Functions of the extracellular matrix in development: Lessons from Caenorhabditis elegans

Cell-extracellular matrix interactions are crucial for the development of an organism from the earliest stages of embryogenesis. The main constituents of the extracellular matrix are collagens, laminins, proteoglycans and glycosaminoglycans that form a network of interactions. The extracellular matrix and its associated molecules provide developmental cues and structural support from the outside of cells during development. The complex nature of the extracellular matrix and its ability for continuous remodeling poses challenges when investigating extracellular matrix-based signaling during development. One way to address these challenges is to employ invertebrate models such as Caenorhabditis elegans, which are easy to genetically manipulate and have an invariant developmental program. C. elegans also expresses fewer extracellular matrix protein isoforms and exhibits reduced redundancy compared to mammalian models, thus providing a simpler platform for exploring development. This review summarizes our current understanding of how the extracellular matrix controls the development of neurons, muscles and the germline in C. elegans.     

Split-wrmScarlet and split-sfGFP: tools for faster,easier fluorescent labeling of endogenous proteins in Caenorhabditis elegans

We create and share a new red fluorophore, along with a set of strains, reagents and protocols, to make it faster and easier to label endogenous Caenorhabditis elegans proteins with fluorescent tags. CRISPR-mediated fluorescent labeling of C. elegans proteins is an invaluable tool, but it is much more difficult to insert fluorophore-size DNA segments than it is to make small gene edits. In principle, high-affinity asymmetrically split fluorescent proteins solve this problem in C. elegans: the small fragment can quickly and easily be fused to almost any protein of interest, and can be detected wherever the large fragment is expressed and complemented. However, there is currently only one available strain stably expressing the large fragment of a split fluorescent protein, restricting this solution to a single tissue (the germline) in the highly autofluorescent green channel. No available C. elegans lines express unbound large fragments of split red fluorescent proteins, and even state-of-the-art split red fluorescent proteins are dim compared to the canonical split-sfGFP protein. In this study, we engineer a bright, high-affinity new split red fluorophore, split-wrmScarlet. We generate transgenic C. elegans lines to allow easy single-color labeling in muscle or germline cells and dual-color labeling in somatic cells. We also describe a novel expression strategy for the germline, where traditional expression strategies struggle. We validate these strains by targeting split-wrmScarlet to several genes whose products label distinct organelles, and we provide a protocol for easy, cloning-free CRISPR/Cas9 editing. As the collection of split-FP strains for labeling in different tissues or organelles expands, we will post updates at doi.org/10.5281/zenodo.3993663     

Wheat quality related differential expressions of albumins and globulins revealed by two-dimensional difference gel electrophoresis (2-D DIGE)

Comparative proteomics analysis offers a new approach to identify differential proteins among different wheat genotypes and developmental stages. In this study, the non-prolamin expression profiles during grain development of two common or bread wheat cultivars (Triticum aestivum L.), Jing 411 and Sunstate, with different quality properties were analyzed using two-dimensional difference gel electrophoresis (2-D DIGE). Five grain developmental stages during the post-anthesis period were sampled corresponding to the cumulative averages of daily temperatures (°C: 156 °C, 250 °C, 354 °C, 447 °C and 749.5 °C). More than 400 differential protein spots detected at one or more of the developmental stages of the two cultivars were monitored, among which 230 proteins were identified by MS. Of the identified proteins, more than 85% were enzymes possessing different physiological functions. A total of 36 differential proteins were characterized between the two varieties, which are likely to be related to wheat quality attributes. About one quarter of the proteins identified expressed in multiple spots with different pIs and molecular masses, implying certain post-translational modifications (PTMs) of proteins such as phosphorylations and glycosylations. The results provide new insights into biochemical mechanisms for grain development and quality.     

Proteomic Characterization of Caenorhabditis elegans Larval Development

The nematode Caenorhabditis elegans is widely used as a model organism to study cell and developmental biology. Quantitative proteomics of C. elegans is still in its infancy and, so far, most studies have been performed on adult worm samples. Here, we used quantitative mass spectrometry to characterize protein level changes across the four larval developmental stages (L1–L4) of C. elegans. In total, we identified 4130 proteins, and quantified 1541 proteins that were present across all four stages in three biological replicates from independent experiments. Using hierarchical clustering and functional ontological analyses, we identified 21 clusters containing proteins with similar protein profiles across the four stages, and highlighted the most overrepresented biological functions in each of these protein clusters. In addition, we used the dataset to identify putative larval stage‐specific proteins in each individual developmental stage, as well as in the early and late developmental stages. In summary, this dataset provides system‐wide analysis of protein level changes across the four C. elegans larval developmental stages, which serves as a useful resource for the C. elegans research community. MS data were deposited in ProteomeXchange ( http://proteomecentral.proteomexchange.org ) via the PRIDE partner repository with the primary accession identifier PXD006676.     

The glycoproteins of Dictyostelium discoideum. Changes during development.

The glycoproteins of D. discoideum have been analyzed by direct binding of radio-iodinated lectins to SDS gels of the successive developmental stages. Compared with the total pattern of proteins, many changes are found in the glycoproteins during development. WGA reacts with few gel bands from the vegetative cells and most of these, including a very intense band at the top of the gel, are lost during the first few hours of development. Approximately half-way through the developmental cycle at least 14 new glycoproteins reacting with WGA begin to appear and progressively accumulate. In contrast, ConA labels many glycoproteins over the complete molecular weight range and most are unaffected during development. Lectins which bind fucose label a single component at the top of the gel of vegetative cells and this decreases rapidly as development begins. No other reactive gel bands are revealed by fucose-binding lectins until the final stages of spore and stalk formation, when four high molecular weight glycoproteins are detected. Lectins specific for terminal galactose residues and for N-acetyl-galactosamine, including the intrinsic lectins produced by D. discoideum during its development, failed to reveal any reactive glycoproteins.     

Study of nuclear basic proteins of Physarum polycephalum: Differential synthesis during plasmodial cell cycle and spherule germination

An electrophoresis system providing a good resolution of all basic proteins and their variants or modified forms extracted from Physarum polycephalum nuclei has been used to study the synthesis of the basic nuclear proteins of this organism during three developmental stages: the S-phase and the G2-phase of the plasmodial cell cycle and the premitotic stage of germinating spherules. Differences have been seen in the synthesis of the different classes of histones and HMG like proteins during these three stages.     

Identification of genes driving lineage divergence from single-cell gene expression data in C. elegans

The nematode Caenorhabditis elegans (C. elegans) is an ideal model organism to study the cell fate specification mechanisms during embryogenesis. It is generally believed that cell fate specification in C. elegans is mainly mediated by lineage-based mechanisms, where the specification paths are driven forward by a succession of asymmetric cell divisions. However, little is known about how each binary decision is made by gene regulatory programs. In this study, we endeavor to obtain a global understanding of cell lineage/fate divergence processes during the early embryogenesis of C. elegans. We reanalyzed the EPIC data set, which traced the expression level of reporter genes at single-cell resolution on a nearly continuous time scale up to the 350-cell stage in C. elegans embryos. We examined the expression patterns for a total of 131 genes from 287 embryos with high quality image recordings, among which 86 genes have replicate embryos. Our results reveal that during early embryogenesis, divergence between sister lineages could be largely explained by a few genes. We predicted genes driving lineage divergence and explored their expression patterns in sister lineages. Moreover, we found that divisions leading to fate divergence are associated with a large number of genes being differentially expressed between sister lineages. Interestingly, we found that the developmental paths of lineages could be differentiated by a small set of genes. Therefore, our results support the notion that the cell fate patterns in C. elegans are achieved through stepwise binary decisions punctuated by cell divisions. Our predicted genes driving lineage divergence provide good starting points for future detailed characterization of their roles in the embryogenesis in this important model organism.     

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Caenorhabditis elegans is a simple genetic organism amenable to large-scale forward and reverse genetic screens and chemical genetic screens. The C. elegans genome includes potential antipsychotic drug (APD) targets conserved in humans, including genes encoding proteins required for neurotransmitter synthesis and for synaptic structure and function. APD exposure produces developmental delay and/or lethality in nematodes in a concentration-dependent manner. These phenotypes are caused, in part, by APD-induced inhibition of pharyngeal pumping^1,2. Thus, the developmental phenotype has a neuromuscular basis, making it useful for pharmacogenetic studies of neuroleptics. Here we demonstrate detailed procedures for testing APD effects on nematode development and pharyngeal pumping. For the developmental assay, synchronized embryos are placed on nematode growth medium (NGM) plates containing APDs, and the stages of developing animals are then scored daily. For the pharyngeal pumping rate assay, staged young adult animals are tested on NGM plates containing APDs. The number of pharyngeal pumps per unit time is recorded, and the pumping rate is calculated. These assays can be used for studying many other types of small molecules or even large molecules.     

Compositional changes in soluble proteins of cerebral mantle,cerebellum, and brain stem of rat brain during development

Soluble proteins from the cerebral mantle, cerebellum, and brain stem of rat brains were analyzed at various developmental stages by a two-dimensional gel electrophoresis technique. The electrophoretic technique resolved the soluble proteins into 100–150 polypeptide spots on two-dimensional gels and gave reproducible and highly resolved profiles of them. Although most of major polypeptides were commonly found in all the three brain regions, some polypeptides were shown to be unique to a specific brain region. Each brain region was different in the electrophoretic profile of soluble proteins at every developmental stage examined, although there was considerable similarity in the profiles of each of the three brain regions in fetal animals (16–17 days), indicating that soluble proteins undergo different compositional changes in each of the three brain regions during postnatal development. In addition, the number of polypeptide spots on the electrophoretic profile increased remarkably during postnatal development in all of the three brain regions, suggesting that soluble proteins become more heterogeneous during postnatal development in each of the three brain regions.     

Interactome analysis of Caenorhabditis elegans synapses by TurboID-based proximity labeling

Proximity labeling provides a powerful in vivo tool to characterize the proteome of subcellular structures and the interactome of specific proteins. The nematode Caenorhabditis elegans is one of the most intensely studied organisms in biology, offering many advantages for biochemistry. Using the highly active biotin ligase TurboID, we optimize here a proximity labeling protocol for C. elegans. An advantage of TurboID is that biotin''s high affinity for streptavidin means biotin-labeled proteins can be affinity-purified under harsh denaturing conditions. By combining extensive sonication with aggressive denaturation using SDS and urea, we achieved near-complete solubilization of worm proteins. We then used this protocol to characterize the proteomes of the worm gut, muscle, skin, and nervous system. Neurons are among the smallest C. elegans cells. To probe the method''s sensitivity, we expressed TurboID exclusively in the two AFD neurons and showed that the protocol could identify known and previously unknown proteins expressed selectively in AFD. The active zones of synapses are composed of a protein matrix that is difficult to solubilize and purify. To test if our protocol could solubilize active zone proteins, we knocked TurboID into the endogenous elks-1 gene, which encodes a presynaptic active zone protein. We identified many known ELKS-1-interacting active zone proteins, as well as previously uncharacterized synaptic proteins. Versatile vectors and the inherent advantages of using C. elegans, including fast growth and the ability to rapidly make and functionally test knock-ins, make proximity labeling a valuable addition to the armory of this model organism.     

Development-Specific Differences in the Proteomics of Angiostrongylus cantonensis

Angiostrongyliasis is an emerging communicable disease. Several different hosts are required to complete the life cycle of Angiostrongylus cantonensis. However, we lack a complete understanding of variability of proteins across different developmental stages and their contribution to parasite survival and progression. In this study, we extracted soluble proteins from various stages of the A. cantonensis life cycle [female adults, male adults, the fifth-stage female larvae (FL5), the fifth-stage male larvae (ML5) and third-stage larvae (L3)], separated those proteins using two-dimensional difference gel electrophoresis (2D-DIGE) at pH 4–7, and analyzed the gel images using DeCyder 7.0 software. This proteomic analysis produced a total of 183 different dominant protein spots. Thirty-seven protein spots were found to have high confidence scores (>95%) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Comparative proteomic analyses revealed that 29 spots represented cytoskeleton-associated proteins and functional proteins. Eight spots were unnamed proteins. Twelve protein spots that were matched to the EST of different-stage larvae of A. cantonensis were identified. Two genes and the internal control 18s were chosen for quantitative real-time PCR (qPCR) and the qPCR results were consistent with those of the DIGE studies. These findings will provide a new basis for understanding the characteristics of growth and development of A. cantonensis and the host–parasite relationship. They may also assist searches for candidate proteins suitable for use in diagnostic assays and as drug targets for the control of eosinophilic meningitis caused by A. cantonensis.     

蛋白质组学研究中的无标记定量方法

蛋白质定量是探索疾病发生发展状况和寻找新药靶标的重要手段.该领域最常用的技术是比较染色后的二维凝胶上蛋白点的光密度值或综合同位素标记后的质谱峰强度方法.但此二者的样品处理方法都比较麻烦,不利于进行大规模蛋白质组的定量研究.最近几年出现了利用质谱数据进行无标记定量的方法, 根据数据类型这些方法可以分为2类：基于鉴定蛋白的肽段数的方法和基于质谱峰强度的方法,在高通量大规模蛋白组定量研究中有很大优势.本综述主要介绍了这2类无标记定量方法的模型及优缺点,并比较了2类方法的灵敏度和准确度.肽段计数方法在检测蛋白丰度变化时更灵敏,而峰面积强度在评估蛋白比率时更准确.     

The first protein map of Synechococcus sp. strain PCC 7942

The first protein map was developed of Synechococcus sp. strain PCC 7942, a model organism for studies of photosynthesis, prokaryotic circadian rhythms, cell division, carbon-concentrating mechanisms, and adaptive responses to a variety of stresses. The proteome was analyzed by two-dimensional gel electrophoresis with subsequent MALDI-TOF mass spectroscopy and database analysis. Of the 140 analyzed protein spots, 110 were successfully identified as 62 different proteins, many of which occurred as multiple spots on the gel. The identified proteins participate in the major metabolic and cellular processes in cyanobacterial cells during the exponential growth phase. In addition, 14 proteins which were previously either unknown or considered to be hypothetical were shown to be true gene products in Synechococcus sp. strain PCC 7942. These results may be helpful for the annotation of the recently sequenced genome of this cyanobacterium, as well as for biochemical and physiological studies of Synechococcus.     

Transfer ribonucleic acid synthesis during sporulation and spore outgrowth in Bacillus subtilis studied by two-dimensional polyacrylamide gel electrophoresis.

The synthesis of transfer ribonucleic acid (tRNA) was examined during spore formation and spore outgrowth in Bacillus subtilis by two-dimensional polyacrylamide gel electrophoresis of in vivo 32P-labeled RNA. The two-dimensional gel system separated the B. subtilis tRNA's into 32 well-resolved spots, with the relative abundances ranging from 0.9 to 17% of the total. There were several spots (five to six) resolved which were not quantitated due to their low abundance. All of the tRNA species resolved by this gel system were synthesized at every stage examined, including vegetative growth, different stages of sporulation, and different stages of outgrowth. Quantitation of the separated tRNA's showed that in general the tRNA species were present in approximately the same relative abundances at the different developmental periods. tRNA turnover and compartmentation occurring during sporulation were examined by labeling during vegetative growth followed by the addition of excess phosphate to block further 32P incorporation. The two-dimensional gels of these samples showed the same tRNA's seen during vegetative growth, and they were in approximately the same relative abundances, indicating minimal differences in the rates of turnover of individual tRNA's. Vegetatively labeled samples, chased with excess phosphate into mature spores, also showed all of the tRNA species seen during vegetative growth, but an additional five to six minor spots were also observed. These are hypothesized to arise from the loss of 3'-terminal residues from preexisting tRNA's.     

Isotopic labeling of mammalian G protein-coupled receptors heterologously expressed in Caenorhabditis elegans

High-resolution structural determination and dynamic characterization of membrane proteins by nuclear magnetic resonance (NMR) require their isotopic labeling. Although a number of labeled eukaryotic membrane proteins have been successfully expressed in bacteria, they lack post-translational modifications and usually need to be refolded from inclusion bodies. This shortcoming of bacterial expression systems is particularly detrimental for the functional expression of G protein-coupled receptors (GPCRs), the largest family of drug targets, due to their inherent instability. In this work, we show that proteins expressed by a eukaryotic organism can be isotopically labeled and produced with a quality and quantity suitable for NMR characterization. Using our previously described expression system in Caenorhabditis elegans, we showed the feasibility of labeling proteins produced by these worms with ¹⁵N,¹³C by providing them with isotopically labeled bacteria. ²H labeling also was achieved by growing C. elegans in the presence of 70% heavy water. Bovine rhodopsin, simultaneously expressed in muscular and neuronal worm tissues, was employed as the “test” GPCR to demonstrate the viability of this approach. Although the worms’ cell cycle was slightly affected by the presence of heavy isotopes, the final protein yield and quality was appropriate for NMR structural characterization.     

Limited microsynteny between the genomes of Pristionchus pacificus and Caenorhabditis elegans

Nematodes are an attractive group of organisms for studying the evolution of developmental processes. Pristionchus pacificus was established as a satellite organism for comparing vulva development and other processes to Caenorhabditis elegans. The generation of a genetic linkage map of P.pacificus has provided a first insight into the structure and organization of the genome of this species. Pristionchus pacificus and C.elegans are separated from one another by >100 000 000 years such that the structure of the genomes of these two nematodes might differ substantially. To evaluate the amount of synteny between the two genomes, we have obtained 126 kb of continuous genomic sequence of P.pacificus, flanking the developmental patterning gene pal-1. Of the 20 predicted open reading frames in this interval, 11 have C.elegans orthologs. Ten of these 11 orthologs are located on C.elegans chromosome III, indicating the existence of synteny. However, most of these genes are distributed over a 12 Mb interval of the C.elegans genome and only three pairs of genes show microsynteny. Thus, intrachromosomal rearrange ments occur frequently in nematodes, limiting the likelihood of identifying orthologous genes of P.pacificus and C.elegans based on positional information within the two genomes.     

Changes in the phosphorylation of non-histone chromatin proteins during the cell cycle of HeLa S 3 cells

The phosphorylation of non-histone chromatin proteins in synchronized HeLa S₃ cells was studied in 5 phases of the cell cycle: mitosis, G₁, early and late S, and G₂. The rate of non-histone chromatin protein phosphorylation was found to be maximal during G₁ and G₂, somewhat decreased during S phase, and almost 90% depressed during mitosis. Analysis of the phosphorylated non-histone chromatin proteins by SDS-acrylamide gel electrophoresis showed a heterogeneous pattern of phosphorylation as measured by labeling with ³²P. Significant variations in the labeling pattern were seen during different stages of the cell cycle, and particular unique species appeared to be phosphorylated selectively during certain stages of the cycle.     

Guidelines for monitoring autophagy in Caenorhabditis elegans

The cellular recycling process of autophagy has been extensively characterized with standard assays in yeast and mammalian cell lines. In multicellular organisms, numerous external and internal factors differentially affect autophagy activity in specific cell types throughout the stages of organismal ontogeny, adding complexity to the analysis of autophagy in these metazoans. Here we summarize currently available assays for monitoring the autophagic process in the nematode C. elegans. A combination of measuring levels of the lipidated Atg8 ortholog LGG-1, degradation of well-characterized autophagic substrates such as germline P granule components and the SQSTM1/p62 ortholog SQST-1, expression of autophagic genes and electron microscopy analysis of autophagic structures are presently the most informative, yet steady-state, approaches available to assess autophagy levels in C. elegans. We also review how altered autophagy activity affects a variety of biological processes in C. elegans such as L1 survival under starvation conditions, dauer formation, aging, and cell death, as well as neuronal cell specification. Taken together, C. elegans is emerging as a powerful model organism to monitor autophagy while evaluating important physiological roles for autophagy in key developmental events as well as during adulthood.     

A Preliminary Study on Proteome Variations Associated with Gall Formation in <Emphasis Type="Italic">Zizania latifolia</Emphasis> Trucs

Zizania latifolia Trucs is a uniquely flavored aquatic vegetable found in southern and eastern Asia. Several physiology and genetic approaches have been employed to increase our knowledge about the physiological basis of gall formation; however, as yet, data at the proteomic level are not available. Protein yield and sodium dodecyl sulfate-polyacrylamide gel electrophoresis were used to determine the most appropriate protein extraction methods for use in this study. Total proteins were extracted from the culm tissue at three relevant developmental stages and separated by two-dimensional gel electrophoresis. The number and abundance of spots varied among the two-dimensional gel electrophoresis gels at the three stages. Proteins with more than 1.7-fold abundance between the different stages were monitored. We identified 10 well-resolved spots by matrix-assisted laser-desorption ionization/time-of-flight peptide mass fingerprinting and tandem mass spectrometry. Some spots linked to signal transduction of the phytohormone could be identified. The expression volume of these spots transiently increased during the expansion phase. In contrast, the spots linked to phytoene dehydrogenase and methionine synthase or pyrophosphate-dependent phosphofructokinase were more abundant during gall formation, showing an increase in spot intensity during development and maximal abundance in mature gall. Higher intensity was also found in the spots linked to stress response. We discuss protein variations, considering their potential role during gall formation and comparing our results with established variations at metabolite-profiling levels.