Protocol for precision editing of endogenous Chlamydomonas reinhardtii genes with CRISPR-Cas.

CRISPR-Cas genome engineering in the unicellular green algal model Chlamydomonas reinhardtii has until recently suffered from low integration efficiencies despite traditional genetics being well established. Here, we present a protocol for efficient homology-directed knockin mutagenesis in all commonly used strains of Chlamydomonas. We describe steps for scarless integration of fusion tags and sequence modifications of almost all proteins without the need for a preceding mutant line.

Efficient precision editing of endogenous genes with CRISPR-Cas.

CRISPR-Cas genome engineering in the unicellular green algal model has until now been primarily applied to targeted gene disruption, whereas scarless knockin transgenesis has generally been considered difficult in practice. We have developed an efficient homology-directed method for knockin mutagenesis in Chlamydomonas by delivering CRISPR-Cas ribonucleoproteins and a linear double-stranded DNA (dsDNA) donor into cells by electroporation. Our method allows scarless integration of fusion tags and sequence modifications of proteins without the need for a preceding mutant line.

Testing the ion-current model for flagellar length sensing and IFT regulation.

Eukaryotic cilia and flagella are microtubule-based organelles whose relatively simple shape makes them ideal for investigating the fundamental question of organelle size regulation. Most of the flagellar materials are transported from the cell body via an active transport process called intraflagellar transport (IFT). The rate of IFT entry into flagella, known as IFT injection, has been shown to negatively correlate with flagellar length.

Conversion of anterograde into retrograde trains is an intrinsic property of intraflagellar transport.

Cilia or eukaryotic flagella are microtubule-based organelles found across the eukaryotic tree of life. Their very high aspect ratio and crowded interior are unfavorable to diffusive transport of most components required for their assembly and maintenance. Instead, a system of intraflagellar transport (IFT) trains moves cargo rapidly up and down the cilium (Figure 1A).

In vivo imaging shows continued association of several IFT-A, IFT-B and dynein complexes while IFT trains U-turn at the tip.

Flagellar assembly depends on intraflagellar transport (IFT), a bidirectional motility of protein carriers, the IFT trains. The trains are periodic assemblies of IFT-A and IFT-B subcomplexes and the motors kinesin-2 and IFT dynein. At the tip, anterograde trains are remodeled for retrograde IFT, a process that in Chlamydomonas involves kinesin-2 release and train fragmentation.

The structure and symmetry of the radial spoke protein complex in flagella.

The radial spoke is a key element in a transducer apparatus controlling the motility of eukaryotic cilia. The transduction biomechanics is a long-standing question in cilia biology. The radial spoke has three regions - a spoke head, a bifurcated neck and a stalk.

The cryo-EM structure of intraflagellar transport trains reveals how dynein is inactivated to ensure unidirectional anterograde movement in cilia.

Movement of cargos along microtubules plays key roles in diverse cellular processes, from signalling to mitosis. In cilia, rapid movement of ciliary components along the microtubules to and from the assembly site is essential for the assembly and disassembly of the structure itself. This bidirectional transport, known as intraflagellar transport (IFT), is driven by the anterograde motor kinesin-2 and the retrograde motor dynein-1b (dynein-2 in mammals).

General and specific promotion of flagellar assembly by a flagellar nucleoside diphosphate kinase.

Nucleoside diphosphate kinases (NDKs) play a central role in diverse cellular processes using the canonical NDK activity or alternative mechanisms that remain poorly defined. Our study of dimeric NDK5 in a flagellar motility control complex, the radial spoke (RS), has revealed new modalities. The flagella in mutant were paralyzed, albeit only deficient in three RS subunits.

Comprehensive and Highly Accurate Measurements of Crane Runways, Profiles and Fastenings.

The process of surveying crane runways has been continually refined due to the competitive situation, modern surveying instruments, additional sensors, accessories and evaluation procedures. Guidelines, such as the International Organization for Standardization (ISO) 12488-1, define target values that must be determined by survey. For a crane runway these are for example the span, the position and height of the rails.

Comparative Analysis of Ciliary Membranes and Ectosomes.

Primary and motile cilia/flagella function as cellular antennae, receiving signals from the environment and subsequently activating signaling pathways that are critical for cellular homeostasis and differentiation [1-3]. Recent work with the green alga Chlamydomonas and the nematode C. elegans demonstrated that ectosomes can be released from the cilium and can mediate the intercellular communication [4-9].

Two classes of short intraflagellar transport train with different 3D structures are present in Chlamydomonas flagella.

Intraflagellar transport (IFT) is responsible for the bidirectional trafficking of molecular components required for the elongation and maintenance of eukaryotic cilia and flagella. Cargo is transported by IFT 'trains', linear rows of multiprotein particles moved by molecular motors along the axonemal doublets. We have previously described two structurally distinct categories of 'long' and 'short' trains.

Proteomic analysis of isolated ciliary transition zones reveals the presence of ESCRT proteins.

The transition zone (TZ) is a specialized region of the cilium characterized by Y-shaped connectors between the microtubules of the ciliary axoneme and the ciliary membrane [1]. Located near the base of the cilium, the TZ is in the prime location to act as a gate for proteins into and out of the ciliary compartment, a role supported by experimental evidence [2-6]. The importance of the TZ has been underscored by studies showing that mutations affecting proteins located in the TZ result in cilia-related diseases, or ciliopathies, presenting symptoms including renal cysts, retinal degeneration, and situs inversus [7-9].

Centrosomal protein CEP104 (Chlamydomonas FAP256) moves to the ciliary tip during ciliary assembly.

The ciliary tip has been implicated in ciliary assembly and disassembly, and signaling, yet information on its protein composition is limited. Using comparative, quantitative proteomics based on the fact that tip proteins will be approximately twice as concentrated in half-length compared with full-length flagella, we have identified FAP256 as a tip protein in Chlamydomonas. FAP256 localizes to the tips of both central pair and outer doublet microtubules (MTs) and it remains at the tip during flagellar assembly and disassembly.

The cilium secretes bioactive ectosomes.

The release of membrane vesicles from the surface of cells into their surrounding environment is now recognized as an important pathway for the delivery of proteins to extracellular sites of biological function. Membrane vesicles of this kind, termed exosomes and ectosomes, are the result of active processes and have been shown to carry a wide array of biological effector molecules that can play roles in cell-to-cell communication and remodeling of the extracellular space. Degradation of the extracellular matrix (ECM) through the regulated release of proteolytic enzymes is a key process for development, morphogenesis, and cell migration in animal and plant cells.

The versatile molecular complex component LC8 promotes several distinct steps of flagellar assembly.

LC8 is present in various molecular complexes. However, its role in these complexes remains unclear. We discovered that although LC8 is a subunit of the radial spoke (RS) complex in Chlamydomonas flagella, it was undetectable in the RS precursor that is converted into the mature RS at the tip of elongating axonemes.

IFT proteins accumulate during cell division and localize to the cleavage furrow in Chlamydomonas.

Intraflagellar transport (IFT) proteins are well established as conserved mediators of flagellum/cilium assembly and disassembly. However, data has begun to accumulate in support of IFT protein involvement in other processes elsewhere in the cell. Here, we used synchronous cultures of Chlamydomonas to investigate the temporal patterns of accumulation and localization of IFT proteins during the cell cycle.

Target-of-rapamycin complex 1 (Torc1) signaling modulates cilia size and function through protein synthesis regulation.

The cilium serves as a cellular antenna by coordinating upstream environmental cues with numerous downstream signaling processes that are indispensable for the function of the cell. This role is supported by the revelation that defects of the cilium underlie an emerging class of human disorders, termed "ciliopathies." Although mounting interest in the cilium has demonstrated the essential role that the organelle plays in vertebrate development, homeostasis, and disease pathogenesis, the mechanisms regulating cilia morphology and function remain unclear.

Cryoelectron tomography of radial spokes in cilia and flagella.

Radial spokes (RSs) are ubiquitous components in the 9 + 2 axoneme thought to be mechanochemical transducers involved in local control of dynein-driven microtubule sliding. They are composed of >23 polypeptides, whose interactions and placement must be deciphered to understand RS function. In this paper, we show the detailed three-dimensional (3D) structure of RS in situ in Chlamydomonas reinhardtii flagella and Tetrahymena thermophila cilia that we obtained using cryoelectron tomography (cryo-ET).

Sequential assembly of flagellar radial spokes.

The unicellular alga Chlamydomonas can assemble two 10 μm flagella in 1 h from proteins synthesized in the cell body. Targeting and transporting these proteins to the flagella are simplified by preassembly of macromolecular complexes in the cell body. Radial spokes are flagellar complexes that are partially assembled in the cell body before entering the flagella.

Subunit interactions within the Chlamydomonas flagellar spokehead.

The radial spoke (RS)/central pair (CP) system in cilia and flagella plays an essential role in the regulation of force generation by dynein, the motor protein that drives cilia/flagella movements. Mechanical and mechanochemicl interactions between the CP and the distal part of the RS, the spokehead, should be crucial for this control; however, the details of interaction are totally unknown. As an initial step toward an understanding of the RS-CP interaction, we examined the protein-protein interactions between the five spokehead proteins (radial spoke protein (RSP)1, RSP4, RSP6, RSP9, and RSP10) and three spoke stalk proteins (RSP2, RSP5, and RSP23), all expressed as recombinant proteins.

Nickel and low CO₂-controlled motility in Chlamydomonas through complementation of a paralyzed flagella mutant with chemically regulated promoters.

Background: Chlamydomonas reinhardtii is a model system for the biology of unicellular green algae. Chemically regulated promoters, such as the nickel-inducible CYC6 or the low CO₂-inducible CAH1 promoter, may prove useful for expressing, at precise times during its cell cycle, proteins with relevant biological functions, or complementing mutants in genes encoding such proteins. To this date, this has not been reported for the above promoters.

CEP290 tethers flagellar transition zone microtubules to the membrane and regulates flagellar protein content.

Mutations in human CEP290 cause cilia-related disorders that range in severity from isolated blindness to perinatal lethality. Here, we describe a Chlamydomonas reinhardtii mutant in which most of the CEP290 gene is deleted. Immunoelectron microscopy indicated that CEP290 is located in the flagellar transition zone in close association with the prominent microtubule-membrane links there.

Centrioles are freed from cilia by severing prior to mitosis.

Cilia are necessary for normal tissue development and homeostasis and are generally present during interphase, but not in mitosis. The precise mechanism of premitotic ciliary loss has been controversial, with data supporting either sequential disassembly through the transition zone or, alternatively, a severing event at the base of the cilia. Here we show by live cell imaging and immunofluorescence microscopy that resorbing flagella of Chlamydomonas leave remnants associated with the mother cell wall.

Electron-tomographic analysis of intraflagellar transport particle trains in situ.

Intraflagellar transport (IFT) is the bidirectional movement of multipolypeptide particles between the ciliary membrane and the axonemal microtubules, and is required for the assembly, maintenance, and sensory function of cilia and flagella. In this paper, we present the first high-resolution ultrastructural analysis of trains of flagellar IFT particles, using transmission electron microscopy and electron-tomographic analysis of sections from flat-embedded Chlamydomonas reinhardtii cells. Using wild-type and mutant cells with defects in IFT, we identified two different types of IFT trains: long, narrow trains responsible for anterograde transport; and short, compact trains underlying retrograde IFT.

The ubiquitin conjugation system is involved in the disassembly of cilia and flagella.

The disassembly of cilia and flagella is linked to the cell cycle and environmental cues. We have found that ubiquitination of flagellar proteins is an integral part of flagellar disassembly. Free ubiquitin and the ubiquitin-conjugating enzyme CrUbc13 are detected in flagella, and several proteins are ubiquitinated in isolated flagella when exogenous ubiquitin and adenosine triphosphatase are added, suggesting that the ubiquitin conjugation system operates in flagella.