@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 {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} }