San Miguel, Adriana et al. (2013) International Worm Meeting "Phenotypic profiling of synaptic sites for subtle mutant identification in automated genetic screens."

Crane, Matthew, Lu, Hang, Shen, Kang, Kurshan, Peri, San Miguel, Adriana

[International Worm Meeting, 2013]

Many neurological diseases, caused by defects in neurotransmission, are suspected to show no obvious morphological alterations in neuronal patterning. Uncovering genes responsible for these disorders by identifying mutants with subtle changes in morphology of micron-sized synapses is a very challenging task that necessitates high-resolution and quantitative phenotyping, making genetic screening virtually impossible. In this work we exploit an integrated approach to perform automated genetic screens in C. elegans that enables the isolation of mutants with extremely subtle phenotypic differences in synaptic morphology. We take advantage of microfluidic devices for easy worm handling, paired with automated control of flow, image analysis, and decision-making. Computer vision algorithms allow the unsupervised detection of fluorescently labeled micron-sized synaptic puncta in an unbiased and quantitative manner. Phenotypic profiling of synaptic sites is performed by quantification of a large number of descriptors relevant for synapse morphology, such as synapse size and intensity. In order to take into account the heterogeneity present from isogenic populations, statistical analyses are performed based on phenotypic profiling of whole populations. Logistic regression models allow discerning between populations of true mutants and wild type animals, and suggest the most relevant features that differentiate them. The phenotypic differences of the mutants found in this work range from smaller, dimmer synaptic puncta, altered number, density and interpunctal distance, all extremely difficult to discern by eye. Performing full profile analysis of the found mutants, we are able to suggest putative genetic pathways in which the newly found alleles belong by analyzing their relationships with a representative collection of mutants of known pathways. Overall, the approach shown here provides a practical, feasible technique to perform extraordinarily difficult genetic screens in an unsupervised and unbiased manner and enables the isolation of mutants with extremely subtle phenotypic differences.

Midkiff, D. et al. (2019) International Worm Meeting "Identifying aging-associated genes by screening for early-onset phenotypes in Caenorhabditis elegans."

San Miguel, A., Bosari, S. Saberi, Midkiff, D.

[International Worm Meeting, 2019]

Many genes and several pathways have been identified through study of the nematode C. elegans to cause a change in lifespan. Previous work has also identified phenotypic characteristics that correlate with lifespan. These can be used as biomarkers of aging. One such biomarker is the formation of protein aggregates in aged C. elegans. At a given age, an inverse relationship has been observed between the level of aggregation and lifespan in both isogenic populations and in lifespan mutants. Aggregates have been observed in young adult worms, indicating that aggregation could be used as a biomarker of aging early in life. Forward genetic screens have been widely used to identify previously undiscovered genes that yield a certain phenotype. Previous forward genetic screens for phenotypes that relate to aging have been cumbersome, given that phenotypes which correlate with age are typically not visible until after the worm's reproductive lifespan is complete. Our objective is to use protein aggregation as a biomarker for C. elegans lifespan in order to perform forward genetic screens for genes that affect aging. We will measure the aggregation of PAB-1, a protein that aggregates naturally with age in the C. elegans pharynx1. We utilized a microfluidic platform previously developed for C. elegans imaging and phenotypic sorting2, and developed an automated screening program for quantifying aggregation of PAB-1 fused to a fluorescent reporter in the anterior and posterior pharynx. We conducted a high-throughput screen for candidate mutants with high levels of PAB-1 aggregation in the pharynx at the end of their egg-laying period. We then further characterized the aggregation levels of candidate mutants to identify the presence of true mutations. Lifespan was then determined for each true mutant. Through this work, we aim to expand the capability of forward genetic screening methods to identify genes that regulate the natural aging process. 1 Lechler et al, 2017. Cell Reports, 18: 464-467 2 San Miguel et al, 2016. Nature Communications, 7: 12990

Huayta, J. et al. (2019) International Worm Meeting "C. elegans lifespan regulation by spatiotemporal activity of DAF-16."

Huayta, J., San Miguel, A.

[International Worm Meeting, 2019]

DAF-16 is a conserved transcription factor that regulates expression of genes involved in lifespan extension and stress response in C. elegans. It is known that environmental factors, such as dietary restriction and oxidative stress, affect the spatiotemporal activity of DAF-16. We aim to elucidate how the lifelong molecular activity of daf-16, driven by environmental stressors, determines lifespan and healthspan in C. elegans. Using the CRISPR/Cas9 genome engineering system, we targeted the endogenous loci of daf-16, and introduced a GFP coding sequence before its stop codon. We used this generated strain to characterize spatial- and temporal-specific changes in DAF-16 activity. This was achieved by quantitative analysis of gene expression by fluorescent imaging performed in vivo. We quantified nuclear and cytoplasmic DAF-16 in a tissue-specific manner. Using a machine-learning algorithm based on semantic segmentation, we tracked expression of daf-16 under various dietary restriction regimes. This imaging processing approach is necessary to evaluate the complex patterns of DAF-16 migration at the tissue and cellular levels. We observed an increased migration of DAF-16 to cell nuclei in tissues (intestine, hypodermis, muscles, neurons, and gonad) of animals under conditions of reduced food intake. This pattern increased the longer the animals were under this condition, but reaching a peak after 6-8 hours. Moreover, under repeated and intermittent exposure of the same C. elegans population to dietary restriction, we identified a decreasing activity of DAF-16 in subsequent days. We seek to characterize lifespan in C. elegans as result of these quantifiable metrics, such as DAF-16 activity and the combination of environmental stressors (food concentration, oxidative stressors, heat-shock, etc.) in a temporal-dependent way.

Crawford, Z. et al. (2019) International Worm Meeting "An inexpensive automated optogenetic platform for controlled neuronal activation regimes in C. elegans."

Crawford, Z., San Miguel, A.

[International Worm Meeting, 2019]

Studying neuronal exercise is vital to understanding the underlying mechanisms of synaptic plasticity, discovering features of learning and memory, and identifying the genes and pathways that play a role in age-associated synaptic plasticity decline. C. elegans are optically transparent, which allows for in vivo imaging of cellular and sub-cellular structures, and neuronal stimulation using optogenetics. The transparency of C. elegans enables in vivo optogenetic stimulation, and this has been applied in several ways. Despite the litany of applications and wide use of optogenetics to test neuronal function in C. elegans, the optogenetic toolset has not yet been applied to create a simple, economical system capable of specific neuronal exercise in large populations. We have developed and used an affordable platform for optogenetic-driven neuronal exercise, using LED lamps, MATLAB, and an Arduino microcontroller to automate neuronal exercise. Our platform is capable of specific and high-fidelity light-induced neuronal activation without a risk of accidental activation, through use of strains containing light-gated cation channel proteins ChR2(H134) and ChIEF. We can analyze the effects of neuronal exercise in liquid media and on plate in a quantitative manner, as well as run automated exercise regimes in a high throughput manner. Through the use of cholinesterase inhibitor aldicarb we have been able to determine the effects of exercise regimes on synaptic transmission in cholinergic neurons. In the future we hope to use this platform to facilitate studies that require repeated and controlled optogenetic stimulation of large populations.

Todd G Nystul et al. (2003) International Worm Meeting "Suspended animation in C. elegans requires the spindle checkpoint"

Mark B Roth, Todd G Nystul, Jesse P Goldmark, Pamela A Padilla

[International Worm Meeting, 2003]

C. elegans are capable of entering into anoxia-induced suspended animation, an extreme form of quiescence in which all microscopically visible movement is ceased. The molecular mechanism by which these organisms are able to coordinate entry into the suspended state is not well understood. We have identified a gene, san-1, that is required for suspended animation in embryos. san-1 is homologous to the spindle checkpoint gene Mad3, and loss of san-1 function results in a failure to arrest the cell cycle at metaphase in anoxia. We have raised antibody to the SAN-1 protein and find that, consistent with a role in the checkpoint, it localizes to the kinetochore. Furthermore, we show that a second spindle checkpoint gene, mdf-2 is also required in suspended animation. As with san-1, loss of mdf-2 function results in an inability to arrest at metaphase when exposed to anoxia. Loss of either san-1 or mdf-2 results in chromosome missegregation in anoxia, as evidenced by anaphase bridging. We quantitated the degree of missegregation in san-1(RNAi) using a strain in which one chromosome contains an insert that facilitates tracking by immunofluorescence. These data provide a molecular model for how chromosome segregation is stopped in anoxia until conditions are favorable for reanimation.

Tejada Vaprio, R. et al. (2019) International Worm Meeting "Microfluidic platform for in vivo characterization of neuronal damage in Alzheimer's disease in C. elegans."

San Miguel, A., Tejada Vaprio, R.

[International Worm Meeting, 2019]

Traumatic brain injury (TBI) has been widely associated with an increased risk of developing neurodegenerative diseases, such as Alzheimer's disease (AD). An important challenge in understanding the links between traumatic brain injury and Alzheimer's disease is a scarcity of experimental models that enable systematic studies of neuronal injury, while quantifying subsequent damage and its interaction with AD. Previous injury studies in C. elegans include laser axotomy to understand the regeneration process; however, this does not resemble the primary injury present in traumatic brain injury. The nematode C. elegans provides a unique experimental platform to perform quantitative analysis of injury, neurodegeneration, and neuronal function in AD models. As the AD model, we have used the C. elegans strain which expresses A?1-42 pan-neuronally. To elucidate the fundamental relationships between traumatic brain injury and AD, capturing pre and post-injury in vivo information of both neurological function and morphology at high-resolution is necessary. For this purpose, we have developed a microfluidic platform that enables performing controlled neuronal injury, and subsequent neuronal integrity characterization in C. elegans populations. This platform integrates microfluidic devices with pressure and fluid control hardware, as well as quantitative image analysis. No morphological differences in AWC were observed in AD-model and wildtype animals prior to injury. To determine if C. elegans would be suitable to study the effects of traumatic injury on neuronal degeneration, we performed manual mechanical injury in the head. After the manual injury, we observed clear morphological changes in the AWC neuron in C. elegans, indicative of neurodegeneration in injured wildtype and AD animals. Almost all injured wild-type and AD animals exhibited beading in neurites and neurite thinning. Using PDMS-deflectable valves as injury features in our microfluidic device, we performed controlled head injury in the animals either one or five times at the same impact pressure. Subtle changes can be observed immediately after injury in the device, such as neurite beading. These results confirm that the current microfluidic device design is able to induce significant injury as assessed by neuronal morphological changes. This platform also allows for further characterization of AWC neurons before and after injury. We performed quantitative analysis of fluorescent images of the AWC neurons using MATLAB in order to identify morphological features indicative of neurodegeneration. By incorporating a C. elegans AD model, with controlled injury and high-throughput platforms, this work will enable systematic studies of traumatic injury and neurodegeneration.

Bhaskar Reddy Gavinolla et al. (2007) International Worm Meeting "Interactions between the microtubule severing protein MEI-2 and spindle checkpoints."

Bhaskar Reddy Gavinolla, Paul E. Mains

[International Worm Meeting, 2007]

The spindle checkpoint protein, SAN-1/MDF-3 is expressed on mitotic centromeres. During anoxia, mutants fail to arrest the cell cycle, leading to chromosome mis-segregation and reduced viability (Nystul et. al 2003). Checkpoint proteins arrest the cell cycle by inhibiting the Anaphase Promoting Complex (APC) and san-1/mdf-3 mutants suppress APC mutants (Stein et al. 2007). We have uncovered a possible link between the microtubule severing complex MEI-1/MEI-2 and the meiotic spindle checkpoint. Like SAN-1/MDF-3, MEI-1 and MEI-2 localize to chromatin and genetically interact with APC mutants. Furthermore, yeast two hybrid data (Li et al. 2004) shows that MEI-2 and SAN-1 interact physically. Both the similar expression patterns and the yeast two-hybrid binding suggested possible genetic interaction between them, so we made double mutants of san-1 and mei-2. This resulted in increased lethality, low brood sizes and spontaneous males, indications of meiotic failure. While enhancement of mei-2 spindle formation defects might be expected by the presence of a compromised spindle checkpoint, the physical interaction and colocalization might indicate a more specific role for MEI-1/MEI-2 in monitoring spindle quality.

Neil M. Stewart et al. (2007) International Worm Meeting "A Synthetic Lethal Screen to Identify Spindle Checkpoint Genetic Interactions in C. elegans."

Pamela A. Padilla, Landon L. Moore, Neil M. Stewart

[International Worm Meeting, 2007]

The spindle checkpoint delays the onset of anaphase until all sister chromatids are aligned properly at the metaphase plate. The genes controlling this process, originally identified in yeast (Mad1, Mad2, Mad3, Bub1, Bub2, Bub3, and Mps1), have been extensively studied in both yeast and mammalian cells. Due to the role these gene products have in cancer and human genetic abnormalities, it is of interest to study the functional role of the spindle checkpoint gene products during development in normal and stressful environments. In Caenorhabditis elegans the genes mdf-1, mdf-2 and san-1/mdf-3 encode key members of the spindle checkpoint. The san-1 component of the spindle checkpoint is required for anoxia-induced suspended animation. Briefly, a reduction in san-1 gene product by RNAi results in a decrease in anoxia survival, a decrease in metaphase blastomeres and an increase in anaphase bridging. We used BLAST analysis to identify genes in the C. elegans genome that have homology to the yeast genes that display a synthetic lethal interaction with the spindle checkpoint genes. We focused our analysis on genes that did not display phenotypes when reduced by RNAi, in a wild-type background. These genes were tested for synthetic lethal interactions with san-1(ok1580). We identified a number of genes that when reduced by RNAi in the san-1(ok1580) background there was a significant reduction in animal viability. To further classify these gene products and understand their function we are using bioinformatics, assaying for susceptibility to microtubule disruption (anoxia exposure), and conducting protein localization analysis. These studies will lead to a greater understanding of the spindle checkpoint pathway in embryos exposed to stressful environmental conditions and potentially identify new gene products in this highly conserved signaling pathway.

Vinita Prabhu et al. (2004) Mid-west Worm Meeting "Analysis of C. elegans Embryos Exposed to Anoxia"

Pamela A Padilla, Vinita Prabhu

[Mid-west Worm Meeting, 2004]

A variety of organisms have adapted mechanism to survive oxygen deprivation. C. elegans , at all stages of their life cycle, are capable of surviving at least one day of anoxia. Embryos exposed to anoxia enter into a state of suspended animation in which there is an arrest of developmental and cell cycle progression. These embryos are capable of surviving anoxia for at least three days. We are interested in understanding the molecular mechanisms C. elegans use to survive oxygen deprivation. Analysis of chromosomal, microtubule, and kinetochore structure in blastomeres of embryos exposed to anoxia was done to investigate the signaling pathway between low oxygen concentrations and cell cycle arrest. Previously, the spindle checkpoint genes, san-1 and mdf-2 , were identified to be required for embryonic anoxia survival. Further phenotype analysis of san-1 (RNAi) and mdf-2 (RNAi) was conducted to understand the signaling pathway from low oxygen concentrations to spindle checkpoint activation. A histone::GFP strain was used to analyze chromosome segregation in N2, san-1 (RNAi) and mdf-2 (RNAi) embryos exposed to anoxia. Additionally, SAN-1 localization in embryos exposed to anoxia was examined and results indicate that the localization is altered in embryos exposed to anoxia. An understanding of how C. elegans survive oxygen deprivation will lead to a greater understanding of the metazoan response to oxygen deprivation and how embryonic development and cell cycle progression is effected by environmental changes.

Vinita Hajeri et al. (2006) Development & Evolution Meeting "Cellular and genetic analysis of anoxia-induced cell cycle arrest in C. elegans"

Pamela Padilla, Vinita Hajeri

[Development & Evolution Meeting, 2006]

Oxygen deprivation is involved with the pathology of disease conditions such as myocardial infraction, pulmonary dysfunction, blood loss due to anemia and resistance of tumor cells to chemotherapy and radiotherapy. C. elegans embryos survive oxygen-deprived conditions (anoxia; < 0.001 kPa O2) by entering into a state of suspended animation, a condition in which development and cell cycle progression is reversibly arrested. Our previous studies determined that cell cycle arrest, at interphase, prophase, and metaphase, is an early response to anoxia. The sub-nuclear changes that occur in response to brief periods of anoxia include condensation of interphase chromatin, relocalization of prophase chromosomes to the nuclear periphery and spindle microtubule depolymerization in metaphase blastomes. We hypothesize that mechanisms that control anoxia-induced cell cycle arrest are activated in response to brief periods of anoxia. Therefore, one approach we are using to understand anoxia-induced cell cycle arrest includes cellular and genetic analysis of embryos exposed to brief periods of anoxia. For example, use of the centrosome localized protein gamma-tubulin is used as a marker to better characterize blastomeres arrested at interphase. Second, we investigated the localization pattern of the nuclear lamina protein, Ce-lamin, and found that brief periods of anoxia alters the localization. Third, analysis of kinetochore-localized proteins in embryos exposed to brief or prolonged periods of anoxia indicates that the structure is not identical in these embryos. Further investigation is needed to determine if these initial responses to anoxia are essential to anoxia-induced cell cycle arrest. Finally, previous genetic analysis showed that the spindle checkpoint protein SAN-1, which is the non-essential homologue to the yeast spindle checkpoint protein mad3p, is required for the arrest of metaphase blastomeres. To identify gene products, like SAN-1, that are required for anoxia survival we are using bioinformatics and RNAi screens. Briefly, we are using RNAi to identify genes that are synthetic lethal with a san-1 deletion allele or are required for anoxia survival. Preliminary evidence indicates that yeast homologue BUB-3 acts with SAN-1. Together, these cellular and genetic approaches are beginning to elucidate the complex process of anoxia-induced cell cycle arrest.