Distribution and bulk flow analyses of the intraflagellar transport (IFT) motor kinesin-2 support an "on-demand" model for Chlamydomonas ciliary length control.

Most cells tightly control the length of their cilia. The regulation likely involves intraflagellar transport (IFT), a bidirectional motility of multi-subunit particles organized into trains that deliver building blocks into the organelle. In Chlamydomonas, the anterograde IFT motor kinesin-2 consists of the motor subunits FLA8 and FLA10 and the nonmotor subunit KAP.

Direct in situ protein tagging in Chlamydomonas reinhardtii utilizing TIM, a method for CRISPR/Cas9-based targeted insertional mutagenesis.

Chlamydomonas reinhardtii is an important model organism for the study of many cellular processes, and protein tagging is an increasingly indispensable tool for these studies. To circumvent the disadvantages of conventional approaches in creating a tagged cell line, which involve transforming either a wild-type or null-mutant cell line with an exogenous DNA construct that inserts randomly into the genome, we developed a strategy to tag the endogenous gene in situ. The strategy utilizes TIM, a CRISPR/Cas9-based method for targeted insertional mutagenesis in C.

Consensus nomenclature for dyneins and associated assembly factors.

Dyneins are highly complex, multicomponent, microtubule-based molecular motors. These enzymes are responsible for numerous motile behaviors in cytoplasm, mediate retrograde intraflagellar transport (IFT), and power ciliary and flagellar motility. Variants in multiple genes encoding dyneins, outer dynein arm (ODA) docking complex subunits, and cytoplasmic factors involved in axonemal dynein preassembly (DNAAFs) are associated with human ciliopathies and are of clinical interest.

Structural organization of the C1b projection within the ciliary central apparatus.

Motile cilia have a '9+2' structure containing nine doublet microtubules and a central apparatus (CA) composed of two singlet microtubules with associated projections. The CA plays crucial roles in regulating ciliary motility. Defects in CA assembly or function usually result in motility-impaired or paralyzed cilia, which in humans causes disease.

Chlamydomonas FAP70 is a component of the previously uncharacterized ciliary central apparatus projection C2a.

Cilia are essential organelles required for cell signaling and motility. Nearly all motile cilia have a '9+2' axoneme composed of nine outer doublet microtubules plus two central microtubules; the central microtubules together with their projections are termed the central apparatus (CA). In Chlamydomonas reinhardtii, a model organism for studying cilia, 30 proteins are known CA components, and ∼36 more are predicted to be CA proteins.

Diffusion rather than intraflagellar transport likely provides most of the tubulin required for axonemal assembly in .

Tubulin enters the cilium by diffusion and motor-based intraflagellar transport (IFT). However, the respective contribution of each route in providing tubulin for axonemal assembly remains unknown. Using , we attenuated IFT-based tubulin transport of GFP-β-tubulin by altering the IFT74N-IFT81N tubulin-binding module and the C-terminal E-hook of tubulin.

TIM, a targeted insertional mutagenesis method utilizing CRISPR/Cas9 in Chlamydomonas reinhardtii.

Generation and subsequent analysis of mutants is critical to understanding the functions of genes and proteins. Here we describe TIM, an efficient, cost-effective, CRISPR-based targeted insertional mutagenesis method for the model organism Chlamydomonas reinhardtii. TIM utilizes delivery into the cell of a Cas9-guide RNA (gRNA) ribonucleoprotein (RNP) together with exogenous double-stranded (donor) DNA.

The unity and diversity of the ciliary central apparatus.

Nearly all motile cilia and flagella (terms here used interchangeably) have a '9+2' axoneme containing nine outer doublet microtubules and two central microtubules. The central pair of microtubules plus associated projections, termed the central apparatus (CA), is involved in the control of flagellar motility and is essential for the normal movement of '9+2' cilia. Research using the green alga , an important model system for studying cilia, has provided most of our knowledge of the protein composition of the CA, and recent work using this organism has expanded the number of known and candidate CA proteins nearly threefold.

Structural organization of the C1a-e-c supercomplex within the ciliary central apparatus.

Nearly all motile cilia contain a central apparatus (CA) composed of two connected singlet microtubules with attached projections that play crucial roles in regulating ciliary motility. Defects in CA assembly usually result in motility-impaired or paralyzed cilia, which in humans causes disease. Despite their importance, the protein composition and functions of the CA projections are largely unknown.

Proteome of the central apparatus of a ciliary axoneme.

Nearly all motile cilia have a "9+2" axoneme containing a central apparatus (CA), consisting of two central microtubules with projections, that is essential for motility. To date, only 22 proteins are known to be CA components. To identify new candidate CA proteins, we used mass spectrometry to compare axonemes of wild-type and a CA-less mutant.

A global analysis of IFT-A function reveals specialization for transport of membrane-associated proteins into cilia.

Intraflagellar transport (IFT), which is essential for the formation and function of cilia in most organisms, is the trafficking of IFT trains (i.e. assemblies of IFT particles) that carry cargo within the cilium.

A microtubule-dynein tethering complex regulates the axonemal inner dynein f (I1).

Motility of cilia/flagella is generated by a coordinated activity of thousands of dyneins. Inner dynein arms (IDAs) are particularly important for the formation of ciliary/flagellar waveforms, but the molecular mechanism of IDA regulation is poorly understood. Here we show using cryoelectron tomography and biochemical analyses of Chlamydomonas flagella that a conserved protein FAP44 forms a complex that tethers IDA f (I1 dynein) head domains to the A-tubule of the axonemal outer doublet microtubule.

The N-terminus of IFT46 mediates intraflagellar transport of outer arm dynein and its cargo-adaptor ODA16.

Cilia are assembled via intraflagellar transport (IFT). The IFT machinery is composed of motors and multisubunit particles, termed IFT-A and IFT-B, that carry cargo into the cilium. Knowledge of how the IFT subunits interact with their cargo is of critical importance for understanding how the unique ciliary domain is established.

IFT trains in different stages of assembly queue at the ciliary base for consecutive release into the cilium.

Intraflagellar transport (IFT) trains, multimegadalton assemblies of IFT proteins and motors, traffic proteins in cilia. To study how trains assemble, we employed fluorescence protein-tagged IFT proteins in . IFT-A and motor proteins are recruited from the cell body to the basal body pool, assembled into trains, move through the cilium, and disperse back into the cell body.

Characterization of a new oda3 allele, oda3-6, defective in assembly of the outer dynein arm-docking complex in Chlamydomonas reinhardtii.

We have used an insertional mutagenesis approach to generate new C. reinhardtii motility mutants. Of 56 mutants isolated, one is a new allele at the ODA3 locus, called oda3-6.

Together, the IFT81 and IFT74 N-termini form the main module for intraflagellar transport of tubulin.

The assembly and maintenance of most cilia and flagella rely on intraflagellar transport (IFT). Recent in vitro studies have suggested that, together, the calponin-homology domain within the IFT81 N-terminus and the highly basic N-terminus of IFT74 form a module for IFT of tubulin. By using Chlamydomonas mutants for IFT81 and IFT74, we tested this hypothesis in vivo.

Intraflagellar transport is essential for mammalian spermiogenesis but is absent in mature sperm.

Drosophila sperm are unusual in that they do not require the intraflagellar transport (IFT) system for assembly of their flagella. In the mouse, the IFT proteins are very abundant in testis, but we here show that mature sperm are completely devoid of them, making the importance of IFT to mammalian sperm development unclear. To address this question, we characterized spermiogenesis and fertility in the Ift88(Tg737Rpw) mouse.

Superresolution Pattern Recognition Reveals the Architectural Map of the Ciliary Transition Zone.

The transition zone (TZ) of primary cilia serves as a diffusion barrier to regulate ciliogenesis and receptor localization for key signaling events such as sonic hedgehog signaling. Its gating mechanism is poorly understood due to the tiny volume accommodating a large number of ciliopathy-associated molecules. Here we performed stimulated emission depletion (STED) imaging of collective samples and recreated superresolved relative localizations of eight representative species of ciliary proteins using position averages and overlapped with representative electron microscopy (EM) images, defining an architectural foundation at the ciliary base.

CFAP54 is required for proper ciliary motility and assembly of the central pair apparatus in mice.

Motile cilia and flagella play critical roles in fluid clearance and cell motility, and dysfunction commonly results in the pediatric syndrome primary ciliary dyskinesia (PCD). CFAP221, also known as PCDP1, is required for ciliary and flagellar function in mice and Chlamydomonas reinhardtii, where it localizes to the C1d projection of the central microtubule apparatus and functions in a complex that regulates flagellar motility in a calcium-dependent manner. We demonstrate that the genes encoding the mouse homologues of the other C.

Reduced tubulin polyglutamylation suppresses flagellar shortness in Chlamydomonas.

Ciliary length control is an incompletely understood process essential for normal ciliary function. The flagella of Chlamydomonas mutants lacking multiple axonemal dyneins are shorter than normal; previously it was shown that this shortness can be suppressed by the mutation suppressor of shortness 1 (ssh1) via an unknown mechanism. To elucidate this mechanism, we carried out genetic analysis of ssh1 and found that it is a new allele of TPG2 (hereafter tpg2-3), which encodes FAP234 functioning in tubulin polyglutamylation in the axoneme.

DRC3 connects the N-DRC to dynein g to regulate flagellar waveform.

The nexin-dynein regulatory complex (N-DRC), which is a major hub for the control of flagellar motility, contains at least 11 different subunits. A major challenge is to determine the location and function of each of these subunits within the N-DRC. We characterized a Chlamydomonas mutant defective in the N-DRC subunit DRC3.

Assembly of IFT trains at the ciliary base depends on IFT74.

Intraflagellar transport (IFT) moves IFT trains carrying cargoes from the cell body into the flagellum and from the flagellum back to the cell body. IFT trains are composed of complexes IFT-A and IFT-B and cargo adaptors such as the BBSome. The IFT-B core proteins IFT74 and IFT81 interact directly through central and C-terminal coiled-coil domains, and recently it was shown that the N termini of these proteins form a tubulin-binding module important for ciliogenesis.

TCTEX1D2 mutations underlie Jeune asphyxiating thoracic dystrophy with impaired retrograde intraflagellar transport.

The analysis of individuals with ciliary chondrodysplasias can shed light on sensitive mechanisms controlling ciliogenesis and cell signalling that are essential to embryonic development and survival. Here we identify TCTEX1D2 mutations causing Jeune asphyxiating thoracic dystrophy with partially penetrant inheritance. Loss of TCTEX1D2 impairs retrograde intraflagellar transport (IFT) in humans and the protist Chlamydomonas, accompanied by destabilization of the retrograde IFT dynein motor.

Cilia and Diseases.

In recent decades, cilia have moved from relative obscurity to a position of importance for understanding multiple complex human diseases. Now termed the ciliopathies, these diseases inflict devastating effects on millions of people worldwide. In this review, written primarily for teachers and students who may not yet be aware of the recent exciting developments in this field, we provide a general overview of our current understanding of cilia and human disease.