Mohan, Swetha et al. (2011) International Worm Meeting "C.elegans rootletin homolog, che-10, is required for intraflagellar transport and cilia maintenance."

Mohan, Swetha, Leroux, Michel

[International Worm Meeting, 2011]

Non-motile or primary cilia are specialized microtubule-based organelles that protrude from many cell types in metazoans. They are adapted to serve many sensory functions, including transducing mechanical, chemical and visual stimuli. The formation and maintenance of all cilia depends on a process termed intraflagellar transport (IFT), which mobilizes ciliary precursor proteins from the base of the cilia (basal body) to the growing end of the ciliary compartment. IFT involves two anterograde molecular motors, heterotrimeric Kinesin-II (KIF3) and homodimeric Kinesin-II (KIF17). The retrograde motor, cytoplasmic dynein 1b/2, is loaded on to the axoneme during anterograde transport and is activated upon arrival at the tip of the cilium, where it is required for retrograde transport of all IFT machinery. Primary cilia are mainly composed of the ciliary axoneme, the basal body from which the axoneme nucleates, the ciliary membrane and a cytoskeleton-like structure called ciliary (or striated) rootlets. Rootlets are associated with the basal body and extend proximally towards the cell nucleus. Striated rootlets are evolutionarily-conserved organelles whose components and function remain unclear. Rootletin is a core structural component of the striated rootlets; in the absence of the protein, the rootlets do not form. Although rootlets are known to be required for the stability of some cilia (e.g. photoreceptor cilia), their mechanism of function remains unknown. Here, we demonstrate that the C. elegans Rootletin homolog, che-10, is required for the maintenance of cilia. In the absence of CHE-10, only some cilia are present post-embryogenesis but these degenerate over time. che-10 mutants have varying lengths of cilia, wherein IFT is absent from shorter (<5 microns) cilia. Furthermore, velocity analyses of IFT proteins suggests that IFT is deregulated in che-10 mutants. The strict regulation of IFT velocities may be compromised due to defects in motor coordination. We hypothesize that CHE-10 (Rootletin) regulates IFT by assembling the IFT components at the base of the cilium, such that disruption of the cytoskeletal protein leads to improper assembly and eventual degeneration of cilia.

Swetha Mohan et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Identifying the players involved in the transport of mitochondria in mechanosensory neurons"

Shaili Johri, Guruprasada Sure, Swetha Mohan, Sandhya Koushika, Michael Nonet

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

Neurons can be long cells that have high-energy requirements for synaptic transmission, which often occurs at sites that are distant from the cell body. Organelle transport is therefore critical in the organization, developmental fate and function of these cells. Mitochondria are involved in aerobic ATP production, calcium homeostasis and apoptosis. Mitochondria, unlike other organelles are transported bi-directionally and remain stationary 75% of the time. In neurodegenerative diseases such as Alzheimer"s and Parkinson"s, abnormal mitochondrial distribution has been observed. We are trying to identify the molecular players involved in the transport of mitochondria using classical genetic approaches. We have used a mitochondrial matrix targeted GFP expressed in the six mechanosensory neurons. Using this transgenic line we have screened 2500 genomes and isolated 15 mutants. These fall into different classes, such as 100% increase in number of mitochondria in axons, 50-75% fewer mitochondria in axons, abnormal accumulation of mitochondria in the minor process, large spherical mitochondria at the region of the axon hillock and mutants that lack mitochondria in synaptic regions. We are also in the process of assessing the role of genes known in other systems to be involved in regulating mitochondrial morphology and transport, such as rab-7 and unc-116

Jensen, Victor L. et al. (2017) International Worm Meeting "Role of metazoan dynein in transition zone formation, ciliary maintenance, and processive intraflagellar transport."

Mohan, Swetha, Timbers, Tiffany A., Leroux, Michel R., Cai, Jerry, Jensen, Victor L.

[International Worm Meeting, 2017]

Cilia are ubiquitous microtubule-based organelles found at surface of metazoan cells. Non-motile (sensory/primary) cilia act to receive signals from the environment, and motile cilia either move cells or the surrounding fluid. Cilia are built and maintained using a cargo-trafficking intraflagellar transport (IFT) machinery powered by kinesin (anterograde) and dynein (retrograde) molecular motors. How IFT-dynein contributes mechanistically to ciliary processes in metazoans remains poorly studied, in part because null mutants exhibit a severe terminal phenotype with IFT protein accumulations in the bulbous tips of highly truncated cilia. We carried out a screen for retrograde IFT transport defects in C. elegans, and identified the first temperature-sensitive IFT mutant in a metazoan. Using this strain, we show that the IFT-dynein (CHE-3) heavy chain is essential for cilium maintenance and correct formation of the transition zone (TZ) ciliary gate. Cilia resorb upon shift to restrictive temperature (inactive IFT-dynein), and are restored upon return to permissive temperature (active IFT-dynein). Importantly, this resorption and regrowth is observed for primary cilia in terminally differentiated, sensory neurons. We next investigated how IFT-dynein enables processive IFT, in contrast to the bidirectional tug-of-war mechanism of transport used by kinesin and dynein elsewhere in the cell. We find that the IFT-dynein heavy, intermediate and two light chains are trafficked by the IFT-subcomplex B, unlike the light intermediate chain, which is transported independent of the canonical IFT complex. This differential transport provides an effective mechanism by which the retrograde transport machinery is maintained in an inactive state during anterograde transport. Together, our findings reveal that IFT-dynein is required for ciliary maintenance and TZ formation, and regulates the processivity of anterograde IFT by spatial separation and thus inactivation of its components.

Reddy Sure, Guruprasada et al. (2009) International Worm Meeting "The complex roles of the JNK signaling scaffold molecule, UNC-16, in regulating mitochondrial distributions in neurons."

Koushika, Sandhya, Romero, Eva, Mohan, Swetha, Reddy Sure, Guruprasada, Awasthi, Anjali, DeviReddy, Swathi

[International Worm Meeting, 2009]

To study the regulation of mitochondrial transport, a transgenic strain, jsIs609 was built in which GFP is targeted to the mitochondrial matrix in all six mechanosensory neurons. Co-localization with mitotracker labeling in mechanosensory neurons in cultures suggest that jsIs609 labels nearly all mitochondria and at least partially co-localize with a synaptic vesicle marker at mechanosensory neuron synapses. Mitochondria number increases with the age/length of the animal/axon. In axons most of the mitochondria are stationary while moving mitochondria exhibit saltatory movements. Mitochondrial flux varies with developmental stages however size and density of mitochondria remain the same through out development. Mutants in unc-116 and km-11 (both together form the Kinesin-I motor) decrease the number of mitochondria in axons while a mutant in dhc-1 does not. Genes such as Milton that link the Kinesin-I motor to mitochondria are absent from the C. elegans genome, suggesting that other proteins may play roles in regulating mitochondrial axonal transport. To understand the role of known interactors of the Kinesin-I complex in mitochondrial transport, genetic analyses with mutants in these components was carried out. Kinesin-I forms a complex with regulatory proteins such as UNC-76, UNC-14, UNC-51, UNC-69 and adaptor proteins like JIP-3/UNC-16 to facilitate vesicle transport. unc-16 mutants show increased number of axonal mitochondria unlike that observed in Kinesin-I motor complex mutants. unc-116 unc-16 and unc-16; klc-2 double mutants show improved anterograde transport of mitochondria compared to unc-116 and klc-2 animals alone. Further, in unc-16 mutants the population of smaller mitochondria in axons increases. Since certain UNC-16 effects on mitochondria are independent on the Kinesin-I motor, we hypothesize that UNC-16 may act in the following ways: (1) recruiting or activating an alternate anterograde mitochondrial motor (2) regulating the size and numbers of axonal mitochondria through fission (3) by decreasing the recruitment of dynein thereby reducing retrograde transport. These possibilities are currently being tested.

Guruprasada R. Sure et al. (2006) East Asia C. elegans Meeting "Identifying the players involved in the transport of mitochondria in Caenorhabditis elegans neurons"

Swetha Mohan, Guruprasada R. Sure, Shaili N Johri, Sandhya P Koushika

[East Asia C. elegans Meeting, 2006]

The growth, maintenance, and survival of neurons rely critically on organelle transport between the cell body and the synapse. Mitochondria are involved in the ATP production, calcium homeostasis and synaptic vesicles recycling. Mitochondria, unlike other organelles are transported bi-directionally and remain stationary 75% of the time. In neurodegenerative diseases such as Alzheimer""s and Parkinson""s, abnormal mitochondrial distribution has been observed. We are trying to identify the molecular players involved in the transport of mitochondria using classical genetic approaches. To address this question we take a classical genetic approach using a transgenic strain jsIs609 in which GFP is expressed in the mechanosensory neurons and targeted to the mitochondrial matrix. We have performed EMS mutagenesis and screened around 3000 genomes and isolated 17 mutants. These fall into different classes, such as 100% increase in number of mitochondria in axons, 50-75% fewer mitochondria in the axons, abnormal accumulation of mitochondria in minor neurites and large spherical mitochondria in the cell bodies of the neurons. We are currently performing complementation analyses of these mutants. We have also analyzed the distribution of mitochondria in mutants with known roles for the transport of organelles and in the dynamics of mitochondria, such as anc-1, unc-16, unc-116, rab-7 and unc-73. In unc-116 Kinesin-I mutants fewer mitochondria are observed in the axons whereas unc-16 ortholog of Sunday driver have more number of mitochondria in the axons. To analyze the mitochondrial behavior/dynamics with respect to metabolism we have performed starvation analyses. We find that the cell bodies of the neurons are fragmented during starvation but the axons are spared and are normal. We are currently imaging mitochondrial motility and dynamics across the developmental stages. # Guruprasada Reddy Sure and Swetha Mohan contributed equally to this work.

Timbers, Tiffany et al. (2015) International Worm Meeting "Accelerating gene discovery by phenotyping the deep-sequenced Million Mutation Project strains and using genome-wide statistical analysis approaches."

Edgley, Mark, Flibotte, Stephane, Leroux, Michel, Lee, Katherine, Moerman, Donald, Timbers, Tiffany, Mohan, Swetha, Garland, Stephanie

[International Worm Meeting, 2015]

Forward genetic screens represent powerful, unbiased approaches to uncover novel components in any biological process. Such screens suffer from a major bottleneck, however, namely the cloning of corresponding genes causing the phenotypic variation. Reverse genetic screens have been employed as a way to circumvent this issue, but can often be limited in scope. We have developed a new methodological approach to accelerate gene discovery that addresses both of these shortcomings and is generally applicable for many biological processes, using C. elegans as an animal model system. We have used a deep-sequenced multi-mutation library, the Million Mutation Project (Thompson et al., 2013), together with the Sequence Kernel Association Test (SKAT; Wu et al., 2011) to rapidly screen for and identify genes associated with a phenotype of interest, namely defects in dye-filling of ciliated sensory neurons. Such anomalies in dye-filling are often associated with the disruption of cilia, organelles which in humans are implicated in sensory physiology (including vision, smell and hearing), development and disease. Beyond identifying several well characterised dye-filling genes, our approach uncovered 18 genes not previously linked to ciliated sensory neuron development or function. From these putative novel dye-filling genes, we confirmed and characterised the involvement of F01D4.9/BGNT-1 in ciliated sensory neuron development. C. elegans BGNT-1 is homologous to the human glycosyltransferase, B3GNT1/B4GAT1, which is associated with the human disease, Walker-Warburg syndrome, a suspected but unconfirmed ciliopathy.We have also begun to explore new methodologies for integrating the genomic data from the Million Mutation Projects strains with multiple phenoytpes collected using the Multi-worm tracker (Swierczek et al., 2011). To do this, sensory-related phenotypes (morphological and behavioural features) are combined to create a phenotypic 'fingerprint' for each strain. Next, we use multi-variate statistical genetic methods to test for genome wide-associations (GWAS) between behavioural and genetic variants to link genes to behaviour. We anticipate that these innovative approaches to integrate phenotype and genomic data will successfully uncover many new components and functional mechanisms of sensory neuron function. We also anticipate that these approaches will be of use to those studying other biological processes in C. elegans that are easily amendable to screening using the Million Mutation Project strains.