Sundaramurthy, Sumana et al. (2021) International Worm Meeting "Probing formin FHOD-1 contributions to body-wall muscle structure and function"

Sundaramurthy, Sumana, Lesanpezeshki, Leila, Pruyne, David, Littlefield, Ryan, Vanapalli, Siva, Yingling, Curtis

[International Worm Meeting, 2021]

In mammalian striated muscle, six actin-organizing formin proteins have been shown to influence sarcomere organization, but their precise molecular roles in this process remain obscure. In C. elegans, only the formin FHOD-1 works in a muscle cell-autonomous manner to influence sarcomere structure in body-wall muscle (BWM), providing a simpler system to determine the mechanism by which these highly conserved proteins influence the muscle cytoskeleton. Worms bearing a fhod-1 allele that is a putative null for effects on actin organization display modest defects in BWM strength, cell growth and sarcomere assembly, arguing against a primary role in assembling the actin-based sarcomeric thin filaments. However, fhod-1 muscle cells assemble fewer sarcomeres, and the muscle-specific myosin heavy chain MYO-3 is subject to elevated proteasome-dependent proteolysis. Moreover, the dense bodies that serve as thin filament-anchoring sites in BWM are malformed. Comparison of the distribution of immature and mature dense bodies in wild-type and fhod-1 BWM cells, and the spatial relationship of FHOD-1 to those structures, suggests FHOD-1 accumulates at specific foci in the BWM cell to assist the formation of new dense bodies there.

Matsunaga, Yohei et al. (2019) International Worm Meeting "UNC-89 Protein Kinase Activity is Required for Normal Mitochondrial Morphology and Function."

Vanapalli, Siva, Moody, Jasmine, Lesanpezeshki, Leila, Matsunaga, Yohei, Qadota, Hiroshi, Kwong, Jennifer, Benian, Guy

[International Worm Meeting, 2019]

Loss of function of unc-89 results in decreased locomotion and a disorganization of the myofilament lattice including lack of M-lines. Through multiple promoters and alternative splicing unc-89 encodes multiple, mostly giant polypeptides as large 900,000 Da, consisting primarily of up to 53 immunoglobulin domains. UNC-89 is localized to the M-line by antibody staining. The human homolog of UNC-89 is obscurin. Several UNC-89 isoforms contain two protein kinase domains, PK1 and PK2. PK1 is predicted to be a pseudokinase, and PK2 is predicted to be an active kinase. In protein kinases a lysine (K) in the small lobe coordinates ATP and helps transfer the g-phosphate. Mutation of the K to alanine (A) inactivates most protein kinases. Using CRISPR/Cas9, we made this K to A inactivating mutation in PK2. These animals, unc-89(sf22), have normal sarcomere organization as detected by immunostaining using antibodies to multiple sarcomeric proteins, and express full-length UNC-89 isoforms by western blot. unc-89(sf22) worms move about 10% faster in swimming and 40% faster in crawling. Using the NemaFlex method, unc-89(sf22) can produce the same amount of maximum force as wild type, whereas 3 other unc-89 mutants produce less force. Using several methods to assess mitochondrial morphology, unc-89(sf22) animals have mitochondria that are more fragmented and more aggregated than in wild type. We find similar abnormal mitochondrial morphology in unc-89(tm752) animals, which lack expression of all kinase-containing isoforms. In contrast, a more normal mitochondrial morphology is seen in two other unc-89 mutants: unc-89(e1460), which expresses kinase-containing isoforms; and in unc-89(su75) which lacks expression of all giant isoforms but expresses the small kinase-containing isoforms. Mitochondria are highly dynamic and are continuously changing their architecture from connected (elongated) to separated (fragmented) through fusion and fission events. As reported in the literature, we find that in drp-1 mutants, muscle mitochondria are more fused and branched. In an unc-89(sf22); drp-1 double mutant, the mitochondria appear like drp-1 itself. This suggests that drp-1 is downstream of UNC-89 PK2. In whole worms, total ATP was increased, and using the biosensor PercevalHR expressed by the myo-3 promoter, the ATP:ADP ratio was increased in body wall muscle in unc-89(sf22) compared to wild type. We hypothesize that the substrate of UNC-89 PK2 is a protein that shuttles between the sarcomere and mitchondria, or a protein that influences mitochondrial architecture.

Rahman, Mizanur et al. (2021) International Worm Meeting "A microfluidics-based chemical screening platform for lifespan and healthspan extension in Caenorhabditis elegans"

Gupta, Siddhartha, Rahman, Mizanur, Szewczyk, Nathaniel, Vanapalli, Siva, Anupom, Taslim, Driscoll, Monica, Edwards, Hunter

[International Worm Meeting, 2021]

C. elegans is a premier model organism used to identify pharmacological interventions that enhance lifespan and healthspan. Generally, animals are cultured on agar surface where drugs are either dissolved in agar or spread over solidified agar. The effects of the drug on lifespan are measured by the change of median or mean lifespan. The agar-based screening method has some severe limitations, including (i) limited throughput, (ii) progeny blocking methods can complicate outcomes, (iii) drug molecules may not be accessible to the animal due to transport limitation, (iv) bacteria can metabolize drug molecules, (vi) animals may avoid the bacterial lawn and therefore minimize drug exposure, and (vii) animals may be lost from the assay due to desiccation or burrowing. We present a microfluidic technology named an "Infinity screening system," where animals are cultured in a confined, micro-structured liquid environment with an integrated fluid processing and imaging hardware. Food and drugs are prepared in a batch (no FuDR) for the entire lifespan experiment for precise control over the consistency of daily dose of food and drug quality. Images of animals moving in the chip pillar environment are acquired each day and processed for live/dead count and locomotion-based cohorts with a software built in-house. This study used seven well-studied anti-aging drugs to benchmark the infinity platform for the drug effectiveness and consumption for whole-life analysis. We used three concentrations for each drug, concentration identified in plate-based assays, 1/10th, and 1/100th of the plate-based dosage. The entire experiments require approximately 3 man-hr/day and 30 days to complete one biological replicates. We found that infinity chip requires approximately 7.5 -750 times less drugs to achieve similar effects to that of agar plates. In this study, we found Thioflavin T as toxic in the microfluidic environment at the highest concentration. The actual dependency of drug type on the effective concentration is still unclear. In general, we observed significant increase in mean lifespan without appreciable changes in maximum lifespan at an effective concentration. Moreover, alpha-Ketoglutarate was able to rescue plate like lifespan enhancement in infinity chip contrasting the lifespan outcome from Lifespan Machine. Although, all the drugs extend lifespan, only a few of them helped maintain a larger fraction of animal highly active in the older age.

Rahman, Mizanur et al. (2017) International Worm Meeting "Microfluidics-based evaluation of neuromuscular healthspan in C.elegans."

Blawzdziewicz, Jerzy, Van-Bussel, Frank, Vanapalli, Siva, Szewczyk, Nathaniel, Edwards, Hunter, Rahman, Mizanur, Driscoll, Monica

[International Worm Meeting, 2017]

Aging studies in C.elegans based on functional metrics and molecular markers are essential to identify genetic and environmental determinants of healthspan. Although functional measures based on locomotion, pharyngeal pumping and oxidative stress have been used to interrogate healthspan of C.elegans, our understanding on maintenance and deterioration of the neuromuscular system with age is far from complete. Here, we propose muscle strength and stimulated reversals as two novel functional measures to report on the neuromuscular health of C.elegans. These measures are possible due to two technological advances made in our laboratory: NemaFlex - an image-based force-sensing micropillars that can quantify muscle strength and NemaLife - a microfluidics-based culturing apparatus that enables longitudinal aging experiments without the need for drug-induced blocking of progeny. We profiled strength across the lifespan of a wild-type population, and our results show that strength increases 5-fold from young adult to mid-life followed by a sharp decline - providing the first direct evidence of muscle strength loss due to age in C. elegans. With respect to stimulated reversals, we find that worms maintain a high reversal speed during their reproductive period and then declines at a rate that is nearly identical to the animal mortality rate. Interestingly, the decline in reversal speed precedes the onset of strength decline by about 3 days indicating that reversal speed is an early predictor of mortality. Moreover, since reversal speed is an indicator of motor activity, the early decline in reversal speed suggests that the presynaptic loss of function occurs early in life, consistent with prior electrophysiological studies. The stochastic nature of aging produces a wide distribution of lifespans in isogenic C. elegans populations. We observed that long lived worms generally maintain muscle strength long after median lifespan although their motor activity significantly declines. We observed four major classes of strength profiles among individual worms, each of which has a unique healthspan and motor activity pattern. For example, individuals starting with high reversal speed do not necessarily have long lifespan but all strong and long lived worms maintain very high motor activity during the late reproductive period. In summary, maintenance of strength together with motor activity is a possible route for increasing locomotory healthspan.

Lesanpezeshki, Leila et al. (2021) International Worm Meeting "Burrrowing chip: A microfluidic platform to visualize and quantitate the burrowing behavior of C. elegans"

Anupom, Taslim, Laranjeiro, Ricardo, Norouzi Darabad, Masoud, Lesanpezeshki, Leila, Driscoll, Monica, Blawzdziewicz, Jerzy, A. Vanapalli, Siva

[International Worm Meeting, 2021]

Caenorhabditis elegans has been widely used to study the genetics of behavior and neuromuscular function. Most of the behavioral studies in C. elegans are limited to two-dimensional (2D) assessment of animal locomotion on agar plates (crawling) or in liquid (swimming), and it was not until recently that C. elegans studies began to focus on 3D motion. 3D locomotion where animals burrow through a matrix is known to be physiologically distinct from 2D crawling and swimming (Bilbao et al. 2018) and 3D movement evokes different gene expression responses (Hewitt et al. 2020). We have previously deployed a Pluronic hydrogel environment to assess the burrowing performance of muscle mutants. The stiffness of the gel can be tuned to challenge the animals to burrow through a higher mechanical resistance environment to increase the assay sensitivity (Lesanpezeshki et al. 2019). However, due to the assay geometry being in a well-plate, the full burrowing trajectory was not observable, and the animals could be only visualized at the top surface, precluding evaluation of the burrowing behavior throughout the gel layers. To address the above issues, we have developed a microfluidic platform called a burrowing chip, that enables visualization and assessment of burrowing behavior in the same Pluronic hydrogel environment. The microfluidic format provides excellent control of the chemotactic gradient and animal/gel loading. The shallow depth of the channel enables use of consumer cameras for visualizing and recording burrowing behavior. We show that the device provides a 3D environment to assess the animal's burrowing performance and the animal distribution along the channel during chemotaxis. Next, we identify two burrowing classes of behaviorally slow and fast movement, based on the time it takes animals to reach the attractant. Slow burrowers weakly maintain their direction toward the attractant. We have discovered pause intervals as a novel phenotype for stimulated burrowing animals, as the burrowing animals exhibit long pauses. Finally, we show that the burrowing chip is capable of characterizing the muscle mutants based on their lower burrowing velocity and higher frequency of pauses compared to wild-type animals. We suggest that the burrowing chip can be used to provide insights into the genetic basis of burrowing behavior and to assess the neuromuscular health in C. elegans, paving the road to identify therapeutic targets for age-related diseases and decline.

Rahman, Mizanur et al. (2015) International Worm Meeting "NemaFlex: A microfluidic tool for phenotyping (neuro)muscular strength in C. elegans."

Driscoll, Monica, Blawzdziewicz, Jerzy, Szewczyk, Nathaniel, Vanapalli, Siva A., Hewitt, Jennifer E., Van Bussel, Frank, Rahman, Mizanur

[International Worm Meeting, 2015]

Maintenance of muscle strength is essential for an individual's health and well-being. In fact, loss of muscle strength is a prognostic indicator for a variety of disorders including sarcopenia, cancer, and neuromuscular diseases. Decline in muscle strength is also a significant issue for space explorers. Therefore, a major challenge for muscle health research is to understand the genetic mechanisms regulating strength. C. elegans is an established genetic model that has muscle with genetic and physiological similarities to human muscle. Aging C. elegans also show muscle deterioration with age much like humans. Thus, prospective life-long muscle health studies can be accomplished in the roundworm C. elegans.To make C. elegans a comprehensive genetic model for muscle health investigations, we engineered NemaFlex-a microfluidic device containing deformable pillars for quantifying muscle strength in C. elegans. Animals crawl through the pillar array, causing pillar displacements from which local force data can be extracted. Since the forces fluctuate depending on the animal's behavior (velocity, body conformation, and position with respect to pillar), reproducible quantitation of animal strength has thus far remained elusive. We establish NemaFlex for high throughput muscle strength assays by developing a robust experimental protocol and analysis workflow to sample the full range of forces and define a metric for maximum strength. We also configure NemaFlex for recording strength across virtually the entire lifespan of animals permitting sarcopenia investigations.We find that forced contractions, such as induced by an acetylcholine agonist, show the same maximum strength as the untreated animals suggesting NemaFlex quantitates maximum strength. We tested mutants with neuronal defects (unc-17 and unc-119) and impaired sarcomeres (unc-52 and unc-112) and observe changes that verify the neuromuscular origin of strength. We profile strength across the lifespan of a wild-type population, and our results show that strength increases 7-fold from the young adult followed by a sharp late-age decline-providing the first direct evidence of muscle strength loss due to aging, similar to sarcopenia in humans. In summary, NemaFlex is a powerful tool to conduct prospective life-long investigations of muscle strength in C. elegans and mutants.

Laranjeiro, Ricardo et al. (2015) International Worm Meeting "Benefits of physical exercise to healthy aging in C. elegans."

Chang, Christina, Burke, Daniel, Vanapalli, Siva A., Royal, Mary Anne, Rahman, Mizanur, Yu, Tianyi, Laranjeiro, Ricardo, Driscoll, Monica

[International Worm Meeting, 2015]

Physical exercise is the most efficient and accessible intervention that can promote healthy aging in humans. In fact, exercise has been reported to prevent, or improve consequences of, a wide range of conditions, such as diabetes, cancer, sarcopenia, cardiovascular disease, and neurodegenerative diseases. However, the molecular mechanisms by which exercise can confer systemic health benefits remain largely unknown.We developed novel exercise training protocols for both juvenile and adult C. elegans based on regular swimming regimens. Exercise-trained animals exhibit a swim performance improvement. Furthermore, we developed a microfluidic device with deformable micro-pillars that can directly measure the force an animal is able to exert ('Nemaflex'), and have made considerable progress on automation of data analysis. Our Nemaflex quantitation reveals that exercise-trained animals can exert significantly higher forces than their untrained counterparts. This direct readout of muscle performance shows that our protocols lead to relevant physiological changes in C. elegans. Animals that just sit in the pool such as those temporarily paralyzed by levamisole do not exhibit training adaptations in strength. Thus, animals must swim to get stronger and the simple exposure to liquid does not confer strength adaptations. We find that two central mediators of exercise signaling in mammals - AMP-activated protein kinase (AMPK) and p38 mitogen-activated protein kinase (p38 MAPK) - are also essential for the muscle exercise benefits observed in C. elegans (aak-2 and pmk-1, respectively). Moreover, mutation of a repressor of mitochondrial function (NCoR1/gei-8) in C. elegans leads to an increased muscle performance even without exercise training ('natural athlete'). These results show that the major exercise signaling mechanisms are conserved from nematodes to humans, making C. elegans an ideal model to dissect the genetic regulation of exercise systemic health benefits. Other than muscle improvement, our swimming protocols in C. elegans promote physiological changes in other tissues (e.g., neurons and pharynx) throughout the aging process. Careful characterization of these systemic benefits of exercise in C. elegans may provide novel insight into critical mechanisms for functional maintenance late into life.

Rahman, Mizanur et al. (2015) International Worm Meeting "A microfluidic platform for parallelized aging and healthspan investigations in C. elegans."

Gabrilska, Rebecca, Rumbaugh, Kendra P., Edwards, Hunter, Szewczyk, Nathaniel, Driscoll, Monica, Birze, Nikolajs, Vanapalli, Siva A., Rahman, Mizanur, Hewitt, Jennifer E., Blawzdziewicz, Jerzy

[International Worm Meeting, 2015]

Caenorhabditis elegans has emerged as a model organism for aging research. Large-scale screens for longevity genes in C. elegans use liquid culture combined with drug-induced blocking of progeny, introducing physiological stress on the animals. Small-scale pilot screens adopt agar-based methods, which necessitates the tedious task of repetitively picking and transferring animals. In both approaches, it is practically impossible to add reagents or remove reagents (e.g., food or drugs) at multiple time points during the lifespan precluding detailed aging investigations. Finally, most screens do not score animals for multidimensional readouts of healthspan.We report a simple and high-throughput microfluidic platform addressing the limitations of current methods. The platform is capable of measuring lifespan and healthspan of wild type and mutants in parallel without a requirement for drug blocking of progeny production. Numerous trials have shown that we can remove progeny efficiently while retaining the adult animals in the device. To reduce physiological stress on the animals, we have optimized device geometry and feeding protocols such that the gait, body size, and lifespan are consistent with aging assays on agar. In addition to standard healthspan readouts such as locomotory prowess and pharyngeal pumping, the device allows recording of novel measures such as animal muscle strength and agility.We test the multifunctional capabilities of the device by conducting a pilot screen that includes wild-type, daf-2, daf-16, age-1 and eat-2 animals. We find that the lifespan curves of the wild-type and genetic mutants are consistent with the literature reports. We also show that in a targeted RNAi screen, the lifespan curves obtained were consistent with those of the genetic mutants. Analysis of muscle strength, agility, and locomotory prowess as a function of lifespan in the long-lived mutants reveal new insights into sarcopenia. In summary, we anticipate that the simplicity of our method, combined with unprecedented capacity to temporally manipulate the environment of the animals and record multidimensional healthspan measures will enable highly parallelized cross-sectional and longitudinal aging experiments.

Kumar, Sandeep et al. (2021) International Worm Meeting "Enhanced functional restoration through axon regeneration by swimming exercise"

Ghosh-Roy, Anindya, Behera, Sibaram, Dey, Shirshendu, Kumar, Sandeep, Basu, Atrayee

[International Worm Meeting, 2021]

Axonal regeneration is a promising approach to overcome impaired functionality due to axonal injury. In mammals, central nervous system has poor regenerative capacity due to both extrinsic and intrinsic factors. The regenerative capacity also declines significantly with ageing. Therefore, functional axon regeneration in adulthood is challenging and needs more understanding. The pharmacological manipulations are not very successful for functional restoration whereas rehabilitation and physical activity shows improvement. As physical exercise has complex systemic effects, understanding the downstream effectors of physical exercise that is relevant for axon regeneration might be useful. Studying this using simple model organism has several advantages. Using posterior gentle touch circuit neuron (PLM) of Caenorhabditis elegans, we are studying effect of swimming exercise on functional restoration after laser assisted axotomy. We found that a single swimming exercise session of 90 minutes, which is an established paradigm of exercise in worm (Laranjeiro et al., 2017; Laranjeiro et al., 2019) improves functional recovery irrespective of age. However multiple swimming session is required for older worms (A5 stage). Anatomical correlation showed that swimming session improves regrowth initiation, regrowth length and functional connections. We found that the energy sensor kinase AMPK/AAK-2 plays an essential role mediating swimming benefits. Characterizing tissue specific requirement, we found that it has both cell autonomous (PLM neuron) and non-autonomous (muscle) requirement. Pharmacological activation of AMPK/AAK-2 showed enhanced functional restoration similar to swimming. We are studying the downstream molecules and their specific roles in various tissues for swimming mediated functional enhancement which will be helpful for better implementation of this approach. References Laranjeiro R, Harinath G, Burke D, Braeckman BP, Driscoll M (2017) Single swim sessions in C. elegans induce key features of mammalian exercise. BMC Biology 15. Laranjeiro R, Harinath G, Hewitt JE, Hartman JH, Royal MA, Meyer JN, Vanapalli SA, Driscoll M (2019) Swim exercise in Caenorhabditis elegans extends neuromuscular and gut healthspan, enhances learning ability, and protects against neurodegeneration. Proc Natl Acad Sci U S A 116:23829-23839.