High-Throughput Metabolite Analysis of Unicellular Microalgae by Orthogonal Hybrid Ionization Label-Free Mass Cytometry.

Microalgae metabolite analysis is fundamental for the rational design of metabolic engineering strategies for the biosynthesis of high-value products. Mass spectrometry (MS) has been utilized for single-cell microalgae analysis. However, limitations in the detection throughput and polarities of detectable substances make it difficult to realize high-throughput screening of high-performance microalgae. Herein, a plasma-assisted label-free mass cytometry, named as PACyESI-MS, was proposed combining the advantages of orthogonal hybrid ionization and high-throughput MS analysis, which realized rapid metabolite detection of single microalgae. The cell detection throughput of PACyESI-MS was up to 52 cells/min. Dozens of the critical primary and secondary metabolites within single microalgae were detected simultaneously, including pigments, lipids, and energy metabolites. Furthermore, metabolite changes of Chlamydomonas reinhardtii and Haematococcus pluvialis under nitrogen deficiency stress were studied. Discrimination of Chlamydomonas under different nutrient conditions was realized using single-cell metabolite profiles obtained by PACyESI-MS. The relationships between the accumulation of bioactive astaxanthin and changes in functional primary metabolites of Haematococcus were investigated. It was demonstrated that PACyESI-MS can detect the flexible change of metabolites in single microalgae cells under different nutritional conditions and during the synthesis of high-value products, which is expected to become an important tool for the design of metabolic engineering-based high-performance microalgae factories.

Utilizing codon degeneracy in the design of donor DNA for CRISPR/Cas9-mediated gene editing to streamline the screening process for single amino acid mutations.

Chlamydomonas reinhardtii, a unicellular green alga, has been widely used as a model organism for studies of algal, plant and ciliary biology. The generation of targeted amino acid mutations is often necessary, and this can be achieved using CRISPR/Cas9 induced homology-directed repair to install genomic modifications from exogenous donor DNA. Due to the low gene editing efficiency, the technical challenge lies in identifying the mutant cells. Direct sequencing is not practical, and pre-screening is required. Here, we report a strategy for generating and screening for amino acid point mutations using the CRISPR/Cas9 gene editing system. The strategy is based on designing donor DNA using codon degeneracy, which enables the design of specific primers to facilitate mutant screening by PCR. An in vitro assembled RNP complex, along with a dsDNA donor and an antibiotic resistance marker, was electroporated into wild-type cells, followed by PCR screening. To demonstrate this principle, we have generated the E102K mutation in centrin and the K40R mutation in α-tubulin. The editing efficiencies at the target sites for Centrin, TUA1, TUA2 were 4, 24 and 8% respectively, based on PCR screening. More than 80% of the mutants with the expected size of PCR products were precisely edited, as revealed by DNA sequencing. Subsequently, the precision-edited mutants were biochemically verified. The introduction of codon degeneracy did not affect the gene expression of centrin and α-tubulins. Thus, this approach can be used to facilitate the identification of point mutations, especially in genes with low editing rates.

Quantitative proteomics reveals insights into the assembly of IFT trains and ciliary assembly.

Intraflagellar transport (IFT) is required for ciliary assembly. The IFT machinery comprises the IFT motors kinesin-2 and IFT dynein plus IFT-A and IFT-B complexes, which assemble into IFT trains in cilia. To gain mechanistic understanding of IFT and ciliary assembly, here, we performed an absolute quantification of IFT machinery in Chlamydomonas reinhardtii cilium. There are ∼756, ∼532, ∼276 and ∼350 molecules of IFT-B, IFT-A, IFT dynein and kinesin-2, respectively, per cilium. The amount of IFT-B is sufficient to sustain rapid ciliary growth in terms of tubulin delivery. The stoichiometric ratio of IFT-B:IFT-A:dynein is ∼3:2:1 whereas the IFT-B:IFT-A ratio in an IFT dynein mutant is 2:1, suggesting that there is a plastic interaction between IFT-A and IFT-B that can be influenced by IFT dynein. Considering diffusion of kinesin-2 during retrograde IFT, it is estimated that one kinesin-2 molecule drives eight molecules of IFT-B during anterograde IFT. These data provide new insights into the assembly of IFT trains and ciliary assembly.

Structure-guided discovery of protein and glycan components in native mastigonemes.

Mastigonemes, the hair-like lateral appendages lining cilia or flagella, participate in mechanosensation and cellular motion, but their constituents and structure have remained unclear. Here, we report the cryo-EM structure of native mastigonemes isolated from Chlamydomonas at 3.0 Å resolution. The long stem assembles as a super spiral, with each helical turn comprising four pairs of anti-parallel mastigoneme-like protein 1 (Mst1). A large array of arabinoglycans, which represents a common class of glycosylation in plants and algae, is resolved surrounding the type II poly-hydroxyproline (Hyp) helix in Mst1. The EM map unveils a mastigoneme axial protein (Mstax) that is rich in heavily glycosylated Hyp and contains a PKD2-like transmembrane domain (TMD). Mstax, with nearly 8,000 residues spanning from the intracellular region to the distal end of the mastigoneme, provides the framework for Mst1 assembly. Our study provides insights into the complexity of protein and glycan interactions in native bio-architectures.

Structure of a TOC-TIC supercomplex spanning two chloroplast envelope membranes.

The TOC and TIC complexes are essential translocons that facilitate the import of the nuclear genome-encoded preproteins across the two envelope membranes of chloroplast, but their exact molecular identities and assembly remain unclear. Here, we report a cryoelectron microscopy structure of TOC-TIC supercomplex from Chlamydomonas, containing a total of 14 identified components. The preprotein-conducting pore of TOC is a hybrid ß-barrel co-assembled by Toc120 and Toc75, while the potential translocation path of TIC is formed by transmembrane helices from Tic20 and YlmG, rather than a classic model of Tic110. A rigid intermembrane space (IMS) scaffold bridges two chloroplast membranes, and a large hydrophilic cleft on the IMS scaffold connects TOC and TIC, forming a pathway for preprotein translocation. Our study provides structural insights into the TOC-TIC supercomplex composition, assembly, and preprotein translocation mechanism, and lays a foundation to interpret the evolutionary conservation and diversity of this fundamental translocon machinery.

Assembly and stability of IFT-B complex and its function in BBSome trafficking.

The machinery of intraflagellar transport (IFT) consists of IFT motors and the ciliary cargo adaptors including IFT-A and IFT-B complexes and BBSome. IFT-B, which is composed of IFT-B1 and IFT-B2 subcomplexes, interacts with IFT motors and IFT-A during anterograde IFT and IFT-A during retrograde IFT while it is also implicated in BBSome trafficking. However, the assembly and stability of IFT-B and its regulation of anterograde IFT and BBSome trafficking remain not clear. Here, we show that IFT38 functions in the regulation of anterograde IFT and retrograde trafficking of BBSome. Deletion of IFT-B1 or IFT-B2 subunits results in differential instability of IFT-B1 and IFT-B2. The stability of IFT-B1 and IFT-B2 is mutually dependent and mediated by the connecting tetramer IFT38/5788/52. The formation of an intact IFT-B1 and IFT-B2 is not altered by the deletion of IFT38 of IFT-B2 and IFT52 of IFT-B1, respectively. Further analysis suggests a modular pathway for IFT-B assembly.

FBB18 participates in preassembly of almost all axonemal dyneins independent of R2TP complex.

Assembly of dynein arms requires cytoplasmic processes which are mediated by dynein preassembly factors (DNAAFs). CFAP298, which is conserved in organisms with motile cilia, is required for assembly of dynein arms but with obscure mechanisms. Here, we show that FBB18, a Chlamydomonas homologue of CFAP298, localizes to the cytoplasm and functions in folding/stabilization of almost all axonemal dyneins at the early steps of dynein preassembly. Mutation of FBB18 causes no or short cilia accompanied with partial loss of both outer and inner dynein arms. Comparative proteomics using 15N labeling suggests partial degradation of almost all axonemal dynein heavy chains (DHCs). A mutant mimicking a patient variant induces particular loss of DHCα. FBB18 associates with 9 DNAAFs and 14 out of 15 dynein HCs but not with IC1/IC2. FBB18 interacts with RuvBL1/2, components of the HSP90 co-chaperone R2TP complex but not the holo-R2TP complex. Further analysis suggests simultaneous formation of multiple DNAAF complexes involves dynein folding/stability and thus provides new insights into axonemal dynein preassembly.

Ciliary transition zone proteins coordinate ciliary protein composition and ectosome shedding.

The transition zone (TZ) of the cilium/flagellum serves as a diffusion barrier that controls the entry/exit of ciliary proteins. Mutations of the TZ proteins disrupt barrier function and lead to multiple human diseases. However, the systematic regulation of ciliary composition and signaling-related processes by different TZ proteins is not completely understood. Here, we reveal that loss of TCTN1 in Chlamydomonas reinhardtii disrupts the assembly of wedge-shaped structures in the TZ. Proteomic analysis of cilia from WT and three TZ mutants, tctn1, cep290, and nphp4, shows a unique role of each TZ subunit in the regulation of ciliary composition, explaining the phenotypic diversity of different TZ mutants. Interestingly, we find that defects in the TZ impair the formation and biological activity of ciliary ectosomes. Collectively, our findings provide systematic insights into the regulation of ciliary composition by TZ proteins and reveal a link between the TZ and ciliary ectosomes.

Identification of Regulators for Ciliary Disassembly by a Chemical Screen.

Cilia are organelles for cellular signaling and motility. They are assembled in G0/G1 and disassembled prior to mitosis. Compared to what is known about ciliary assembly, less is understood about ciliary disassembly. To uncover new mechanisms of ciliary disassembly, we performed an unbiased chemical screen. Chlamydomonas reinhardtii cells were experimentally induced for ciliary disassembly by treatment with sodium pyrophosphate. An FDA approved drug library (HY-L022P-1, MedChemExpress) was used for the screening. Primary screening with further experiments has identified microtubule stabilizer taxanes, CDK4/6 inhibitor abemaciclib and Raf inhibitor dabrafenib being effective in inhibiting ciliary disassembly induced experimentally but also under physiological conditions. In addition, their effects on ciliary disassembly in mammalian cells has also been confirmed. Thus, our studies have not only revealed new mechanisms in ciliary disassembly but also provided new tools for studying ciliary disassembly. These discovered drugs may be used for therapeutic interventions of disorders involving ciliary degeneration such as retinopathies.

IFT54 directly interacts with kinesin-II and IFT dynein to regulate anterograde intraflagellar transport.

The intraflagellar transport (IFT) machinery consists of the anterograde motor kinesin-II, the retrograde motor IFT dynein, and the IFT-A and -B complexes. However, the interaction among IFT motors and IFT complexes during IFT remains elusive. Here, we show that the IFT-B protein IFT54 interacts with both kinesin-II and IFT dynein and regulates anterograde IFT. Deletion of residues 342-356 of Chlamydomonas IFT54 resulted in diminished anterograde traffic of IFT and accumulation of IFT motors and complexes in the proximal region of cilia. IFT54 directly interacted with kinesin-II and this interaction was strengthened for the IFT54Δ342-356 mutant in vitro and in vivo. The deletion of residues 261-275 of IFT54 reduced ciliary entry and anterograde traffic of IFT dynein with accumulation of IFT complexes near the ciliary tip. IFT54 directly interacted with IFT dynein subunit D1bLIC, and deletion of residues 261-275 reduced this interaction. The interactions between IFT54 and the IFT motors were also observed in mammalian cells. Our data indicate a central role for IFT54 in binding the IFT motors during anterograde IFT.

Functional exploration of heterotrimeric kinesin-II in IFT and ciliary length control in Chlamydomonas.

Heterodimeric motor organization of kinesin-II is essential for its function in anterograde IFT in ciliogenesis. However, the underlying mechanism is not well understood. In addition, the anterograde IFT velocity varies significantly in different organisms, but how this velocity affects ciliary length is not clear. We show that in Chlamydomonas motors are only stable as heterodimers in vivo, which is likely the key factor for the requirement of a heterodimer for IFT. Second, chimeric CrKinesin-II with human kinesin-II motor domains functioned in vitro and in vivo, leading to a ~ 2.8 fold reduced anterograde IFT velocity and a similar fold reduction in IFT injection rate that supposedly correlates with ciliary assembly activity. However, the ciliary length was only mildly reduced (~15%). Modeling analysis suggests a nonlinear scaling relationship between IFT velocity and ciliary length that can be accounted for by limitation of the motors and/or its ciliary cargoes, e.g. tubulin.

Potassium channel KCN11 is required for maintaining cellular osmolarity during nitrogen starvation to control proper cell physiology and TAG accumulation in Chlamydomonas reinhardtii.

BACKGROUND: Nitrogen (N) starvation in algae induces a variety of structural and metabolic changes including accumulation of triacylglycerol (TAG). Given the promising prospect of using algae as feedstock for biofuel production, accumulation of TAG upon N starvation becomes an ideal system to study TAG biosynthesis. Under nitrogen-depleted conditions, algae also accumulate compatible solutes such as sugar and certain amino acids, which is expected to elevate osmolarity in the cytoplasm. However, how osmoregulation is maintained and how it impacts on carbon metabolism, especially TAG accumulation under N starvation, are not well understood. RESULTS: We show here that potassium channel KCN11 localized in the contractile vacuole (CV) mediates osmoregulation during N starvation and loss of KCN11 profoundly affects cell physiology and TAG biosynthesis. KCN11 level is increased and the CV pulsation is accelerated. Loss of KCN11 induces aberrant CV cycle, inhibition of cell growth, increase of cell size, inhibition of chlorophyll loss and TAG accumulation. These effects are rescued by addition of sucrose to raise osmolarity in the culture medium, indicating that osmoregulation is required for cell adaptation to N starvation. Metabolomic analysis shows reduction of acetyl-CoA and accumulation of glyceraldehyde-3-phosphate in kcn11 mutant relative to the control under N starvation, indicating that defects in acetyl-CoA biosynthesis and some metabolic steps from glyceraldehyde-3-phosphate to TAG contribute to the decreased TAG accumulation due to loss of osmoregulation. CONCLUSIONS: This work provides novel insight of osmoregulation during N starvation in the control of cell physiology and metabolism especially TAG accumulation. According to these findings, we propose that osmolarity should be carefully monitored during the industrial production of biodiesel.

Single-Cell Mass Spectrometry Analysis of Metabolites Facilitated by Cell Electro-Migration and Electroporation.

Single-cell metabolite analysis plays an important role in biological study. While mass spectrometry is a powerful tool for identification and quantitation of metabolites, the low absolute analyte amounts in single cell and difficulty in sampling represent significant challenges in single cell analysis. In this study, we developed an effective method with a simple sampling procedure for analyzing single cells. A single cell was driven to a capillary tip through electro-migration, followed by releasing the cell contents through electroporation, into a sealed small volume (∼1.5 pL) to prevent dilution. Subsequent mass spectrometry analysis was performed directly with nanoelectrospray ionization. This method was applied for analyzing a variety of cells and monitoring the metabolic changes in response to perturbed cell culturing conditions. This method opens a new avenue for easy and rapid analysis of single cells with high sensitivity.

FLS2 is a CDK-like kinase that directly binds IFT70 and is required for proper ciliary disassembly in Chlamydomonas.

Intraflagellar transport (IFT) is required for ciliary assembly and maintenance. While disruption of IFT may trigger ciliary disassembly, we show here that IFT mediated transport of a CDK-like kinase ensures proper ciliary disassembly. Mutations in flagellar shortening 2 (FLS2), encoding a CDK-like kinase, lead to retardation of cilia resorption and delay of cell cycle progression. Stimulation for ciliary disassembly induces gradual dephosphorylation of FLS2 accompanied with gradual inactivation. Loss of FLS2 or its kinase activity induces early onset of kinesin13 phosphorylation in cilia. FLS2 is predominantly localized in the cell body, however, it is transported to cilia upon induction of ciliary disassembly. FLS2 directly interacts with IFT70 and loss of this interaction inhibits its ciliary transport, leading to dysregulation of kinesin13 phosphorylation and retardation of ciliary disassembly. Thus, this work demonstrates that IFT plays active roles in controlling proper ciliary disassembly by transporting a protein kinase to cilia to regulate a microtubule depolymerizer.

Nourseothricin N-acetyl transferase (NAT), a new selectable marker for nuclear gene expression in Chlamydomonas.

BACKGROUND: Chlamydomonas reinhardtii is a unicellular green alga, which is a most commonly used model organism for basic research and biotechnological applications. Generation of transgenic strains, which usually requires selectable markers, is instrumental in such studies/applications. Compared to other organisms, the number of selectable markers is limited in this organism. Nourseothricin (NTC) N-acetyl transferase (NAT) has been reported as a selectable marker in a variety of organisms but not including C. reinhardtii. Thus, we investigated whether NAT was useful and effective for selection of transgenic strains in C. reinhardtii. The successful use of NAT would provide alterative choice for selectable markers in this organism and likely in other microalgae. RESULTS: C. reinhardtii was sensitive to NTC at concentrations as low as 5 µg/ml. There was no cross-resistance to nourseothricin in strains that had been transformed with hygromycin B and/or paromomycin resistance genes. A codon-optimized NAT from Streptomyces noursei was synthesized and assembled into different expression vectors followed by transformation into Chlamydomonas. Around 500 transformants could be obtained by using 50 ng DNA on selection with 10 µg/ml NTC. The transformants exhibited normal growth rate and were stable at least for 10 months on conditions even without selection. We successfully tested that NAT could be used as a selectable marker for ectopic expression of IFT54-HA in strains with paromomycin and hygromycin B resistance markers. We further showed that the selection rate for IFT54-HA positive clones was greatly increased by fusing IFT54-HA to NAT and processing with the FMDV 2A peptide. CONCLUSIONS: This work represents the first demonstration of stable expression of NAT in the nuclear genome of C. reinhardtii and provides evidence that NAT can be used as an effective selectable marker for transgenic strains. It provides alterative choice for selectable markers in C. reinhardtii. NAT is compatible with paromomycin and hygromycin B resistance genes, which allows for multiple selections.

Regulation of flagellar assembly and length in Chlamydomonas by LF4, a MAPK-related kinase.

Members of the MAPK superfamily are known as key regulators of ciliogenesis. Long flagellar (LF) 4, a MAPK-related kinase in Chlamydomonas, is the first kinase that was implicated in ciliary assembly and length. However, little is known about its cellular properties, regulation, and molecular functions. LF4 is localized both in the flagella and cell body with enrichment at the 2 basal bodies, shown by super-resolution microscopy. LF4 is constitutively phosphorylated at T159 at the kinase activation loop and remains at the basal bodies during flagellar assembly. Gene mutations that affect the kinase activity or T159 phosphorylation alter the localization of LF4 at the basal bodies, and the mutants fail to rescue lf4-3, a null mutant. LF4 does not affect the velocities of intraflagellar transport (IFT). However, LF4 null mutation induces accumulation of IFT proteins in the flagellum and reduces the phosphorylation of the kinesin-II subunit FLA8/KIF3B, indicating that LF4 negatively regulates IFT entry. Furthermore, LF2, a cell cycle-related kinase, and LF3, a novel protein, are required for LF4 phosphorylation. Our study demonstrates that LF4 is likely a constitutively active kinase that is regulated by LF2 and regulates IFT entry at the basal bodies to control flagellar assembly and length.-Wang, Y., Ren, Y., Pan, J. Regulation of flagellar assembly and length in Chlamydomonas by LF4, a MAPK-related kinase.

[Bardet-Biedl syndrome protein-8 is involved in flagellar membrane protein transport in Chlamydomonas reinhardtii].

Cilia and flagella on eukaryotic cells are polarized organelles extending from the surfaces of cells, which participate not only in cell motility, but also in signal transduction and other processes. Structural or functional abnormalities of cilia can cause various human diseases, termed ciliopathies. Bardet-Biedl syndrome (BBS) is a ciliopathic human genetic disorder, and the pathogenesis is that mutated BBS genes result in abnormal cilia function. In order to study the pathogenic genes BBS8, we screened bbs8 mutant in Chlamydomonas reinhardtii and did a lot of physiology and biochemistry experiments. We affirmed that BBS8 protein was a cilia protein and had specific localization in the basal body by immunofluorescence (IF). The bbs8 mutant lost photokinesis, and it was defective in flagella shortening with drug induction. The results of silver staining and mass spectrometric analysis showed aberrant accumulation of flagellar proteins in the mutant flagella. We concluded that the BBS8 protein plays a significant role in flagellar membrane proteins transport, and the BBS8 protein might mediate retrograde transport to exert physiological function in the process.

Chlamydomonas WDR92 in association with R2TP-like complex and multiple DNAAFs to regulate ciliary dynein preassembly.

The motility of cilia or eukaryotic flagella is powered by the axonemal dyneins, which are preassembled in the cytoplasm by proteins termed dynein arm assembly factors (DNAAFs) before being transported to and assembled on the ciliary axoneme. Here, we characterize the function of WDR92 in Chlamydomonas. Loss of WDR92, a cytoplasmic protein, in a mutant wdr92 generated by DNA insertional mutagenesis resulted in aflagellate cells or cells with stumpy or short flagella, disappearance of axonemal dynein arms, and diminishment of dynein arm heavy chains in the cytoplasm, suggesting that WDR92 is a DNAAF. Immunoprecipitation of WDR92 followed by mass spectrometry identified inner dynein arm heavy chains and multiple DNAAFs including RuvBL1, RPAP3, MOT48, ODA7, and DYX1C. The PIH1 domain-containing protein MOT48 formed a R2TP-like complex with RuvBL1/2 and RPAP3, while PF13, another PIH1 domain-containing protein with function in dynein preassembly, did not. Interestingly, the third PIH1 domain-containing protein TWI1 was not related to flagellar motility. WDR92 physically interacted with the R2TP-like complex and the other identified DNNAFs. Our data suggest that WDR92 functions in association with the HSP90 co-chaperone R2TP-like complex as well as linking other DNAAFs in dynein preassembly.