Quantitative Proteome Analysis Reveals RNA Processing Factors As Modulators of Ionizing Radiation-Induced Apoptosis in the C. elegans Germline

The nematode Caenorhabditis elegans is an organism most recognized for forward and reverse genetic and functional genomic approaches. Proteomic analyses of DNA damage-induced apoptosis have not been shown because of a limited number of cells undergoing apoptosis. We applied mass spectrometry-based quantitative proteomics to evaluate protein changes induced by ionizing radiation (IR) in isolated C. elegans germlines. For this purpose, we used isobaric peptide termini labeling (IPTL) combined with the data analysis tool IsobariQ, which utilizes MS/MS spectra for relative quantification of peak pairs formed during fragmentation. Using stringent statistical critera, we identified 48 proteins to be significantly up- or down-regulated, most of which are part of a highly interconnected protein-protein interaction network dominated by proteins involved in translational control. RNA-mediated depletion of a selection of the IR-regulated proteins revealed that the conserved CAR-1/CGH-1/CEY-3 germline RNP complex acts as a novel negative regulator of DNA-damage induced apoptosis. Finally, a central role of nucleolar proteins in orchestrating these responses was confirmed as the H/ACA snRNP protein GAR-1 was required for IR-induced apoptosis in the C. elegans germline.     

Model-Independent Phenotyping of C. elegans Locomotion Using Scale-Invariant Feature Transform

To uncover the genetic basis of behavioral traits in the model organism C. elegans, a common strategy is to study locomotion defects in mutants. Despite efforts to introduce (semi-)automated phenotyping strategies, current methods overwhelmingly depend on worm-specific features that must be hand-crafted and as such are not generalizable for phenotyping motility in other animal models. Hence, there is an ongoing need for robust algorithms that can automatically analyze and classify motility phenotypes quantitatively. To this end, we have developed a fully-automated approach to characterize C. elegans’ phenotypes that does not require the definition of nematode-specific features. Rather, we make use of the popular computer vision Scale-Invariant Feature Transform (SIFT) from which we construct histograms of commonly-observed SIFT features to represent nematode motility. We first evaluated our method on a synthetic dataset simulating a range of nematode crawling gaits. Next, we evaluated our algorithm on two distinct datasets of crawling C. elegans with mutants affecting neuromuscular structure and function. Not only is our algorithm able to detect differences between strains, results capture similarities in locomotory phenotypes that lead to clustering that is consistent with expectations based on genetic relationships. Our proposed approach generalizes directly and should be applicable to other animal models. Such applicability holds promise for computational ethology as more groups collect high-resolution image data of animal behavior.     

Directional Locomotion of C. elegans in the Absence of External Stimuli

Many organisms respond to food deprivation by altering their pattern of movement, often in ways that appear to facilitate dispersal. While the behavior of the nematode C. elegans in the presence of attractants has been characterized, long-range movement in the absence of external stimuli has not been examined in this animal. Here we investigate the movement pattern of individual C. elegans over times of ∼1 hour after removal from food, using two custom imaging set-ups that allow us to track animals on large agar surfaces of 22 cm×22 cm. We find that a sizeable fraction of the observed trajectories display directed motion over tens of minutes. Remarkably, this directional persistence is achieved despite a local orientation memory that decays on the scale of about one minute. Furthermore, we find that such trajectories cannot be accounted for by simple random, isotropic models of animal locomotion. This directional behavior requires sensory neurons, but appears to be independent of known sensory signal-transduction pathways. Our results suggest that long-range directional behavior of C. elegans may not be driven by sensory cues.     

Quantitative Analysis of Cytokinesis In Situ during C. elegans Postembryonic Development

The physical separation of a cell into two daughter cells during cytokinesis requires cell-intrinsic shape changes driven by a contractile ring. However, in vivo, cells interact with their environment, which includes other cells. How cytokinesis occurs in tissues is not well understood. Here, we studied cytokinesis in an intact animal during tissue biogenesis. We used high-resolution microscopy and quantitative analysis to study the three rounds of division of the C. elegans vulval precursor cells (VPCs). The VPCs are cut in half longitudinally with each division. Contractile ring breadth, but not the speed of ring closure, scales with cell length. Furrowing speed instead scales with division plane dimensions, and scaling is consistent between the VPCs and C. elegans blastomeres. We compared our VPC cytokinesis kinetics data with measurements from the C. elegans zygote and HeLa and Drosophila S2 cells. Both the speed dynamics and asymmetry of ring closure are qualitatively conserved among cell types. Unlike in the C. elegans zygote but similar to other epithelial cells, Anillin is required for proper ring closure speed but not asymmetry in the VPCs. We present evidence that tissue organization impacts the dynamics of cytokinesis by comparing our results on the VPCs with the cells of the somatic gonad. In sum, this work establishes somatic lineages in post-embryonic C. elegans development as cell biological models for the study of cytokinesis in situ.     

Track-A-Worm,An Open-Source System for Quantitative Assessment of C. elegans Locomotory and Bending Behavior

A major challenge of neuroscience is to understand the circuit and gene bases of behavior. C. elegans is commonly used as a model system to investigate how various gene products function at specific tissue, cellular, and synaptic foci to produce complicated locomotory and bending behavior. The investigation generally requires quantitative behavioral analyses using an automated single-worm tracker, which constantly records and analyzes the position and body shape of a freely moving worm at a high magnification. Many single-worm trackers have been developed to meet lab-specific needs, but none has been widely implemented for various reasons, such as hardware difficult to assemble, and software lacking sufficient functionality, having closed source code, or using a programming language that is not broadly accessible. The lack of a versatile system convenient for wide implementation makes data comparisons difficult and compels other labs to develop new worm trackers. Here we describe Track-A-Worm, a system rich in functionality, open in source code, and easy to use. The system includes plug-and-play hardware (a stereomicroscope, a digital camera and a motorized stage), custom software written to run with Matlab in Windows 7, and a detailed user manual. Grayscale images are automatically converted to binary images followed by head identification and placement of 13 markers along a deduced spline. The software can extract and quantify a variety of parameters, including distance traveled, average speed, distance/time/speed of forward and backward locomotion, frequency and amplitude of dominant bends, overall bending activities measured as root mean square, and sum of all bends. It also plots worm travel path, bend trace, and bend frequency spectrum. All functionality is performed through graphical user interfaces and data is exported to clearly-annotated and documented Excel files. These features make Track-A-Worm a good candidate for implementation in other labs.     

Changes in Postural Syntax Characterize Sensory Modulation and Natural Variation of C. elegans Locomotion

Locomotion is driven by shape changes coordinated by the nervous system through time; thus, enumerating an animal''s complete repertoire of shape transitions would provide a basis for a comprehensive understanding of locomotor behaviour. Here we introduce a discrete representation of behaviour in the nematode C. elegans. At each point in time, the worm’s posture is approximated by its closest matching template from a set of 90 postures and locomotion is represented as sequences of postures. The frequency distribution of postural sequences is heavy-tailed with a core of frequent behaviours and a much larger set of rarely used behaviours. Responses to optogenetic and environmental stimuli can be quantified as changes in postural syntax: worms show different preferences for different sequences of postures drawn from the same set of templates. A discrete representation of behaviour will enable the use of methods developed for other kinds of discrete data in bioinformatics and language processing to be harnessed for the study of behaviour.     

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson''s disease (PD), which involves the degeneration of dopaminergic (DA) neurons ¹. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds ^2,3.Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans'' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") ⁴. Reversing the field''s polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response ⁵. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae ⁴. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype ⁶.We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae ⁷. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel ⁸.Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.     

Quantitative analysis of germline mitosis in adult C. elegans

Certain aspects of the distal gonad of C. elegans are comparable to niche/stem cell systems in other organisms. The distal tip cell (DTC) caps a blind-ended tube; only the distal germ cells maintain proliferation in response to signaling from the DTC via the GLP-1/Notch signaling pathway in the germ line. Fruitful comparison between this system and other stem cell systems is limited by a lack of basic information regarding germ cell division behavior in C. elegans. Here, we explore the spatial pattern of cell division frequency in the adult C. elegans germ line relative to distance from the distal tip. We mapped the positions of actively dividing germline nuclei in over 600 fixed gonad preparations including the wild type and a gain-of-function ligand-responsive GLP-1 receptor mutant with an extended mitotic zone. One particularly surprising observation from these data is that the frequency of cell divisions is lower in distal-most cells-cells that directly contact the distal tip cell body-relative to cells further proximal, a difference that persists in the gain-of-function GLP-1 mutant. These results suggest that cell division frequency in the distal-most cells may be suppressed or otherwise controlled in a complex manner. Further, our data suggest that the presence of an active cell division influences the probability of observing simultaneous cell divisions in the same gonad arm, and that simultaneous divisions tend to cluster spatially. We speculate that this system behaves similarly to niche/stem cell/transit amplifying cell systems in other organisms.     

计算机视觉在线虫进食行为自动分析中的应用

秀丽隐杆线虫被广泛地用作研究基因与行为关系的绝佳模式生物.线虫的咽部神经元回路控制着复杂的进食行为.为了研究进食行为的分子机制,有必要对线虫进食行为表型分析鉴定.然而,目前为止,几乎所有的线虫进食行为表型鉴定都是通过人眼来判断.因为其泵入食物的肌肉运动频率高,该行为的分析是很困难而且效率低下的.为解决这个问题,我们设计了基于计算机视觉技术的自动化成像系统来高通量分析线虫进食行为表型.此成像系统对进食表型的检测准确率达到98%以上,并使得连续可靠地分析其表型细微变化成为可能.同时,在保证高准确率的前提下单位时间内分析数据的效率比人工分析提高了3倍.     

Analysis of C. elegans VIG-1 expression

Double-stranded RNA (dsRNA) induces gene silencing in a sequence-specific manner by a process known as RNA interference (RNAi). The RNA-induced silencing complex (RISC) is a multi-subunit ribonucleoprotein complex that plays a key role in RNAi. VIG (Vasa intronic gene) has been identified as a component of Drosophila RISC; however, the role VIG plays in regulating RNAi is poorly understood. Here, we examined the spatial and temporal expression patterns of VIG-1, the C. elegans ortholog of Drosophila VIG, using a vig-1::gfp fusion construct. This construct contains the 908-bp region immediately upstream of vig-1 gene translation initiation site. Analysis by confocal microscopy demonstrated GFP-VIG-1 expression in a number of tissues including the pharynx, body wall muscle, hypodermis, intestine, reproductive system, and nervous system at the larval and adult stages. Furthermore, western blot analysis showed that VIG-1 is present in each developmental stage examined. To investigate regulatory sequences for vig-1 gene expression, we generated constructs containing deletions in the upstream region. It was determined that the GFP expression pattern of a deletion construct (delta-908 to -597) was generally similar to that of the non-deletion construct. In contrast, removal of a larger segment (delta-908 to -191) resulted in the loss of GFP expression in most cell types. Collectively, these results indicate that the 406-bp upstream region (-596 to -191) contains essential regulatory sequences required for VIG-1 expression.     

Environmental Programming of Adult Foraging Behavior in C. elegans

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Evolution: an ecological context for C. elegans

Despite low global diversity among natural populations of Caenorhabditis elegans, neighboring populations can be as genetically distinct as strains from different continents, probably owing to transient bottlenecks and ongoing dispersal as a dauer larva. Selfing predominates in the wild, but rare outcrossing may also play an important role.     

Monoaminergic Orchestration of Motor Programs in a Complex C. elegans Behavior

Monoamines provide chemical codes of behavioral states. However, the neural mechanisms of monoaminergic orchestration of behavior are poorly understood. Touch elicits an escape response in Caenorhabditis elegans where the animal moves backward and turns to change its direction of locomotion. We show that the tyramine receptor SER-2 acts through a Gα_o pathway to inhibit neurotransmitter release from GABAergic motor neurons that synapse onto ventral body wall muscles. Extrasynaptic activation of SER-2 facilitates ventral body wall muscle contraction, contributing to the tight ventral turn that allows the animal to navigate away from a threatening stimulus. Tyramine temporally coordinates the different phases of the escape response through the synaptic activation of the fast-acting ionotropic receptor, LGC-55, and extrasynaptic activation of the slow-acting metabotropic receptor, SER-2. Our studies show, at the level of single cells, how a sensory input recruits the action of a monoamine to change neural circuit properties and orchestrate a compound motor sequence.     

Genetic Characterization of smg-8 Mutants Reveals No Role in C. elegans Nonsense Mediated Decay

The nonsense mediated decay (NMD) pathway degrades mRNAs bearing premature translation termination codons. In mammals, SMG-8 has been implicated in the NMD pathway, in part by its association with SMG-1 kinase. Here we use four independent assays to show that C. elegans smg-8 is not required to degrade nonsense-containing mRNAs. We examine the genetic requirement for smg-8 to destabilize the endogenous, natural NMD targets produced by alternative splicing of rpl-7a and rpl-12. We test smg-8 for degradation of the endogenous, NMD target generated by unc-54(r293), which lacks a normal polyadenylation site. We probe the effect of smg-8 on the exogenous NMD target produced by myo-3::GFP, which carries a long 3′ untranslated region that destabilizes mRNAs. None of these known NMD targets is influenced by smg-8 mutations. In addition, smg-8 animals lack classical Smg mutant phenotypes such as a reduced brood size or abnormal vulva. We conclude that smg-8 is unlikely to encode a component critical for NMD.     

A Novel Dictionary Based Computer Vision Method for the Detection of Cell Nuclei

Cell nuclei detection in fluorescent microscopic images is an important and time consuming task in a wide range of biological applications. Blur, clutter, bleed through and partial occlusion of nuclei make individual nuclei detection a challenging task for automated image analysis. This paper proposes a novel and robust detection method based on the active contour framework. Improvement over conventional approaches is achieved by exploiting prior knowledge of the nucleus shape in order to better detect individual nuclei. This prior knowledge is defined using a dictionary based approach which can be formulated as the optimization of a convex energy function. The proposed method shows accurate detection results for dense clusters of nuclei, for example, an F-measure (a measure for detection accuracy) of 0.96 for the detection of cell nuclei in peripheral blood mononuclear cells, compared to an F-measure of 0.90 achieved by state-of-the-art nuclei detection methods.