Rosetta gen. nov. (Chlorophyta): Resolving the identity of red snow algal rosettes.

Thick-walled rosette-like snow algae were long thought to be a life stage of various other species of snow algae. Rosette-like cells have not been cultured, but by manually isolating cells from 38 field samples in southern British Columbia, we assigned a variety of rosette morphologies to DNA sequence. Phylogenetic analysis of Rubisco large-subunit (rbcL) gene, ribosomal internal transcribed spacer 2 (ITS2) rRNA region, and 18S rRNA gene revealed that the rosette-like cells form a new clade within the phylogroup Chloromonadinia. Based on these data, we designate a new genus, Rosetta, which comprises five novel species: R. castellata, R. floranivea, R. stellaria, R. rubriterra, and R. papavera. In a survey of 762 snow samples from British Columbia, we observed R. floranivea exclusively on snow overlying high-elevation glaciers, whereas R. castellata was observed at lower elevations, near the tree line. The other three species were rarely observed. Spherical red cells enveloped in a thin translucent sac were conspecific with Rosetta, possibly a developmental stage. These results highlight the unexplored diversity among snow algae and emphasize the utility of single-cell isolation to advance the centuries-old problem of disentangling life stages and cryptic species.

Satellite mapping of red snow on North American glaciers.

Red snow caused by blooms of microalgae darkens the surface of summer snowfields, increasing snowmelt. To assess the contribution of red snow to supraglacial snowmelt in northwestern North America, we systematically mapped the 2019-2022 distribution of blooms by applying supervised classification to 6158 satellite images. Blooms occurred on 5% of the total glaciated area, heavily affecting many glaciers in years of prolonged snow cover duration. Individual glaciers had up to 65% of their surface area affected by bloom in one melt season, which we estimate caused as much as 3 cm of snow meltwater equivalent averaged across the glacier surface. These results demonstrate appreciable snowmelt caused by red snow albedo over vast areas of North American glaciers.

A metagenomic study of the bacteria in snow algae microbiomes.

The bacterial communities found in snow algae blooms have been described in terms of their 16S rRNA gene community profiles, but little information exists on their metabolic potential. Previously, we reported that several bacterial taxa are common across snow algae blooms in the southwestern mountains of the Coast Range in British Columbia, Canada. Here, we further this work by reporting a partial bacterial metagenome from the same snow algal microbiomes. Using shotgun metagenomic data, we constructed metagenomically assembled bacterial genomes (MAGs). Of the total 54 binned MAGs, 28 were bacterial and estimated to be at least 50% complete based on single copy core genes. The 28 MAGs fell into five classes: Actinomycetia, Alphaproteobacteria, Bacteroidia, Betaproteobacteria, and Gammaproteobacteria. All MAGs were assigned to a class, 27 to an order, 25 to a family, 18 to a genus, and none to species. MAGs showed the potential to support algal growth by synthesizing B-vitamins and growth hormones. There was also widespread adaptation to the low oxygen environment of biofilms, including synthesis of high-affinity terminal oxidases and anaerobic pathways for cobalamin synthesis. Also notable were the absence of N2 fixation, and the presence of incomplete denitrification pathways suggestive of NO signalling within the microbiome.

The underlying green biciliate morphology of the orange snow alga Sanguina aurantia.

In the summer, blooms of microalgae appear on alpine and polar snowfields, creating expanses of red snow sometimes called 'watermelon snow'1. These blooms are attracting research attention because they decrease snow albedo, thereby accelerating the effects of global warming on snowmelt2. Currently, meltwater from alpine snowfields provides one-sixth of the world's population with water for drinking, agriculture, and the generation of hydroelectric power3. Each spring, the surface of new snow is colonized by microscopic organisms from unknown sources. One possibility is that when the melt begins, ciliated cells swim up from the substrate below to populate the snow surface. However, Sanguina, a cosmopolitan genus that frequently dominates high-alpine and arctic blooms4,5, are thick-walled, red or orange in colour, and immotile. Here, we describe a culture of motile green biciliate cells isolated from a sample of red snow. Using cross-referenced Bayesian and maximum-likelihood phylogenetic methods for two genetic markers, ITS2 and rbcL, we establish the green biciliate as belonging to the genus Sanguina. Compensatory-base-change analysis of ITS2 rRNA structure delimits the green culture as S. aurantia, conspecific with individual, thick-walled immotile orange cells, picked from field samples collected in British Columbia and Svalbard. Using single cells was invaluable for comparing sequences derived from thick-walled red and orange Sanguina cells, which do not exist in culture, with the cultured green biciliates.

A Molecular Analysis of Microalgae from Around the Globe to Revise Raphidonema (Trebouxiophyceae, Chlorophyta).

We isolated five microalgal strains from alpine snow near Vancouver, Canada, which display morphological features suggestive of the genera Koliella and Raphidonema. Due to variations in cell size and shape, we could not make a clear delimitation based on morphology. We proceeded to a molecular analysis and included 22 strains from the CCCryo culture collection, previously identified as members of four closely related genera: Raphidonema, Koliella, Stichococcus, and Pseudochlorella. For greater taxonomic context in our phylogenetic analysis, we also obtained authentic strains for the type species of Koliella and Pseudochlorella, but were unable to find one for Raphidonema. To examine generic boundaries, we did a phylogenetic analysis on the rbcL gene for all strains, establishing distinct lineages. Our novel isolates fell within Raphidonema, and so we analyzed the ITS2 gene of all Raphidonema strains to delimit species. To support species delimitations, we did a Compensatory Base Change analysis using the secondary structure of the ITS2 gene to assist in aligning the sequence. We also computed a maximum likelihood phylogenetic tree to examine species clades of Raphidonema. We assigned epitypes for two Raphidonema species based on the best morphological match to strains in the ITS2 clades. We then amended their diagnoses so they can be more reliably identified using DNA sequence data. We also propose two new species, R. catena and R. monicae, that formed their own species clades according to our ITS2 analysis.

Alpine Snow Algae Microbiome Diversity in the Coast Range of British Columbia.

Snow algae blooms contain bacteria, fungi, and other microscopic organisms. We surveyed 55 alpine snow algae blooms, collecting a total of 68 samples, from 12 mountains in the Coast Range of British Columbia, Canada. We used microscopy and rDNA metabarcoding to document biodiversity and query species and taxonomic associations. Across all samples, we found 173 algal, 2,739 bacterial, 380 fungal, and 540 protist/animalia operational taxonomic units (OTUs). In a previous study, we reported that most algal species were distributed along an elevational gradient. In the current study, we were surprised to find no corresponding distribution in any other taxa. We also tested the hypothesis that certain bacterial and fungal taxa co-occur with specific algal taxa. However, despite previous evidence that particular genera co-occur, we found no significant correlations between taxa across our 68 samples. Notably, seven bacterial, one fungal, and two cercozoan OTUs were widely distributed across our study regions. Taken together, these data suggest that any mutualisms with algae may not be taxon specific. We also report evidence of snow algae predation by rotifers, tardigrades, springtails, chytrid fungi, and ciliates, establishing the framework for a complex food web.

Variation in Snow Algae Blooms in the Coast Range of British Columbia.

Snow algae blooms cover vast areas of summer snowfields worldwide, reducing albedo and increasing snow melt. Despite their global prevalence, little is known about the algae species that comprise these blooms. We used 18S and rbcL metabarcoding and light microscopy to characterize algae species composition in 31 snow algae blooms in the Coast Range of British Columbia, Canada. This study is the first to thoroughly document regional variation between blooms. We found all blooms were dominated by the genera Sanguina, Chloromonas, and Chlainomonas. There was considerable variation between blooms, most notably species assemblages above treeline were distinct from forested sites. In contrast to previous studies, the snow algae genus Chlainomonas was abundant and widespread in snow algae blooms. We found few taxa using traditional 18S metabarcoding, but the high taxonomic resolution of rbcL revealed substantial diversity, including OTUs that likely represent unnamed species of snow algae. These three cross-referenced datasets (rbcL, 18S, and microscopy) reveal that alpine snow algae blooms are more diverse than previously thought, with different species of algae dominating different elevations.

Cilia in cystic kidney and other diseases.

Epithelial cells lining the ducts and tubules of the kidney nephron and collecting duct have a single non-motile cilium projecting from their surface into the lumen of the tubule. These organelles were long considered vestigial remnants left as a result of evolution from a ciliated ancestor, but we now recognize them as critical sensory antennae. In the kidney, the polycystins and fibrocystin, products of the major human polycystic kidney disease genes, localize to this organelle. The polycystins and fibrocystin, through an unknown mechanism, monitor the diameter of the kidney tubules and regulate the proliferation and differentiation of the cells lining the tubule. When the polycystins, fibrocystin or cilia themselves are defective, the cell perceives this as a pro-proliferative signal, which leads to tubule dilation and cystic disease. In addition to critical roles in preventing cyst formation in the kidney, cilia are also important in cystic and fibrotic diseases of the liver and pancreas, and ciliary defects lead to a variety of developmental abnormalities that cause structural birth defects in most organs.

A Forward Genetic Screen and Whole Genome Sequencing Identify Deflagellation Defective Mutants in Chlamydomonas, Including Assignment of ADF1 as a TRP Channel.

With rare exception, ciliated cells entering mitosis lose their cilia, thereby freeing basal bodies to serve as centrosomes in the formation of high-fidelity mitotic spindles. Cilia can be lost by shedding or disassembly, but either way, it appears that the final release may be via a coordinated severing of the nine axonemal outer doublet microtubules linking the basal body to the ciliary transition zone. Little is known about the mechanism or regulation of this important process. The stress-induced deflagellation response of Chlamydomonas provides a basis to identifying key players in axonemal severing. In an earlier screen we uncovered multiple alleles for each of three deflagellation genes, ADF1, FA1, and FA2 Products of the two FA genes localize to the site of axonemal severing and encode a scaffolding protein and a member of the NIMA-related family of ciliary-cell cycle kinases. The identity of the ADF1 gene remained elusive. Here, we report a new screen using a mutagenesis that yields point mutations in Chlamydomonas, an enhanced screening methodology, and whole genome sequencing. We isolated numerous new alleles of the three known genes, and one or two alleles each of at least four new genes. We identify ADF1 as a TRP ion channel, which we suggest may reside at the flagellar transition zone.

Cilia assembly: a role for F-actin in IFT recruitment.

Ciliary growth rates are limited by the availability of precursors at the growing tip. A new paper reveals that the early rapid growth of nascent cilia is supported by F-actin-facilitated delivery of IFT proteins to basal bodies.

The kinases LF4 and CNK2 control ciliary length by feedback regulation of assembly and disassembly rates.

BACKGROUND: Many of the diverse functions of cilia depend upon tight control of their length. Steady-state length reflects a balance between rates of ciliary assembly and disassembly, two parameters likely controlled by a length sensor of unknown identity or mechanism. RESULTS: A null mutation in Chlamydomonas CNK2, a member of the evolutionarily conserved family of NIMA-related kinases, reveals feedback regulation of assembly and disassembly rates. cnk2-1 mutant cells have a mild long-flagella (lf) phenotype as a consequence of reduced rates of flagellar disassembly. This is in contrast to the strong lf mutant lf4-7, which exhibits an aberrantly high rate of assembly. Cells carrying both mutations have even longer flagella than lf4-7 single mutants do. In addition to their high rate of assembly, lf4-7 mutants have a CNK2-dependent increase in disassembly rate. Finally, cnk2-1 cells have a decreased rate of turnover of flagellar subunits at the tip of the flagellum, demonstrating that the effects on disassembly are compensated by a reduced rate of assembly. CONCLUSIONS: We propose a model wherein CNK2 and LF4 modulate rates of disassembly and assembly respectively in a feedback loop that is activated when flagella exceed optimal length.

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. We postulated that the remnants are the product of severing of doublet microtubules between the basal bodies and the flagellar transition zone, thereby freeing the centrioles to participate in spindle organization. We show via TEM that flagellar remnants are indeed flagellar transition zones encased in vesicles derived from the flagellar membrane. This transition zone vesicle can be lodged within the cell wall or it can be expelled into the environment. This process is observable in Chlamydomonas, first because the released flagellar remnants can remain associated with the cell by virtue of attachments to the cell wall, and second because the Chlamydomonas transition zone is particularly rich with electron-dense structure. However, release of basal bodies for spindle-associated function is likely to be conserved among the eukaryotes.

Individuals with mutations in XPNPEP3, which encodes a mitochondrial protein, develop a nephronophthisis-like nephropathy.

The autosomal recessive kidney disease nephronophthisis (NPHP) constitutes the most frequent genetic cause of terminal renal failure in the first 3 decades of life. Ten causative genes (NPHP1-NPHP9 and NPHP11), whose products localize to the primary cilia-centrosome complex, support the unifying concept that cystic kidney diseases are "ciliopathies". Using genome-wide homozygosity mapping, we report here what we believe to be a new locus (NPHP-like 1 [NPHPL1]) for an NPHP-like nephropathy. In 2 families with an NPHP-like phenotype, we detected homozygous frameshift and splice-site mutations, respectively, in the X-prolyl aminopeptidase 3 (XPNPEP3) gene. In contrast to all known NPHP proteins, XPNPEP3 localizes to mitochondria of renal cells. However, in vivo analyses also revealed a likely cilia-related function; suppression of zebrafish xpnpep3 phenocopied the developmental phenotypes of ciliopathy morphants, and this effect was rescued by human XPNPEP3 that was devoid of a mitochondrial localization signal. Consistent with a role for XPNPEP3 in ciliary function, several ciliary cystogenic proteins were found to be XPNPEP3 substrates, for which resistance to N-terminal proline cleavage resulted in attenuated protein function in vivo in zebrafish. Our data highlight an emerging link between mitochondria and ciliary dysfunction, and suggest that further understanding the enzymatic activity and substrates of XPNPEP3 will illuminate novel cystogenic pathways.

Sensorium: the original raison d'etre of the motile cilium?

The role of non-motile (primary) cilia as sensory antennae critical for metazoan development and physiology has surfaced over the last decade, long after the function of motile cilia in propelling cells or moving fluids across tissues was well established. A new study of motile cilia from respiratory airways raises the possibility that transducing sensory cues from the environment is a universal characteristic of cilia and may have been the original raison d'être of the ancestral cilium.

The NIMA-related kinase NEK1 cycles through the nucleus.

Mutations in NEK1 in mice are causal for cystic kidneys, and model the ciliopathy polycystic kidney disease caused by abnormal ciliary structure or signaling. NEK1 has previously been shown to localize near centrosomes and to play a role in centrosomal stability and ciliogenesis. Recent data suggest that the etiology of kidney cysts involves aberrant signaling from the primary cilium to the nucleus. Here we demonstrate that NEK1 contains functional nuclear localization signals, is exported from the nucleus via a nuclear export signal-dependent pathway and that the protein cycles through the nucleus. Our data suggest that NEK1 is a candidate to transduce messages from the ciliary-basal body region to the regulation of nuclear gene expression.

Functional interactions between the ciliopathy-associated Meckel syndrome 1 (MKS1) protein and two novel MKS1-related (MKSR) proteins.

Meckel syndrome (MKS) is a ciliopathy characterized by encephalocele, cystic renal disease, liver fibrosis and polydactyly. An identifying feature of MKS1, one of six MKS-associated proteins, is the presence of a B9 domain of unknown function. Using phylogenetic analyses, we show that this domain occurs exclusively within a family of three proteins distributed widely in ciliated organisms. Consistent with a ciliary role, all Caenorhabditis elegans B9-domain-containing proteins, MKS-1 and MKS-1-related proteins 1 and 2 (MKSR-1, MKSR-2), localize to transition zones/basal bodies of sensory cilia. Their subcellular localization is largely co-dependent, pointing to a functional relationship between the proteins. This localization is evolutionarily conserved, because the human orthologues also localize to basal bodies, as well as cilia. As reported for MKS1, disrupting human MKSR1 or MKSR2 causes ciliogenesis defects. By contrast, single, double and triple C. elegans mks/mksr mutants do not display overt defects in ciliary structure, intraflagellar transport or chemosensation. However, we find genetic interactions between all double mks/mksr mutant combinations, manifesting as an increased lifespan phenotype, which is due to abnormal insulin-IGF-I signaling. Our findings therefore demonstrate functional interactions between a novel family of proteins associated with basal bodies or cilia, providing new insights into the molecular etiology of a pleiotropic human disorder.

Ciliary ion channels: location, location, location.

The elegant waveforms of motile cilia derive from temporal and spatial regulation of dynein-driven microtubule sliding. A new study reveals the surprising localization of the channel responsible for waveform switch.