Mondal, Sudip et al. (2011) International Worm Meeting "A simple microfluidic device for long-term in vivo imaging of mitochondrial dynamics in C. elegans."

Mondal, Sudip, Koushika, Sandhya

[International Worm Meeting, 2011]

To track development and stability of mitochondrial distributions in identified animals, we developed a growth cum imaging microfluidic device for C. elegans. In wild type animals, mitochondria numbers co-relate linearly with neuronal process length of the touch receptor neurons. In short time-scales we observe that all large mitochondria are stationary and only about 15-20% of mitochondria, all small in size, move. Distribution of mitochondria along the neuronal process is regulated such that few mitochondria are placed less then 3 microns from each other. We wished to investigate how these large stationary mitochondria arise by tracking individual organisms throughout their development. The growth and imaging device utilizes a deformable PDMS membrane to immobilize C. elegans for high magnification mitochondrial matrix::GFP fluorescence tracking, with wide enough channel geometry to track developmental parameters such as body length and body diameter inside the device at low magnification. The worm is fed using a simple hydrostatically driven E. coli supply in liquid culture media using 200 mL pipette tips. Preliminary observation suggests that C. elegans grown in the microfluidic device shows similar developmental and behavioural parameters as compared to animals grown on NGM plates. We tracked mitochondrial positions over 10 hours in animals grown in a microfluidic device. This tracking shows greater mitochondrial dynamics in the first 100 mm of the neuronal process as compared to the middle of the neuronal process. Currently we are optimizing imaging conditions to avoid fluorescence bleaching to be able to track individual animals for all developmental stages.

Sure, Guruprasad et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Mitochondria distributions in C elegans are regulated by microtubules but not by molecular motors"

Sure, Guruprasad, Koushika, Sandhya, Awasthi, Anjali, Romero, Eva M, Mondal, Sudip

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

Mitochondria are important for neuronal function partly due to their ability to generate ATP and buffer calcium at synapses. However, the volume of mitochondria at synaptic regions is smaller than the volume along the axon, highlighting the importance of axonal mitochondria. We studied the distribution of mitochondria in touch receptor neurons of C. elegans using a transgenic strain that marks the matrix of all mitochondria with GFP. Using this strain we observed a linear correlation of mitochondrial number with the length of the neuronal process and established t hat distributions of mitochondria along the neuronal process are not random. The distributions are regulated such that two neighboring mitochondria maintain a certain minimum distance between them. We show that number of mitochondria along the axon is regulated by the activity of the Kinesin-I motor. The distributions of these fewer mitochondria in Kinesin-I mutants remain close to those observed in wild type neurons. By comparison, in microtubule mutants the number of mitochondria in the axon increases and their distributions become more random. Analysis of electron micrographs of wild type touch receptor neurons reveals the presence of filamentous structures that connect mitochondria to both microtubules as well as to the plasma membrane. These filamentous structures may underlie the regulated distributions observed in neurons. We also observe that mutants that have more random distributions of mitochondria in axons have quicker desensitization to gentle touch stimulation , suggesting a role for axonal mitochondria in neuronal function

Lim, Yunki et al. (2021) International Worm Meeting "High-throughput phenotypic screening to identify neurotoxic chemicals causing neuro-degeneration"

Moore, Jacob, Hegarty, Evan, Kagias, Konstantinos, Mondal, Sudip, Lim, Yunki, Laing, Adam, Ben-Yakar, Adela

[International Worm Meeting, 2021]

Direct exposure of humans to different chemicals becomes more prevalent as society is progressing further into industrialization. In addition, an increasing number of medical drugs are being developed at different non-clinical and clinical stages aiming to treat a variety of conditions. In order to protect the public from potential deleterious effects of these chemicals, toxicology analysis is necessary to ensure early identification of toxic effects. The most common type of chemically-induced toxicity is neurotoxicity. Therefore, screening for chemical compounds, which can cause specific neuronal damage, can help identify toxic chemicals and potentially help elucidate specific neurodegeneration mechanisms, which can lead to the development of novel targeted therapeutic approaches. Current neurotoxicity assays rely mainly on mammalian models' mortality tests and are associated with high screening costs and long experimental times. To overcome these limitations, we developed a high throughput in vivo neurotoxicity assay using C. elegans. We screened animals treated with a number of well-characterized reference chemicals using Newormics' proprietary microfluidic device (vivoChip), which enabled us to perform high-resolution imaging and multi-parametric structural analysis of GFP-labeled neurons in a high-throughput manner. We characterized the chemical-induced neurotoxicity in the dopaminergic, cholinergic, GABAergic, and serotonergic neurons generating a complete set of imaging data for each reference chemical. Semi-automatic analysis of this dataset identified the cellular and sub-cellular neuronal defects and created neuron-specific degeneration metrics for the reference chemicals. We hope to gain valuable insights into potential mechanisms of action for these neurotoxic compounds while at the same time progressing towards a more complete screen through an increasing number of chemicals using our high-content and high-throughput system.