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.

Lichty, James et al. (2021) International Worm Meeting "Elucidating Alzheimer's Disease related interactions between amyloid beta and the pathogen P. gingivalis in the model organism C. elegans"

Lichty, James, San Miguel, Adriana

[International Worm Meeting, 2021]

Alzheimer's Disease (AD) is a neurodegenerative disease which causes progressive loss of function in patients. AD affects about 5.8 million people in the US and is currently the sixth leading cause of death, with an estimated 122,000 deaths in 2018. AD is characterized by abnormal, widespread aggregation of the proteins amyloid beta (Abeta) and microtubule-associated protein tau (tau), neurodegeneration, immune system activation, inflammation, and oxidative stress. Abeta localization and aggregation have been linked to a number of AD traits but lack a clear causative role in the disease. Recently, infection by several pathogens have been identified as possible initiators for AD. One pathogen in particular, P. gingivalis (PG), has exhibited a number of features capable of inducing AD-like symptoms but its direct interactions with Abeta are poorly understood. We aim to identify and study these Abeta-PG interactions utilizing a novel Abeta expression system in the model organism C. elegans. This approach uses the C99 fragment of the amyloid precursor protein, the endogenous C. elegans gamma-gamma secretase, and a neuron-derived C. elegans signal peptide. Abeta is expressed as a part of C99, which is directed to the neuron membrane and cleaved, releasing Abeta extracellularly. This strain was compared to an intracellular Abeta expression strain to observe localization-dependent effects of Abeta on the organisms. We then infected the strains with PG, observing neuron health and overall health over the worms' lifespan. We seek to further characterize the detrimental effects, connecting them to AD traits seen in humans. Additionally, we will screen for genes relevant to the Abeta-PG interactions, such as C. elegans immunity pathways.

Huayta, Javier et al. (2021) International Worm Meeting "Quantitative Analysis of DAF-16 Lifelong Spatiotemporal Activity under Dietary Restriction as a Predictor of C. elegans lifespan"

San Miguel, Adriana, Huayta, Javier

[International Worm Meeting, 2021]

Several environmental factors affect longevity in C. elegans. Most of these function through the DAF-16 transcription factor, which regulates expression of genes involved in aging and stress response. Current methods to track DAF-16 activity have a destructive nature, and use strains with multiple copies of daf-16. Thus, we created a single copy GFT-tagged strain at the endogenous locus of daf-16. This strain, along with quantitative analysis of fluorescence imaging enables lifelong tracking of DAF-16 activity in response to environmental perturbations in vivo. Thus, the relationships between environmental perturbations, longitudinal gene activity, and lifespan can be explored. We aim to elucidate how the lifelong molecular activity of daf-16, driven by environmental interventions, determines lifespan in C. elegans. Using a custom image-processing algorithm, we tracked DAF-16 under various dietary restriction (DR) regimes. In particular, the image processing approach enabled evaluation of complex patterns of DAF-16 nuclear migration at the tissue and cellular levels. To characterize DAF-16 activity, we developed a strain that labels the endogenous DAF-16 protein with GFP using the CRISPR/Cas9 system. We observed migration of DAF-16 to cell nuclei of nematodes under DR conditions. This pattern increased the longer the animals were under this condition, reaching a peak after 12 hours of DR and decreasing thereafter. Moreover, under repeated and intermittent exposure of the same C. elegans population to DR, we identified a decreasing activity of DAF-16 in subsequent days. Additionally, we have observed migration of DAF-16 to nucleoli, a phenomena not described previously, and an increased response of DAF-16 in neurons when compared to other tissues. We aim to determine how tissue specific activity of DAF-16 contributes to longevity in this nematode. Lifespan measurements were performed for animals under the same DR regimes, enabling assessing how lifelong DAF-16 activity, , measured as cumulative intensity at the cellular level, correlates with lifespan. We characterized lifespan in C. elegans, while correlating this metric to quantifiable endogenous activity of DAF-16 under various DR regimes. Furthermore, our findings show that DAF-16 activity is tissue specific and its lifelong activity is correlated with longevity. These results will help understand the fundamental mechanisms by which this transcription factor regulates the aging process.

Saberi Bosari , Sahand et al. (2019) International Worm Meeting "Deep phenotyping degeneration of PVD neuron using Convolutional Neuronal Network machine learning technique."

San Miguel , Adriana, Saberi Bosari , Sahand, Flores , Kevin

[International Worm Meeting, 2019]

Aging is known to trigger decline in neuronal function and morphology. Age-induced changes in PVD neuron, a multidenderic sensory neuron responsible for mechanosensation and thermosensation have been extensively characterized in C. elegans. Some hallmarks of aging in PVD neurons include disorganized and dislocated dendrites, as well as neurite beading and protrusions. In a majority of these aging studies, the analysis has been limited to qualitative assessments of these phenotypes. However, considering the intricate nature of PVD neurons, qualitative evaluation of images is not sufficient to track complex age-induced changes. Previous work has identified neurite beading at old age, which is affected by expression of antimicrobial peptides. In this work, we sought to quantitatively analyze the intricate aging phenotypes of neurite beading in PVD using computer-assisted quantitative analysis. Due to the complexity of these phenotypes, traditional image processing techniques lack the power for accurate segmentation. To tackle this problem, we sought to integrate state of art machine learning techniques to conduct semantic segmentation using a Convolutional Neuronal Network algorithm. This approach enabled us to perform high-throughput, unbiased, and accurate image segmentation. To train this model, 19 ground truth masks with more than 2000 positive objects were created from images acquired throughout confocal fluorescence microscopy of a population's lifespan. The accuracy of the model (True positive/ (True Positive+ False Negative)) was ~ 91.5% based on validation using 12 test images. The output of the model was then used to extract and characterize metrics such as number of beads, mean bead surface area, bead localization, and mean distance between beads. In this work we were able to address the main issues of qualitative assessment in studying age-induced phenotypes in PVD neuron by integrating cutting edge semantic segmentation using a Convolutional Neuronal Network. By integrating this tool, we were be able to quantify the dynamic morphological changes in an aging population, which revealed interesting bead formation and disappearance patterns. Using this novel method for deep phenotyping complex aging morphologies, we will also be able to characterize the neurodegenerative effects induced by other factors such as acute cold shock, oxidative stress, dietary restriction, and genetic manipulation on PVD neurite beading process and compare the phenotypic profiles caused by each of these stressors.

Saberi Bosari, Sahand et al. (2017) International Worm Meeting "Microfluidic platforms for aging studies: longitudinal high-resolution phenotyping and post-reproductive screening."

Saberi Bosari, Sahand, San Miguel, Adriana, Midkiff, Daniel

[International Worm Meeting, 2017]

C. elegans has been fundamental to our current understanding of aging and the molecular mechanisms that play a role in determining healthspan and longevity. Aging studies, however, pose technical challenges that are typically solved by labor-intensive picking and transfer of populations, or by the use of reproduction-inhibiting agents. In this work, we are developing microfluidic platforms that allow performing aging studies in the absence of reproduction-inhibitng agents, and with far less manual involvement. Our platforms are focused on two main areas which have not been fully develepoed due to these limitations. First, we have developed a platform that allows for lifelong longitudinal, high-resolution (sub-cellular) phenotyping of C. elegans populations. This platform allows tracking subcellular processes in the same population for hundreds of animals, and we are currently aiming to quantify the morphological changes that neurons and synapses undergo during the aging process in a variety of conditions. In our second platform, our goal is to perform genetic screens in aged individuals (i.e., post-reproduction). We aim to identify mutations that result in "late-onset" phenotypes, which are only exhibited late in life. Our platform allows monitoring of individual mutagenized animals, cultured in individual microfluidic chambers, while isolating their progeny by interfacing with a deep-well system. Our platforms dedicated to challenging aging studies can result in the identification of genes and morphological changes associated with the aging process under natural conditions.

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.

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 A Hajeri et al. (2005) International Worm Meeting "Subcellular responses to anoxia-induced suspended animation in C. elegans"

Vinita A Hajeri, Pamela A Padilla, Jesus Trejo

[International Worm Meeting, 2005]

While several animal model systems are used to understand the physiological response organisms have to oxygen deprivation, the subcellular response to oxygen deprivation, in developing embryos, is less understood. We are using the C. elegans embryo to study the molecular response to oxygen deprivation. C. elegans exposed to anoxia (<.001 kPa; 0% O2) enter into a state of suspended animation in which developmental and cell cycle progression reversibly arrests. To further characterize the subcellular response to anoxia we analyzed nuclear structures in blastomeres of embryos exposed to brief as well as prolonged periods of anoxia. Embryos were exposed to various periods of anoxia (30 minutes, 6, 12, 24 and 72 hours) and nuclear components such as chromatin structure, nuclear pore proteins, histone modifications, microtubules and the kinetochore were analyzed. Embryos exposed to at least 30 minutes of anoxia contain blastomeres that arrest at interphase, prophase, prometaphase and metaphase but not anaphase. Prophase and interphase blastomeres, of embryos exposed to 30 minutes of anoxia, contain chromatin in close proximity to the nuclear envelope, suggesting that an early subcellular response to anoxia is the nuclear reorganization of chromatin structure. The spindle checkpoint protein SAN-1, which is required for anoxia survival, localizes to the kinetochore in wild type embryos exposed to 30 minutes of anoxia. The detection of SAN-1 at the kinetochore is greatly reduced in metaphase blastomeres of embryos exposed to 24 or 72 hours of anoxia. The san-1 (RNAi) embryos exposed to 30 minutes of anoxia contain blastomeres with anaphase bridges and abnormal nuclei. This data supports the idea that the spindle checkpoint activity of SAN-1 is at the kinetochore, SAN-1 is required for the early response to anoxia, and an oxygen-sensing pathway regulates the spindle checkpoint components. In other systems the spindle checkpoint components monitor kinetochore-microtubule attachment by delaying the metaphase to anaphase transition until all chromatids are attached to microtubules. We determined that detectable depolymerization of the spindle microtubules occurs in blastomeres of embryos exposed to 6 hours of anoxia at 20<sym05>C. Spindle microtubule depolymerization was not detected in embryos exposed to short periods (30 minutes) of anoxia however, we cannot rule out the possibility that only a few, undetected, microtubules depolymerize. Identifying the early and late responses to anoxia will lead to the understanding and differentiation of mechanisms required to initiate and maintain anoxia induced suspended animation.