this Review will spur further work, shedding light not only on ciliogenesis and ciliary signaling, but also on intracellular transport in general.