Editing of endogenous tubulins reveals varying effects of tubulin posttranslational modifications on axonal growth and regeneration.

Tubulin posttranslational modifications (PTMs) modulate the dynamic properties of microtubules and their interactions with other proteins. However, the effects of tubulin PTMs were often revealed indirectly through the deletion of modifying enzymes or the overexpression of tubulin mutants. In this study, we directly edited the endogenous tubulin loci to install PTM-mimicking or -disabling mutations and studied their effects on microtubule stability, neurite outgrowth, axonal regeneration, cargo transport, and sensory functions in the touch receptor neurons of Caenorhabditis elegans. We found that the status of ß-tubulin S172 phosphorylation and K252 acetylation strongly affected microtubule dynamics, neurite growth, and regeneration, whereas α-tubulin K40 acetylation had little influence. Polyglutamylation and detyrosination in the tubulin C-terminal tail had more subtle effects on microtubule stability likely by modulating the interaction with kinesin-13. Overall, our study systematically assessed and compared several tubulin PTMs for their impacts on neuronal differentiation and regeneration and established an in vivo platform to test the function of tubulin PTMs in neurons.

MACSPI enables tissue-selective proteomic and interactomic analyses in multicellular organisms.

Multicellular organisms are composed of many tissue types that have distinct morphologies and functions, which are largely driven by specialized proteomes and interactomes. To define the proteome and interactome of a specific type of tissue in an intact animal, we developed a localized proteomics approach called Methionine Analog-based Cell-Specific Proteomics and Interactomics (MACSPI). This method uses the tissue-specific expression of an engineered methionyl-tRNA synthetase to label proteins with a bifunctional amino acid 2-amino-5-diazirinylnonynoic acid in selected cells. We applied MACSPI in Caenorhabditis elegans, a model multicellular organism, to selectively label, capture, and profile the proteomes of the body wall muscle and the nervous system, which led to the identification of tissue-specific proteins. Using the photo-cross-linker, we successfully profiled HSP90 interactors in muscles and neurons and identified tissue-specific interactors and stress-related interactors. Our study demonstrates that MACSPI can be used to profile tissue-specific proteomes and interactomes in intact multicellular organisms.

Using an ER-specific optogenetic mechanostimulator to understand the mechanosensitivity of the endoplasmic reticulum.

The ability of cells to perceive and respond to mechanical cues is essential for numerous biological activities. Emerging evidence indicates important contributions of organelles to cellular mechanosensitivity and mechanotransduction. However, whether and how the endoplasmic reticulum (ER) senses and reacts to mechanical forces remains elusive. To fill the knowledge gap, after developing a light-inducible ER-specific mechanostimulator (LIMER), we identify that mechanostimulation of ER elicits a transient, rapid efflux of Ca2+ from ER in monkey kidney COS-7 cells, which is dependent on the cation channels transient receptor potential cation channel, subfamily V, member 1 (TRPV1) and polycystin-2 (PKD2) in an additive manner. This ER Ca2+ release can be repeatedly stimulated and tuned by varying the intensity and duration of force application. Moreover, ER-specific mechanostimulation inhibits ER-to-Golgi trafficking. Sustained mechanostimuli increase the levels of binding-immunoglobulin protein (BiP) expression and phosphorylated eIF2α, two markers for ER stress. Our results provide direct evidence for ER mechanosensitivity and tight mechanoregulation of ER functions, placing ER as an important player on the intricate map of cellular mechanotransduction.

Single-cell phylotranscriptomics of developmental and cell type evolution.

Single-cell phylotranscriptomics is an emerging tool to reveal the molecular and cellular mechanisms of evolution. We summarize its utility in studying the hourglass pattern of ontogenetic evolution and for understanding the evolutionary history of cell types. The developmental hourglass model suggests that the mid-embryonic stage is the most conserved period of development across species, which is supported by morphological and molecular studies. Single-cell phylotranscriptomic analysis has revealed previously underappreciated heterogeneity in transcriptome ages among lineages and cell types throughout development, and has identified the lineages and tissues that drive the whole-organism hourglass pattern. Single-cell transcriptome age analyses also provide important insights into the origin of germ layers, the different selective forces on tissues during adaptation, and the evolutionary relationships between cell types.

Metabolic rescue of α-synuclein-induced neurodegeneration through propionate supplementation and intestine-neuron signaling in C. elegans.

Microbial metabolites that can modulate neurodegeneration are promising therapeutic targets. Here, we found that the short-chain fatty acid propionate protects against α-synuclein-induced neuronal death and locomotion defects in a Caenorhabditis elegans model of Parkinson's disease (PD) through bidirectional regulation between the intestine and neurons. Both depletion of dietary vitamin B12, which induces propionate breakdown, and propionate supplementation suppress neurodegeneration and reverse PD-associated transcriptomic aberrations. Neuronal α-synuclein aggregation induces intestinal mitochondrial unfolded protein response (mitoUPR), which leads to reduced propionate levels that trigger transcriptional reprogramming in the intestine and cause defects in energy production. Weakened intestinal metabolism exacerbates neurodegeneration through interorgan signaling. Genetically enhancing propionate production or overexpressing metabolic regulators downstream of propionate in the intestine rescues neurodegeneration, which then relieves mitoUPR. Importantly, propionate supplementation suppresses neurodegeneration without reducing α-synuclein aggregation, demonstrating metabolic rescue of neuronal proteotoxicity downstream of protein aggregates. Our study highlights the involvement of small metabolites in the gut-brain interaction in neurodegenerative diseases.

Young duplicate genes show developmental stage- and cell type-specific expression and function in Caenorhabditis elegans.

Gene duplication produces the material that fuels evolutionary innovation. The "out-of-testis" hypothesis suggests that sperm competition creates selective pressure encouraging the emergence of new genes in male germline, but the somatic expression and function of the newly evolved genes are not well understood. We systematically mapped the expression of young duplicate genes throughout development in Caenorhabditis elegans using both whole-organism and single-cell transcriptomic data. Based on the expression dynamics across developmental stages, young duplicate genes fall into three clusters that are preferentially expressed in early embryos, mid-stage embryos, and late-stage larvae. Early embryonic genes are involved in protein degradation and develop essentiality comparable to the genomic average. In mid-to-late embryos and L4-stage larvae, young genes are enriched in intestine, epidermal cells, coelomocytes, and amphid chemosensory neurons. Their molecular functions and inducible expression indicate potential roles in innate immune response and chemosensory perceptions, which may contribute to adaptation outside of the sperm.

Transcriptome age of individual cell types in Caenorhabditis elegans.

The phylotranscriptomic analysis of development in several species revealed the expression of older and more conserved genes in midembryonic stages and younger and more divergent genes in early and late embryonic stages, which supported the hourglass mode of development. However, previous work only studied the transcriptome age of whole embryos or embryonic sublineages, leaving the cellular basis of the hourglass pattern and the variation of transcriptome ages among cell types unexplored. By analyzing both bulk and single-cell transcriptomic data, we studied the transcriptome age of the nematode Caenorhabditis elegans throughout development. Using the bulk RNA-seq data, we identified the morphogenesis phase in midembryonic development as the phylotypic stage with the oldest transcriptome and confirmed the results using whole-embryo transcriptome assembled from single-cell RNA-seq data. The variation in transcriptome ages among individual cell types remained small in early and midembryonic development and grew bigger in late embryonic and larval stages as cells and tissues differentiate. Lineages that give rise to certain tissues (e.g., hypodermis and some neurons) but not all recapitulated the hourglass pattern across development at the single-cell transcriptome level. Further analysis of the variation in transcriptome ages among the 128 neuron types in C. elegans nervous system found that a group of chemosensory neurons and their downstream interneurons expressed very young transcriptomes and may contribute to adaptation in recent evolution. Finally, the variation in transcriptome age among the neuron types, as well as the age of their cell fate regulators, led us to hypothesize the evolutionary history of some neuron types.

Whole-cell FRET monitoring of transcription factor activities enables functional annotation of signal transduction systems in living bacteria.

Bacteria adapt to their constantly changing environments largely by transcriptional regulation through the activities of various transcription factors (TFs). However, techniques that monitor TF-promoter interactions in situ in living bacteria are lacking. Herein, we developed a whole-cell TF-promoter binding assay based on the intermolecular FRET between an unnatural amino acid, l-(7-hydroxycoumarin-4-yl) ethylglycine, which labels TFs with bright fluorescence through genetic encoding (donor fluorophore) and the live cell nucleic acid stain SYTO 9 (acceptor fluorophore). We show that this new FRET pair monitors the intricate TF-promoter interactions elicited by various types of signal transduction systems, including one-component (CueR) and two-component systems (BasSR and PhoPQ), in bacteria with high specificity and sensitivity. We demonstrate that robust CouA incorporation and FRET occurrence is achieved in all these regulatory systems based on either the crystal structures of TFs or their simulated structures, if 3D structures of the TFs were unavailable. Furthermore, using CueR and PhoPQ systems as models, we demonstrate that the whole-cell FRET assay is applicable for the identification and validation of complex regulatory circuit and novel modulators of regulatory systems of interest. Finally, we show that the FRET system is applicable for single-cell analysis and monitoring TF activities in Escherichia coli colonizing a Caenorhabditis elegans host. In conclusion, we established a tractable and sensitive TF-promoter binding assay, which not only complements currently available approaches for DNA-protein interactions but also provides novel opportunities for functional annotation of bacterial signal transduction systems and studies of the bacteria-host interface.

890-nm-excited SHG and fluorescence imaging enabled by an all-fiber mode-locked laser.

We demonstrate second-harmonic generation (SHG) microscopy excited by the ∼890-nm light frequency-doubled from a 137-fs, 19.4-MHz, and 300-mW all-fiber mode-locked laser centered at 1780 nm. The mode-locking at the 1.7-µm window is realized by controlling the emission peak of the gain fiber, and uses the dispersion management technique to broaden the optical spectrum up to 30 nm. The spectrum is maintained during the amplification and the pulse is compressed by single-mode fibers. The SHG imaging performance is showcased on a mouse skull, leg, and tail. Two-photon fluorescence imaging is also demonstrated on C. elegans labeled with green and red fluorescent proteins. The frequency-doubled all-fiber laser system provides a compact and efficient tool for SHG and fluorescence microscopy.

Using Caenorhabditis elegans to Model Therapeutic Interventions of Neurodegenerative Diseases Targeting Microbe-Host Interactions.

Emerging evidence from both clinical studies and animal models indicates the importance of the interaction between the gut microbiome and the brain in the pathogenesis of neurodegenerative diseases (NDs). Although how microbes modulate neurodegeneration is still mostly unclear, recent studies have started to probe into the mechanisms for the communication between microbes and hosts in NDs. In this review, we highlight the advantages of using Caenorhabditis elegans (C. elegans) to disentangle the microbe-host interaction that regulates neurodegeneration. We summarize the microbial pro- and anti-neurodegenerative factors identified using the C. elegans ND models and the effects of many are confirmed in mouse models. Specifically, we focused on the role of bacterial amyloid proteins, such as curli, in promoting proteotoxicity and neurodegeneration by cross-seeding the aggregation of endogenous ND-related proteins, such as α-synuclein. Targeting bacterial amyloid production may serve as a novel therapeutic strategy for treating NDs, and several compounds, such as epigallocatechin-3-gallate (EGCG), were shown to suppress neurodegeneration at least partly by inhibiting curli production. Because bacterial amyloid fibrils contribute to biofilm formation, inhibition of amyloid production often leads to the disruption of biofilms. Interestingly, from a list of 59 compounds that showed neuroprotective effects in C. elegans and mouse ND models, we found that about half of them are known to inhibit bacterial growth or biofilm formation, suggesting a strong correlation between the neuroprotective and antibiofilm activities. Whether these potential therapeutics indeed protect neurons from proteotoxicity by inhibiting the cross-seeding between bacterial and human amyloid proteins awaits further investigations. Finally, we propose to screen the long list of antibiofilm agents, both FDA-approved drugs and novel compounds, for their neuroprotective effects and develop new pharmaceuticals that target the gut microbiome for the treatment of NDs. To this end, the C. elegans ND models can serve as a platform for fast, high-throughput, and low-cost drug screens that target the microbe-host interaction in NDs.

The Expression and Function of Tubulin Isotypes in Caenorhabditis elegans.

Microtubules, made from the polymerization of the highly conserved α/ß-tubulin heterodimers, serve as important components of the cytoskeleton in all eukaryotic cells. The existence of multiple tubulin isotypes in metazoan genomes and a dazzling variety of tubulin posttranslational modifications (PTMs) prompted the "tubulin code" hypothesis, which proposed that microtubule structure and functions are determined by the tubulin composition and PTMs. Evidence for the tubulin code has emerged from studies in several organisms with the characterization of specific tubulins for their expression and functions. The studies of tubulin PTMs are accelerated by the discovery of the enzymes that add or remove the PTMs. In tubulin research, the use of simple organisms, such as Caenorhabditis elegans, has been instrumental for understanding the expression and functional specialization of tubulin isotypes and the effects of their PTMs. In this review, we summarize the current understanding of the expression patterns and cellular functions of the nine α-tubulin and six ß-tubulin isotypes. Expression studies are greatly facilitated by the CRISPR/Cas9-mediated endogenous GFP knock-in reporters and the organism-wide single cell transcriptomic studies. Meanwhile, functional studies benefit from the ease of genetic manipulation and precise gene replacement in C. elegans. These studies identified both ubiquitously expressed tubulin isotypes and tissue-specific isotypes. The isotypes showed functional redundancy, as well as functional specificity, which is likely caused by the subtle differences in their amino acid sequences. Many of these differences concentrate at the C-terminal tails that are subjected to several PTMs. Indeed, tubulin PTM, such as polyglutamylation, is shown to modulate microtubule organization and properties in both ciliated and non-ciliated neurons. Overall, studies from C. elegans support the distinct expression and function patterns of tubulin isotypes and the importance of their PTMs and offer the promise of cracking the tubulin code at the whole-genome and the whole-organism level.

Nervous system-wide analysis of Hox regulation of terminal neuronal fate specification in Caenorhabditis elegans.

Hox genes encode evolutionarily conserved transcription factors that specify regional identities along the anterior-posterior (A-P) axis. Although some Hox genes are known to regulate the differentiation of certain neurons, to what extent Hox genes are involved in the terminal specification of the entire nervous system is unclear. Here, we systematically mapped the expression of all six Hox genes in C. elegans nervous system and found Hox expression in 97 (32%) of the 302 neurons in adult hermaphrodites. Our results are generally consistent with previous high-throughput expression mapping and single-cell transcriptomic studies. Detailed analysis of the fate markers for these neurons revealed that Hox genes regulate the differentiation of 29 (25%) of the 118 classes of C. elegans neurons. Hox genes not only regulate the specification of terminal neuronal fates through multiple mechanisms but also control subtype diversification along the A-P axis. The widespread involvement of Hox genes in neuronal differentiation indicates their roles in establishing complex nervous systems.

Genome-wide screen identifies curli amyloid fibril as a bacterial component promoting host neurodegeneration.

Growing evidence indicates that gut microbiota play a critical role in regulating the progression of neurodegenerative diseases such as Parkinson's disease. The molecular mechanism underlying such microbe-host interaction is unclear. In this study, by feeding Caenorhabditis elegans expressing human α-syn with Escherichia coli knockout mutants, we conducted a genome-wide screen to identify bacterial genes that promote host neurodegeneration. The screen yielded 38 genes that fall into several genetic pathways including curli formation, lipopolysaccharide assembly, and adenosylcobalamin synthesis among others. We then focused on the curli amyloid fibril and found that genetically deleting or pharmacologically inhibiting the curli major subunit CsgA in E. coli reduced α-syn-induced neuronal death, restored mitochondrial health, and improved neuronal functions. CsgA secreted by the bacteria colocalized with α-syn inside neurons and promoted α-syn aggregation through cross-seeding. Similarly, curli also promoted neurodegeneration in C. elegans models of Alzheimer's disease, amyotrophic lateral sclerosis, and Huntington's disease and in human neuroblastoma cells.

Large genetic diversity and strong positive selection in F-box and GPCR genes among the wild isolates of Caenorhabditis elegans.

The F-box and chemosensory GPCR (csGPCR) gene families are greatly expanded in nematodes, including the model organism Caenorhabditis elegans, compared to insects and vertebrates. However, the intraspecific evolution of these two gene families in nematodes remain unexamined. In this study, we analyzed the genomic sequences of 330 recently sequenced wild isolates of C. elegans using a range of population genetics approaches. We found that F-box and csGPCR genes, especially the Srw family csGPCRs, showed much more diversity than other gene families. Population structure analysis and phylogenetic analysis divided the wild strains into eight non-Hawaiian and three Hawaiian subpopulations. Some Hawaiian strains appeared to be more ancestral than all other strains. F-box and csGPCR genes maintained a great amount of the ancestral variants in the Hawaiian subpopulation and their divergence among the non-Hawaiian subpopulations contributed significantly to population structure. F-box genes are mostly located at the chromosomal arms and high recombination rate correlates with their large polymorphism. Moreover, using both neutrality tests and Extended Haplotype Homozygosity analysis, we identified signatures of strong positive selection in the F-box and csGPCR genes among the wild isolates, especially in the non-Hawaiian population. Accumulation of high-frequency derived alleles in these genes was found in non-Hawaiian population, leading to divergence from the ancestral genotype. In summary, we found that F-box and csGPCR genes harbour a large pool of natural variants, which may be subjected to positive selection. These variants are mostly mapped to the substrate-recognition domains of F-box proteins and the extracellular and intracellular regions of csGPCRs, possibly resulting in advantages during adaptation by affecting protein degradation and the sensing of environmental cues, respectively.

Epistatic, synthetic, and balancing interactions among tubulin missense mutations affecting neurite growth in Caenorhabditis elegans.

Mutations in tubulins affect microtubule (MT) dynamics and functions during neuronal differentiation and their genetic interaction provides insights into the regulation of MT functions. We previously used Caenorhabditis elegans touch receptor neurons to analyze the cellular impact of tubulin mutations and reported the phenotypes of 67 tubulin missense mutations, categorized into three classes: loss-of-function (lf), antimorphic (anti), and neomorphic (neo) alleles. In this study, we isolated 54 additional tubulin alleles through suppressor screens in sensitized backgrounds that caused excessive neurite growth. These alleles included 32 missense mutations not analyzed before, bringing the total number of mutations in our collection to 99. Phenotypic characterization of these newly isolated mutations identified three new types of alleles: partial lf and weak neo alleles of mec-7/ß-tubulin that had subtle effects and strong anti alleles of mec-12/α-tubulin. We also discovered complex genetic interactions among the tubulin mutations, including the suppression of neo mutations by intragenic lf and anti alleles, additive and synthetic effects between mec-7 neo alleles, and unexpected epistasis, in which weaker neo alleles masked the effects of stronger neo alleles in inducing ectopic neurite growth. We also observed balancing between neo and anti alleles, whose respective MT-hyperstablizing and -destabilizing effects neutralized each other.

Mid-infrared metabolic imaging with vibrational probes.

Understanding metabolism is indispensable in unraveling the mechanistic basis of many physiological and pathological processes. However, in situ metabolic imaging tools are still lacking. Here we introduce a framework for mid-infrared (MIR) metabolic imaging by coupling the emerging high-information-throughput MIR microscopy with specifically designed IR-active vibrational probes. We present three categories of small vibrational tags including azide bond, 13C-edited carbonyl bond and deuterium-labeled probes to interrogate various metabolic activities in cells, small organisms and mice. Two MIR imaging platforms are implemented including broadband Fourier transform infrared microscopy and discrete frequency infrared microscopy with a newly incorporated spectral region (2,000-2,300 cm-1). Our technique is uniquely suited to metabolic imaging with high information throughput. In particular, we performed single-cell metabolic profiling including heterogeneity characterization, and large-area metabolic imaging at tissue or organ level with rich spectral information.

Opposing effects of an F-box protein and the HSP90 chaperone network on microtubule stability and neurite growth in Caenorhabditis elegans.

Molecular chaperones often work collaboratively with the ubiquitylation-proteasome system (UPS) to facilitate the degradation of misfolded proteins, which typically safeguards cellular differentiation and protects cells from stress. In this study, however, we report that the Hsp70/Hsp90 chaperone machinery and an F-box protein, MEC-15, have opposing effects on neuronal differentiation, and that the chaperones negatively regulate neuronal morphogenesis and functions. Using the touch receptor neurons (TRNs) of Caenorhabditis elegans, we find that mec-15(-) mutants display defects in microtubule formation, neurite growth, synaptic development and neuronal functions, and that these defects can be rescued by the loss of Hsp70/Hsp90 chaperones and co-chaperones. MEC-15 probably functions in a Skp-, Cullin- and F-box- containing complex to degrade DLK-1, which is an Hsp90 client protein stabilized by the chaperones. The abundance of DLK-1, and likely other Hsp90 substrates, is fine-tuned by the antagonism between MEC-15 and the chaperones; this antagonism regulates TRN development, as well as synaptic functions of GABAergic motor neurons. Therefore, a balance between the UPS and the chaperones tightly controls neuronal differentiation.

Inhibition of cell fate repressors secures the differentiation of the touch receptor neurons of Caenorhabditis elegans.

Terminal differentiation generates the specialized features and functions that allow postmitotic cells to acquire their distinguishing characteristics. This process is thought to be controlled by transcription factors called 'terminal selectors' that directly activate a set of downstream effector genes. In Caenorhabditis elegans, the differentiation of both the mechanosensory touch receptor neurons (TRNs) and the multidendritic nociceptor FLP neurons uses the terminal selectors UNC-86 and MEC-3. The FLP neurons fail to activate TRN genes, however, because a complex of two transcriptional repressors (EGL-44/EGL-46) prevents their expression. Here, we show that the ZEB family transcriptional factor ZAG-1 promotes TRN differentiation not by activating TRN genes but by preventing the expression of EGL-44/EGL-46. As EGL-44/EGL-46 also inhibits the production of ZAG-1, these proteins form a bistable, negative-feedback loop that regulates the choice between the two neuronal fates.

Optical imaging of metabolic dynamics in animals.

Direct visualization of metabolic dynamics in living animals with high spatial and temporal resolution is essential to understanding many biological processes. Here we introduce a platform that combines deuterium oxide (D2O) probing with stimulated Raman scattering (DO-SRS) microscopy to image in situ metabolic activities. Enzymatic incorporation of D2O-derived deuterium into macromolecules generates carbon-deuterium (C-D) bonds, which track biosynthesis in tissues and can be imaged by SRS in situ. Within the broad vibrational spectra of C-D bonds, we discover lipid-, protein-, and DNA-specific Raman shifts and develop spectral unmixing methods to obtain C-D signals with macromolecular selectivity. DO-SRS microscopy enables us to probe de novo lipogenesis in animals, image protein biosynthesis without tissue bias, and simultaneously visualize lipid and protein metabolism and reveal their different dynamics. DO-SRS microscopy, being noninvasive, universally applicable, and cost-effective, can be adapted to a broad range of biological systems to study development, tissue homeostasis, aging, and tumor heterogeneity.