@article {109935, title = {A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans.}, journal = {J Vis Exp}, year = {2022}, month = {2022 Apr 11}, abstract = {

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding these biological processes often requires long-term and repeated imaging of the same animal. Long recovery times associated with conventional immobilization methods done on agar pads have detrimental effects on animal health making it inappropriate to repeatedly image the same animal over long periods of time. This paper describes a microfluidic chip design, fabrication method, on-chip C. elegans culturing protocol, and three examples of long-term imaging to study developmental processes in individual animals. The chip, fabricated with polydimethylsiloxane and bonded on a cover glass, immobilizes animals on a glass substrate using an elastomeric membrane that is deflected using nitrogen gas. Complete immobilization of C. elegans enables robust time-lapse imaging of cellular and sub-cellular events in an anesthetic-free manner. A channel geometry with a large cross-section allows the animal to move freely within two partially sealed isolation membranes permitting growth in the channel with a continuous food supply. Using this simple chip, imaging of developmental phenomena such as neuronal process growth, vulval development, and dendritic arborization in the PVD sensory neurons, as the animal grows inside the channel, can be performed. The long-term growth and imaging chip operates with a single pressure line, no external valves, inexpensive fluidic consumables, and utilizes standard worm handling protocols that can easily be adapted by other laboratories using C. elegans.

}, keywords = {Animals, Biological Phenomena, Caenorhabditis elegans, Diagnostic Imaging, Microfluidics}, issn = {1940-087X}, doi = {10.3791/63136}, author = {Dubey, Jyoti and Mondal, Sudip and Koushika, Sandhya P} } @article {94156, title = {Tracking mitochondrial density and positioning along a growing neuronal process in individual neuron using a long-term growth and imaging microfluidic device.}, journal = {eNeuro}, year = {2021}, month = {2021 May 21}, abstract = {

The long cellular architecture of neurons requires regulation in part through transport and anchoring events to distribute intracellular organelles. During development, cellular and sub-cellular events such as organelle additions and their recruitment at specific sites on the growing axons occur over different time scales and often show inter-animal variability thus making it difficult to identify specific phenomena in population averages. To measure the variability in sub-cellular events such as organelle positions, we developed a microfluidic device to feed and immobilize for high-resolution imaging over several days. The microfluidic device enabled long-term imaging of individual animals and allowed us to investigate organelle density using mitochondria as a testbed in a growing neuronal process Sub-cellular imaging of an individual neuron in multiple animals, over 36 hours in our microfluidic device, shows the addition of new mitochondria along the neuronal process and an increase in the accumulation of synaptic vesicles at synapses. Long-term imaging of individual touch receptor neurons shows that the addition of new mitochondria takes place along the entire neuronal process length at a rate of \~{}0.6 mitochondria per hour. The threshold for the addition of a new mitochondrion occurs when the average separation between the two pre-existing mitochondria exceeds 24 {\textmu}m. Our assay provides a new opportunity to move beyond simple observations obtained from assays to allow the discovery of genes that regulate positioning of mitochondria in neurons.Axonal transport of mitochondria is required for the normal function and health of a developing animal with continuously growing axonal processes. Existing technologies are unable to monitor the addition of a new mitochondrion in a growing axon , as it requires continuous or intermittent tracking of the same individual neuron over several hours to days. We have developed a microfluidic device that enables long-term high-resolution imaging of individual in an anesthetic-free setting. Using this device, we observe that the addition of a new mitochondrion can occur anywhere along the entire neuronal process, likely mediated by actively transported mitochondria, and at docking sites that occur with high probability when the separation between adjacent mitochondria crosses 24 {\textmu}m threshold.

}, issn = {2373-2822}, doi = {10.1523/ENEURO.0360-20.2021}, author = {Mondal, Sudip and Dubey, Jyoti and Awasthi, Anjali and Sure, Guruprasad Reddy and Vasudevan, Amruta and Koushika, Sandhya P} } @article {102260, title = {UNC-16 alters DLK-1 localization and negatively regulates actin and microtubule dynamics in Caenorhabditis elegans regenerating neurons}, journal = { Genetics}, volume = {219}, year = {2021}, month = {11/2021}, pages = {iyab139}, abstract = {

Neuronal regeneration after injury depends on the intrinsic growth potential of neurons. Our study shows that UNC-16, a Caenorhabditis elegans JIP3 homolog, inhibits axonal regeneration by regulating initiation and rate of regrowth. This occurs through the inhibition of the regeneration-promoting activity of the long isoform of DLK-1 and independently of the inhibitory short isoform of DLK-1. We show that UNC-16 promotes DLK-1 punctate localization in a concentration-dependent manner limiting the availability of the long isoform of DLK-1 at the cut site, minutes after injury. UNC-16 negatively regulates actin dynamics through DLK-1 and microtubule dynamics partially via DLK-1. We show that post-injury cytoskeletal dynamics in unc-16 mutants are also partially dependent on CEBP-1. The faster regeneration seen in unc-16 mutants does not lead to functional recovery. Our data suggest that the inhibitory control by UNC-16 and the short isoform of DLK-1 balances the intrinsic growth-promoting function of the long isoform of DLK-1 in vivo. We propose a model where UNC-16{\textquoteright}s inhibitory role in regeneration occurs through both a tight temporal and spatial control of DLK-1 and cytoskeletal dynamics.

}, doi = {10.1093/genetics/iyab139 }, author = {Kulkarni, SS and Sabharwal, V. and Sheoran, S. and Basu, A. and Matsumoto, K. and Hisamoto, N. and Ghosh-Roy, A. and Koushika, Sandhya P} } @article {30721, title = {Cargo crowding at actin-rich regions along axons causes local traffic jams.}, journal = {Traffic}, volume = {19}, year = {2018}, month = {2018 Mar}, pages = {166-181}, abstract = {

Steady axonal cargo flow is central to the functioning of healthy neurons. However, a substantial fraction of cargo in axons remains stationary up to several minutes. We examine the transport of precursors of synaptic vesicles (pre-SVs), endosomes and mitochondria in Caenorhabditis elegans touch receptor neurons, showing that stationary cargo are predominantly present at actin-rich regions along the neuronal process. Stationary vesicles at actin-rich regions increase the propensity of moving vesicles to stall at the same location, resulting in traffic jams arising from physical crowding. Such local traffic jams at actin-rich regions are likely to be a general feature of axonal transport since they also occur in Drosophila neurons. Repeated touch stimulation of C. elegans reduces the density of stationary pre-SVs, indicating that these traffic jams can act as both sources and sinks of vesicles. This suggests that vesicles trapped in actin-rich regions are functional reservoirs that may contribute to maintaining robust cargo flow in the neuron. A video abstract of this article can be found at: Video S1; Video S2.

}, issn = {1600-0854}, doi = {10.1111/tra.12544}, author = {Sood, Parul and Murthy, Kausalya and Kumar, Vinod and Nonet, Michael L and Menon, Gautam I and Koushika, Sandhya P} } @article {45446, title = {UNC-16/JIP3 and UNC-76/FEZ1 limit the density of mitochondria in C. elegans neurons by maintaining the balance of anterograde and retrograde mitochondrial transport.}, journal = {Sci Rep}, volume = {8}, year = {2018}, month = {2018 Jun 12}, pages = {8938}, abstract = {

We investigate the role of axonal transport in regulating neuronal mitochondrial density. We show that the density of mitochondria in the touch receptor neuron (TRN) of adult Caenorhabditis elegans is constant. Mitochondrial density and transport are controlled both by the Kinesin heavy chain and the Dynein-Dynactin complex. However, unlike in other models, the presence of mitochondria in C. elegans TRNs depends on a Kinesin light chain as well. Mutants in the three C. elegans miro genes do not alter mitochondrial density in the TRNs. Mutants in the Kinesin-1 associated proteins, UNC-16/JIP3 and UNC-76/FEZ1, show increased mitochondrial density and also have elevated levels of both the Kinesin Heavy and Light Chains in neurons. Genetic analyses suggest that, the increased mitochondrial density at the distal end of the neuronal process in unc-16 and unc-76 depends partly on Dynein. We observe a net anterograde bias in the ratio of anterograde to retrograde mitochondrial flux in the neuronal processes of unc-16 and unc-76, likely due to both increased Kinesin-1 and decreased Dynein in the neuronal processes. Our study shows that UNC-16 and UNC-76 indirectly limit mitochondrial density in the neuronal process by maintaining a balance in anterograde and retrograde mitochondrial axonal transport.

}, issn = {2045-2322}, doi = {10.1038/s41598-018-27211-9}, author = {Sure, Guruprasada Reddy and Chatterjee, Anusheela and Mishra, Nikhil and Sabharwal, Vidur and Devireddy, Swathi and Awasthi, Anjali and Mohan, Swetha and Koushika, Sandhya P} } @article {26282, title = {UNC-16/JIP3 regulates early events in synaptic vesicle protein trafficking via LRK-1/LRRK2 and AP complexes.}, journal = {PLoS Genet}, volume = {13}, year = {2017}, month = {2017 Nov 16}, pages = {e1007100}, abstract = {

JIP3/UNC-16/dSYD is a MAPK-scaffolding protein with roles in protein trafficking. We show that it is present on the Golgi and is necessary for the polarized distribution of synaptic vesicle proteins (SVPs) and dendritic proteins in neurons. UNC-16 excludes Golgi enzymes from SVP transport carriers and facilitates inclusion of specific SVPs into the same transport carrier. The SVP trafficking roles of UNC-16 are mediated through LRK-1, whose localization to the Golgi is reduced in unc-16 animals. UNC-16, through LRK-1, also enables Golgi-localization of the μ-subunit of the AP-1 complex. AP1 regulates the size but not the composition of SVP transport carriers. Additionally, UNC-16 and LRK-1 through the AP-3 complex regulates the composition but not the size of the SVP transport carrier. These early biogenesis steps are essential for dependence on the synaptic vesicle motor, UNC-104 for axonal transport. Our results show that UNC-16 and its downstream effectors, LRK-1 and the AP complexes function at the Golgi and/or post-Golgi compartments to control early steps of SV biogenesis. The UNC-16 dependent steps of exclusion, inclusion and motor recruitment are critical for polarized distribution of neuronal cargo.

}, issn = {1553-7404}, doi = {10.1371/journal.pgen.1007100}, author = {Choudhary, Bikash and Kamak, Madhushree and Ratnakaran, Neena and Kumar, Jitendra and Awasthi, Anjali and Li, Chun and Nguyen, Ken and Matsumoto, Kunihiro and Hisamoto, Naoki and Koushika, Sandhya P} }