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.

The Ancient Gamete Fusogen HAP2 Is a Eukaryotic Class II Fusion Protein.

Sexual reproduction is almost universal in eukaryotic life and involves the fusion of male and female haploid gametes into a diploid cell. The sperm-restricted single-pass transmembrane protein HAP2-GCS1 has been postulated to function in membrane merger. Its presence in the major eukaryotic taxa-animals, plants, and protists (including important human pathogens like Plasmodium)-suggests that many eukaryotic organisms share a common gamete fusion mechanism. Here, we report combined bioinformatic, biochemical, mutational, and X-ray crystallographic studies on the unicellular alga Chlamydomonas reinhardtii HAP2 that reveal homology to class II viral membrane fusion proteins. We further show that targeting the segment corresponding to the fusion loop by mutagenesis or by antibodies blocks gamete fusion. These results demonstrate that HAP2 is the gamete fusogen and suggest a mechanism of action akin to viral fusion, indicating a way to block Plasmodium transmission and highlighting the impact of virus-cell genetic exchanges on the evolution of eukaryotic life.

RABL4/IFT27 in a nucleotide-independent manner promotes phospholipase D ciliary retrieval via facilitating BBSome reassembly at the ciliary tip.

Certain ciliary transmembrane and membrane-associated signaling proteins export from cilia as intraflagellar transport (IFT) cargoes in a BBSome-dependent manner. Upon reaching the ciliary tip via anterograde IFT, the BBSome disassembles before being reassembled to form an intact entity for cargo phospholipase D (PLD) coupling. During this BBSome remodeling process, Chlamydomonas Rab-like 4 GTPase IFT27, by binding its partner IFT25 to form the heterodimeric IFT25/27, is indispensable for BBSome reassembly. Here, we show that IFT27 binds IFT25 in an IFT27 nucleotide-independent manner. IFT25/27 and the IFT subcomplexes IFT-A and -B are irrelevant for maintaining the stability of one another. GTP-loading onto IFT27 enhances the IFT25/27 affinity for binding to the IFT-B subcomplex core IFT-B1 entity in cytoplasm, while GDP-bound IFT27 does not prevent IFT25/27 from entering and cycling through cilia by integrating into IFT-B1. Upon at the ciliary tip, IFT25/27 cycles on and off IFT-B1 and this process is irrelevant with the nucleotide state of IFT27. During BBSome remodeling at the ciliary tip, IFT25/27 promotes BBSome reassembly independent of IFT27 nucleotide state, making postremodeled BBSomes available for PLD to interact with. Thus, IFT25/27 facilitates BBSome-dependent PLD export from cilia via controlling availability of intact BBSomes at the ciliary tip, while IFT27 nucleotide state does not participate in this regulatory event.

Chlamydomonas CHT7 Is Required for an Effective Quiescent State by Regulating Nutrient-Responsive Cell Cycle Gene Expression.

COMPROMISED HYDROLYSIS OF TRIACYLGLYCEROLS7 (CHT7) in Chlamydomonas (Chlamydomonas reinhardtii) was previously shown to affect the transcription of a subset of genes during nitrogen (N)-replete growth and following N refeeding. Here, we show that an extensive derepression of genes involved in DNA metabolism and cell cycle-related processes, as well as downregulation of genes encoding oxidoreductases and nutrient transporters, occurs in the cht7 mutant during N deprivation. Cellular mutant phenotypes are consistent with the observed transcriptome misregulation, as cht7 cells fail to properly arrest growth, nuclear replication, and cell division following N deprivation. Reduction in cht7 colony formation following N refeeding is explained by its compromised viability during N deprivation and by the occurrence of abortive divisions during N refeeding. Surprisingly, the largely unstructured C-terminal half of CHT7 with predicted protein binding domains, but not the canonical CXC DNA binding domain, is essential for the ability of CHT7 to form stable complexes and reverse the cellular phenotypes and transcription levels in the cht7 mutant. Hence, although lacking the presumed DNA binding domain, CHT7 modulates the expression of cell cycle genes in response to N availability, which is essential for establishing an effective quiescent state and the coordinated resumption of growth following N refeeding.

Cleavage-furrow formation without F-actin in Chlamydomonas.

It is widely believed that cleavage-furrow formation during cytokinesis is driven by the contraction of a ring containing F-actin and type-II myosin. However, even in cells that have such rings, they are not always essential for furrow formation. Moreover, many taxonomically diverse eukaryotic cells divide by furrowing but have no type-II myosin, making it unlikely that an actomyosin ring drives furrowing. To explore this issue further, we have used one such organism, the green alga Chlamydomonas reinhardtii We found that although F-actin is associated with the furrow region, none of the three myosins (of types VIII and XI) is localized there. Moreover, when F-actin was eliminated through a combination of a mutation and a drug, furrows still formed and the cells divided, although somewhat less efficiently than normal. Unexpectedly, division of the large Chlamydomonas chloroplast was delayed in the cells lacking F-actin; as this organelle lies directly in the path of the cleavage furrow, this delay may explain, at least in part, the delay in cytokinesis itself. Earlier studies had shown an association of microtubules with the cleavage furrow, and we used a fluorescently tagged EB1 protein to show that microtubules are still associated with the furrows in the absence of F-actin, consistent with the possibility that the microtubules are important for furrow formation. We suggest that the actomyosin ring evolved as one way to improve the efficiency of a core process for furrow formation that was already present in ancestral eukaryotes.

Formation of Spherical Palmelloid Colony with Enhanced Lipid Accumulation by Gel Encapsulation of Chlamydomonas debaryana NIES-2212.

Microalgae such as Chlamydomonas reinhardtii accumulate triacylglycerol (TAG), which is a potential source of biofuels, under stress conditions such as nitrogen deprivation, whereas Chlamydomonas debaryana NIES-2212 has previously been identified and characterized as one of the rare species of Chlamydomonas, which massively accumulates TAG in the stationary phase without external stress. As the high density of the cells in the stationary phase was supposed to act as a trigger for the accumulation of TAG in C. debaryana, in this study, C. debaryana was encapsulated in a Ca2+-alginate gel for the culture with high cell density. We discovered that the growth of the encapsulated cells resulted in the formation of spherical palmelloid colonies with high cell density, where daughter cells with truncated flagella remained wrapped within the mother cell walls. Interestingly, gel encapsulation markedly promoted proliferation of C. debaryana cells, and the encapsulated cells reached the stationary phase earlier than that of the free-living cells. Gel encapsulation also enhanced TAG accumulation. Gene expression analysis revealed that two genes of acyltransferases, DGAT1 and DGTT3, were upregulated in the stationary phase of free-living C. debaryana. In addition, the gene expression of these acyltransferases increased earlier in the encapsulated cells than that in the free-living cells. The enhanced production of TAG by alginate gel encapsulation was not found in C. reinhardtii which is known to use a different repertoire of acyltransferases in lipid accumulation.

Ciliary and cytoskeletal functions of an ancient monooxygenase essential for bioactive amidated peptide synthesis.

Many secreted peptides used for cell-cell communication require conversion of a C-terminal glycine to an amide for bioactivity. This reaction is catalyzed only by the integral membrane protein peptidylglycine α-amidating monooxygenase (PAM). PAM has been highly conserved and is found throughout the metazoa; PAM-like sequences are also present in choanoflagellates, filastereans, unicellular and colonial chlorophyte green algae, dinoflagellates and haptophytes. Recent studies have revealed that in addition to playing a key role in peptidergic signaling, PAM also regulates ciliogenesis in vertebrates, planaria and chlorophyte algae, and is required for the stability of actin-based microvilli. Here we briefly introduce the basic principles involved in ciliogenesis, the sequential reactions catalyzed by PAM and the trafficking of PAM through the secretory and endocytic pathways. We then discuss the multi-faceted roles this enzyme plays in the formation and maintenance of cytoskeleton-based cellular protrusions and propose models for how PAM protein and amidating activity might contribute to ciliogenesis. Finally, we consider why some ciliated organisms lack PAM, and discuss the potential ramifications of ciliary localized PAM for the endocrine features commonly observed in patients with ciliopathies.

An Animal-Like Cryptochrome Controls the Chlamydomonas Sexual Cycle.

Cryptochromes are known as flavin-binding blue light receptors in bacteria, fungi, plants, and insects. The animal-like cryptochrome (aCRY) of the green alga Chlamydomonas reinhardtii has extended our view on cryptochromes, because it responds also to other wavelengths of the visible spectrum, including red light. Here, we have investigated if aCRY is involved in the regulation of the sexual life cycle of C. reinhardtii, which is controlled by blue and red light at the steps of gametogenesis along with its restoration and germination. We show that aCRY is differentially expressed not only during the life cycle but also within the cell as part of the soluble and/or membrane-associated protein fraction. Moreover, localization of aCRY within the algal cell body varies between vegetative cells and the different cell types of gametogenesis. aCRY is significantly (early day) or to a small extent (late night) enriched in the nucleus in vegetative cells. In pregametes, gametes and dark-inactivated gametes, aCRY is localized over the cell body. aCRY plays an important role in the sexual life cycle of C. reinhardtii: It controls the germination of the alga, under which the zygote undergoes meiosis, in a positive manner, similar to the regulation by the blue light receptors phototropin and plant cryptochrome (pCRY). However, aCRY acts in combination with pCRY as a negative regulator for mating ability as well as for mating maintenance, opposite to the function of phototropin in these processes.

Control of Autophagy in Chlamydomonas Is Mediated through Redox-Dependent Inactivation of the ATG4 Protease.

Autophagy is a major catabolic pathway by which eukaryotic cells deliver unnecessary or damaged cytoplasmic material to the vacuole for its degradation and recycling in order to maintain cellular homeostasis. Control of autophagy has been associated with the production of reactive oxygen species in several organisms, including plants and algae, but the precise regulatory molecular mechanisms remain unclear. Here, we show that the ATG4 protease, an essential protein for autophagosome biogenesis, plays a central role for the redox regulation of autophagy in the model green alga Chlamydomonas reinhardtii Our results indicate that the activity of C. reinhardtii ATG4 is regulated by the formation of a single disulfide bond with a low redox potential that can be efficiently reduced by the NADPH/thioredoxin system. Moreover, we found that treatment of C. reinhardtii cells with norflurazon, an inhibitor of carotenoid biosynthesis that generates reactive oxygen species and triggers autophagy in this alga, promotes the oxidation and aggregation of ATG4. We propose that the activity of the ATG4 protease is finely regulated by the intracellular redox state, and it is inhibited under stress conditions to ensure lipidation of ATG8 and thus autophagy progression in C. reinhardtii.

The Chlamydomonas heat stress response.

Heat waves occurring at increased frequency as a consequence of global warming jeopardize crop yield safety. One way to encounter this problem is to genetically engineer crop plants toward increased thermotolerance. To identify entry points for genetic engineering, a thorough understanding of how plant cells perceive heat stress and respond to it is required. Using the unicellular green alga Chlamydomonas reinhardtii as a model system to study the fundamental mechanisms of the plant heat stress response has several advantages. Most prominent among them is the suitability of Chlamydomonas for studying stress responses system-wide and in a time-resolved manner under controlled conditions. Here we review current knowledge on how heat is sensed and signaled to trigger temporally and functionally grouped sub-responses termed response elements to prevent damage and to maintain cellular homeostasis in plant cells.

The Chlamydomonas cell cycle.

The position of Chlamydomonas within the eukaryotic phylogeny makes it a unique model in at least two important ways: as a representative of the critically important, early-diverging lineage leading to plants; and as a microbe retaining important features of the last eukaryotic common ancestor (LECA) that has been lost in the highly studied yeast lineages. Its cell biology has been studied for many decades and it has well-developed experimental genetic tools, both classical (Mendelian) and molecular. Unlike land plants, it is a haploid with very few gene duplicates, making it ideal for loss-of-function genetic studies. The Chlamydomonas cell cycle has a striking temporal and functional separation between cell growth and rapid cell division, probably connected to the interplay between diurnal cycles that drive photosynthetic cell growth and the cell division cycle; it also exhibits a highly choreographed interaction between the cell cycle and its centriole-basal body-flagellar cycle. Here, we review the current status of studies of the Chlamydomonas cell cycle. We begin with an overview of cell-cycle control in the well-studied yeast and animal systems, which has yielded a canonical, well-supported model. We discuss briefly what is known about similarities and differences in plant cell-cycle control, compared with this model. We next review the cytology and cell biology of the multiple-fission cell cycle of Chlamydomonas. Lastly, we review recent genetic approaches and insights into Chlamydomonas cell-cycle regulation that have been enabled by a new generation of genomics-based tools.

Insertion of Vertically Aligned Nanowires into Living Cells by Inkjet Printing of Cells.

Effective insertion of vertically aligned nanowires (NWs) into cells is critical for bioelectrical and biochemical devices, biological delivery systems, and photosynthetic bioenergy harvesting. However, accurate insertion of NWs into living cells using scalable processes has not yet been achieved. Here, NWs are inserted into living Chlamydomonas reinhardtii cells (Chlamy cells) via inkjet printing of the Chlamy cells, representing a low-cost and large-scale method for inserting NWs into living cells. Jetting conditions and printable bioink composed of living Chlamy cells are optimized to achieve stable jetting and precise ink deposition of bioink for indentation of NWs into Chlamy cells. Fluorescence confocal microscopy is used to verify the viability of Chlamy cells after inkjet printing. Simple mechanical considerations of the cell membrane and droplet kinetics are developed to control the jetting force to allow penetration of the NWs into cells. The results suggest that inkjet printing is an effective, controllable tool for stable insertion of NWs into cells with economic and scale-related advantages.

Taxonomic revision of Chlamydomonas subg. Amphichloris (Volvocales, Chlorophyceae), with resurrection of the genus Dangeardinia and descriptions of Ixipapillifera gen. nov. and Rhysamphichloris gen. nov.

Chlamydomonas (Cd.) is one of the largest but most polyphyletic genera of freshwater unicellular green algae. It consists of 400-600 morphological species and requires taxonomic revision. Toward reclassification, each morphologically defined classical subgenus (or subgroup) should be examined using culture strains. Chlamydomonas subg. Amphichloris is characterized by a central nucleus between two axial pyrenoids, however, the phylogenetic structure of this subgenus has yet to be examined using molecular data. Here, we examined 12 strains including six newly isolated strains, morphologically identified as Chlamydomonas subg. Amphichloris, using 18S rRNA gene phylogeny, light microscopy, and mitochondria fluorescent microscopy. Molecular phylogenetic analyses revealed three independent lineages of the subgenus, separated from the type species of Chlamydomonas, Cd. reinhardtii. These three lineages were further distinguished from each other by light and fluorescent microscopy-in particular by the morphology of the papillae, chloroplast surface, stigmata, and mitochondria-and are here assigned to three genera: Dangeardinia emend., Ixipapillifera gen. nov., and Rhysamphichloris gen. nov. Based on the molecular and morphological data, two to three species were recognized in each genus, including one new species, I. pauromitos. In addition, Cd. deasonii, which was previously assigned to subgroup "Pleiochloris," was included in the genus Ixipapillifera as I. deasonii comb. nov.

Scaling body size fluctuations.

The size of an organism matters for its metabolic, growth, mortality, and other vital rates. Scale-free community size spectra (i.e., size distributions regardless of species) are routinely observed in natural ecosystems and are the product of intra- and interspecies regulation of the relative abundance of organisms of different sizes. Intra- and interspecies distributions of body sizes are thus major determinants of ecosystems' structure and function. We show experimentally that single-species mass distributions of unicellular eukaryotes covering different phyla exhibit both characteristic sizes and universal features over more than four orders of magnitude in mass. Remarkably, we find that the mean size of a species is sufficient to characterize its size distribution fully and that the latter has a universal form across all species. We show that an analytical physiological model accounts for the observed universality, which can be synthesized in a log-normal form for the intraspecies size distributions. We also propose how ecological and physiological processes should interact to produce scale-invariant community size spectra and discuss the implications of our results on allometric scaling laws involving body mass.

Increase in peroxisome number and the gene expression of putative glyoxysomal enzymes in Chlamydomonas cells supplemented with acetate.

We cultured Chlamydomonas reinhardtii cells in a minimal culture medium supplemented with various concentrations of acetate, fatty acids, ethanol, fatty alcohols, or sucrose. The presence of acetate (0.5 or 1.0%, w/v) was advantageous for cell growth. To determine whether peroxisomes are involved in fatty acid and fatty alcohol metabolism, we investigated the dynamics of peroxisomes, including changes in their number and size, in the presence of acetate, ethanol, and sucrose. The total volume of peroxisomes increased when cells were grown with acetate, but did not change when cells were grown with ethanol or sucrose. We analyzed cell growth on minimal culture medium supplemented with various fatty acids (carbon chain length ranging from one to ten) to investigate which fatty acids are metabolized by C. reinhardtii. Among them, acetate caused the greatest increase in growth when added to minimal culture media. We analyzed the transcript levels of genes encoding putative glyoxysomal enzymes. The transcript levels of genes encoding malate synthase, malate dehydrogenase, isocitrate lyase, and citrate synthase increased when Chlamydomonas cells were grown on minimal culture medium supplemented with acetate. Our results suggest that Chlamydomonas peroxisomes are involved in acetate metabolism via the glyoxylate cycle.

Equations of interdoublet separation during flagella motion reveal mechanisms of wave propagation and instability.

The motion of flagella and cilia arises from the coordinated activity of dynein motor protein molecules arrayed along microtubule doublets that span the length of axoneme (the flagellar cytoskeleton). Dynein activity causes relative sliding between the doublets, which generates propulsive bending of the flagellum. The mechanism of dynein coordination remains incompletely understood, although it has been the focus of many studies, both theoretical and experimental. In one leading hypothesis, known as the geometric clutch (GC) model, local dynein activity is thought to be controlled by interdoublet separation. The GC model has been implemented as a numerical simulation in which the behavior of a discrete set of rigid links in viscous fluid, driven by active elements, was approximated using a simplified time-marching scheme. A continuum mechanical model and associated partial differential equations of the GC model have remained lacking. Such equations would provide insight into the underlying biophysics, enable mathematical analysis of the behavior, and facilitate rigorous comparison to other models. In this article, the equations of motion for the flagellum and its doublets are derived from mechanical equilibrium principles and simple constitutive models. These equations are analyzed to reveal mechanisms of wave propagation and instability in the GC model. With parameter values in the range expected for Chlamydomonas flagella, solutions to the fully nonlinear equations closely resemble observed waveforms. These results support the ability of the GC hypothesis to explain dynein coordination in flagella and provide a mathematical foundation for comparison to other leading models.

Slow axonemal dynein e facilitates the motility of faster dynein c.

We highly purified the Chlamydomonas inner-arm dyneins e and c, considered to be single-headed subspecies. These two dyneins reside side-by-side along the peripheral doublet microtubules of the flagellum. Electron microscopic observations and single particle analysis showed that the head domains of these two dyneins were similar, whereas the tail domain of dynein e was short and bent in contrast to the straight tail of dynein c. The ATPase activities, both basal and microtubule-stimulated, of dynein e (kcat = 0.27 s(-1) and kcat,MT = 1.09 s(-1), respectively) were lower than those of dynein c (kcat = 1.75 s(-1) and kcat,MT = 2.03 s(-1), respectively). From in vitro motility assays, the apparent velocity of microtubule translocation by dynein e was found to be slow (Vap = 1.2 ± 0.1 µm/s) and appeared independent of the surface density of the motors, whereas dynein c was very fast (Vmax = 15.8 ± 1.5 µm/s) and highly sensitive to decreases in the surface density (Vmin = 2.2 ± 0.7 µm/s). Dynein e was expected to be a processive motor, since the relationship between the microtubule landing rate and the surface density of dynein e fitted well with first-power dependence. To obtain insight into the in vivo roles of dynein e, we measured the sliding velocity of microtubules driven by a mixture of dynein e and c at various ratios. The microtubule translocation by the fast dynein c became even faster in the presence of the slow dynein e, which could be explained by assuming that dynein e does not retard motility of faster dyneins. In flagella, dynein e likely acts as a facilitator by holding adjacent microtubules to aid dynein c's power stroke.

VMP1-deficient Chlamydomonas exhibits severely aberrant cell morphology and disrupted cytokinesis.

BACKGROUND: The versatile Vacuole Membrane Protein 1 (VMP1) has been previously investigated in six species. It has been shown to be essential in macroautophagy, where it takes part in autophagy initiation. In addition, VMP1 has been implicated in organellar biogenesis; endo-, exo- and phagocytosis, and protein secretion; apoptosis; and cell adhesion. These roles underly its proven involvement in pancreatitis, diabetes and cancer in humans. RESULTS: In this study we analyzed a VMP1 homologue from the green alga Chlamydomonas reinhardtii. CrVMP1 knockdown lines showed severe phenotypes, mainly affecting cell division as well as the morphology of cells and organelles. We also provide several pieces of evidence for its involvement in macroautophagy. CONCLUSION: Our study adds a novel role to VMP1's repertoire, namely the regulation of cytokinesis. Though the directness of the observed effects and the mechanisms underlying them remain to be defined, the protein's involvement in macroautophagy in Chlamydomonas, as found by us, suggests that CrVMP1 shares molecular characteristics with its animal and protist counterparts.

Turbulent fluid acceleration generates clusters of gyrotactic microorganisms.

The motility of microorganisms is often biased by gradients in physical and chemical properties of their environment, with myriad implications on their ecology. Here we show that fluid acceleration reorients gyrotactic plankton, triggering small-scale clustering. We experimentally demonstrate this phenomenon by studying the distribution of the phytoplankton Chlamydomonas augustae within a rotating tank and find it to be in good agreement with a new, generalized model of gyrotaxis. When this model is implemented in a direct numerical simulation of turbulent flow, we find that fluid acceleration generates multifractal plankton clustering, with faster and more stable cells producing stronger clustering. By producing accumulations in high-vorticity regions, this process is fundamentally different from clustering by gravitational acceleration, expanding the range of mechanisms by which turbulent flows can impact the spatial distribution of active suspensions.