Pause, Arnim et al. (2015) International Worm Meeting "Loss of Folliculin confers osmotic stress resistance via AMPK-dependent remodeling of carbohydrate stores in C. elegans."

Coull, Barry, Flamand, Mathieu, Possik, Elite, Vijayaraghavan, Tarika, Ajisebutu, Andrew, Manteghi, Sanaz, Hall, David, Pause, Arnim, van Stensel, Maurice

[International Worm Meeting, 2015]

Mechanisms of adaptation to environmental changes in osmolarity are fundamental for cellular life. When exposed to hyperosmotic stress, cells and organisms utilize conserved strategies to prevent water loss and maintain cellular integrity and viability. Here we identify a novel AMPK-dependent pathway of resistance to hypertonic stress mediated by the AMPK regulator flcn-1 in C. elegans. FLCN-1 is the worm homologue of the tumor suppressor Folliculin (FLCN), responsible for the Birt-Hogg-Dube hereditary cancer disorder. We show that loss of flcn-1 increases glycogen stores in an AMPK dependent manner and that the glycogen reserves are rapidly degraded upon hypertonic stress, leading to a remarkable accumulation of the organic osmolyte glycerol, promoting resistance to hyperosmotic stress. Importantly, loss of AMPK, glycogen synthase or glycogen phospharylase, which are critical enzymes in glycogen metabolism, strongly suppressed the increased osmotic stress resistance in flcn mutant animals. We further demonstrate that glycerol 3-phosphate dehydrogenase-1 is strongly induced in flcn-1 animals upon hyperosmotic stress and that simultaneous loss of gpdh-1 and gpdh-2 abolished the flcn-1/AMPK-mediated osmotic stress phenotype. Importantly, we show that glycogen accumulates in kidneys from mice lacking FLCN and in kidneys and renal tumors from a BHD patient. Since BHD is a renal hyperproliferation disorder, a mechanism of osmotic stress resistance in kidney hyperosmotic environments might explain tumorigenesis in BHD patients. Overall, our data indicate that FLCN is an evolutionary conserved regulator of glycogen metabolism, that might be acting as a tumor suppressor via AMPK-dependent accumulation of glycogen and organic osmolytes, resulting in an advantageous increase in proliferation and survival in hyperosmotic environments. .

Gramlich MW et al. (2021) Sci Rep "Distinguishing synaptic vesicle precursor navigation of microtubule ends with a single rate constant model."

Parkes M, Balseiro-Gomez S, Yogev S, Gramlich MW, Tabei SMA

[Sci Rep, 2021]

Axonal motor driven cargo utilizes the microtubule cytoskeleton in order to direct cargo, such as synaptic vesicle precursors (SVP), to where they are needed. This transport requires vesicles to travel up to microns in distance. It has recently been observed that finite microtubule lengths can act as roadblocks inhibiting SVP and increasing the time required for transport. SVPs reach the end of a microtubule and pause until they can navigate to a neighboring microtubule in order to continue transport. The mechanism(s) by which axonal SVPs navigate the end of a microtubule in order to continue mobility is unknown. In this manuscript we model experimentally observed vesicle pausing at microtubule ends in C. elegans. We show that a single rate-constant model reproduces the time SVPs pause at MT-ends. This model is based on the time an SVP must detach from its current microtubule and re-attach to a neighboring microtubule. We show that vesicle pause times are different for anterograde and retrograde motion, suggesting that vesicles utilize different proteins at plus and minus end sites. Last, we show that vesicles do not likely utilize a tug-of-war like mechanism and reverse direction in order to navigate microtubule ends.

Vasudevan A et al. (2024) Genetics "Preferential transport of synaptic vesicles across neuronal branches is regulated by the levels of the anterograde motor UNC-104/KIF1A in vivo."

Koushika SP, Hall DH, Ratnakaran N, Kumari S, Murthy K, Vasudevan A

[Genetics, 2024]

Asymmetric transport of cargo across axonal branches is a field of active research. Mechanisms contributing to preferential cargo transport along specific branches in vivo in wild type neurons are poorly understood. We find that anterograde synaptic vesicles preferentially enter the synaptic branch or pause at the branch point in C. elegans PLM neurons. The synaptic vesicle anterograde kinesin motor UNC-104/KIF1A regulates this vesicle behaviour at the branch point. Reduced levels of functional UNC-104 cause vesicles to predominantly pause at the branch point and lose their preference for turning into the synaptic branch. SAM-4/Myrlysin, which aids in recruitment/activation of UNC-104 on synaptic vesicles, regulates vesicle behaviour at the branch point similar to UNC-104. Increasing the levels of UNC-104 increases the preference of vesicles to go straight towards the asynaptic end. This suggests that the neuron optimises UNC-104 levels on the cargo surface to maximise the fraction of vesicles entering the branch and minimise the fraction going to the asynaptic end.

Shawn Lockery et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "A Predictive Model of Locomotory State Transitions in C. elegans"

Steven Augustine, Rebecca Anderson, Shawn Lockery, Kristy Lawton

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

Locomotory state in C. elegans is regulated by a network of command neurons in two reciprocally connected pools-the forward neurons AVB and PVC and the reverse neurons AVA and AVD-but how this network functions is poorly understood. We propose a model in which the network functions as a stochastic, bi-stable switch. The model makes three simple assumptions: (A) forward command neurons act as a single unit, as do reverse command neurons; (B) unit activation switches stochastically between two states: 0 (off) and 1 (on); (C) the stochastic processes underlying the state changes of the forward and reverse units are uncorrelated. Accordingly, the network can exist in four states: both units off (00); forward on (10), reverse on (01), and both on (11). Previous neuronal ablations suggest that states 10, 01, and 00 correspond, respectively, to forward locomotion, reverse locomotion, and a pause state; we propose that state 11 is also a pause state because co-activation of forward and reverse motor systems could cause the body musculature to lock up. A key consequence of (C) is that only one unit changes state at a time. This means that transitions between the forward and reverse states (01 to 10, or 10 to 01) must pass through the intermediate state 00 or 11. Thus the model predicts pauses during transitions between forward and reverse locomotion. The model also predicts pauses during apparently continuous bouts of forward or reverse locomotion. To test these predictions, we constructed a novel tracking system capable of recording the worm's speed with a precision of 19 um/sec at a rate of 30 samples/sec. We found clear evidence for pauses, which were typified by a precipitous drop in speed and, after a variable delay, a precipitous rise in speed. Mean dwell time in the pause state was 0.15 sec (SEM = 0.01). Pauses were observed during all transitions between forward and reverse, and also during bouts of forward and reverse locomotion that appeared continuous to the naked eye. These results support the stochastic switch model and suggest revised definitions of fundamental locomotory states in C. elegans.

Shawn R Lockery et al. (2005) International Worm Meeting "Kinetic analysis of locomotory switching in C. elegans"

Shawn R Lockery, Steven B Augustine, Kristy J Lawton

[International Worm Meeting, 2005]

Locomotory state in C. elegans is regulated by a network of command neurons in two functional pools: the forward neurons AVB and PVC, and the reverse neurons AVA and AVD. These two pools are linked by reciprocal synaptic connections, but how this network functions to regulate locomotion is not well understood.In one model, the network functions as a stochastic, bi-stable switch. This model embodies two main assumptions: (1) Forward command neurons act as a single unit, as do reverse command neurons, (2) Units switch stochastically between the on (1) and off (0) states. According to these assumptions, the network can exist in four different states: both units off (00); forward unit on, reverse unit off (10); forward unit off, reverse unit on (01); both units on (11). Previous neuronal ablation studies suggest that state 10 is forward locomotion, state 01 is reverse locomotion, and state 00 is a pause state. Here it is further assumed that state 11 is also a pause state, because co-activation of forward and reverse motor systems might cause the body musculature to lock-up.A key consequence of assumption (2) is that only one unit changes state at a time, because the probability of simultaneous random events is zero. This means that transitions in either direction between the forward and reverse state (10<sym17>01 and 01<sym17>10, respectively) must involve the intermediate states 00 or 11. Thus the model predicts that a worm pauses briefly during transitions between forward and reverse. The model also predicts that brief pauses occur during apparently continuous bouts of forward or reverse locomotion.To test these predictions we videotaped wild type worms moving on foodless agar plates. Frame-by-frame analysis (30 frames/sec) revealed the presence of brief but measurable pauses (mean SD

Yogev S et al. (2016) Neuron "Microtubule Organization Determines Axonal Transport Dynamics."

Cooper R, Fetter R, Shen K, Yogev S, Horowitz M

[Neuron, 2016]

Axonal microtubule (MT) arrays are the major cytoskeleton substrate for cargo transport. How MTorganization, i.e., polymer length, number, and minus-end spacing, is regulated and how it impinges on axonal transport are unclear. We describe a method for analyzing neuronal MT organization usinglight microscopy. This method circumvents the needfor electron microscopy reconstructions and is compatible with live imaging of cargo transport and MT dynamics. Examination of a C.elegans motor neuron revealed how age, MT-associated proteins, and signaling pathways control MT length, minus-end spacing, and coverage. In turn, MT organization determines axonal transport progression: cargoes pause at polymer termini, suggesting that switching MT tracks is rate limiting for efficient transport. Cargo run length is set by MT length, and higher MT coverage correlates with shorter pauses. These results uncover the principles and mechanisms of neuronal MT organization and its regulation of axonal cargo transport.

Wu YE et al. (2013) Neuron "The balance between capture and dissociation of presynaptic proteins controls the spatial distribution of synapses."

Feng W, Maeder CI, Wu YE, Huo L, Shen K

[Neuron, 2013]

The location, size, and number of synapses critically influence the specificity and strength of neural connections. In axons, synaptic vesicle (SV) and active zone (AZ) proteins are transported by molecular motors and accumulate at discrete presynaptic loci. Little is known about the mechanisms coordinating presynaptic protein transport and deposition to achieve proper distribution of synaptic material. Here we show that SV and AZ proteins exhibit extensive cotransport and undergo frequent pauses. At the axonal and synaptic pause sites, the balance between the capture and dissociation of mobile transport packets determines the extent of presynaptic assembly. The small G protein ARL-8 inhibits assembly by promoting dissociation, while a JNK kinase pathway and AZ assembly proteins inhibit dissociation. Furthermore, ARL-8 directly binds to the UNC-104/KIF1A motor to limit the capture efficiency. Together, molecular regulation of the dichotomy between axonal trafficking and local assembly controls vital aspects of synapse formation and maintenance.

Bird DM (1991) International C. elegans Meeting "THE 5'-END OF ama-1 AND THE dpy-13 CONNECTION."

Bird DM

[International C. elegans Meeting, 1991]

Examination of the sequence extending 4,499 bp 5' from ama-l to col- 34 has revealed an absence of any obvious promotor elements (except for col-34, and which run in the opposite direction). A primer extension from Poly-A+ RNA using an ama-l anti-sense oligo revealed two bands corresponding to a 5'-untranslated region of ~120 bp, with a 'pause' at ~40 bp upstream from the ATG. Alignment of an ama-l cDNA sequence with the genome revealed a deviation of the sequences at position -40, corresponding to the 'pause.' This appears not to be as the result of an intron, as the genomic sequence at this point bears no resemblance to a typical splice acceptor. This should also preclude trans-splicing (this sequence is not SLl or SL2). A database search revealed that 29 nucleotides of the ama-l cDNA are identical with residues 951-980 of dpy-13, and encode a helix to non- helix transition near the carboxyl terminus ofthis collagen; the remaining 11 residues at the ama-l 5'end do not share dpy-13 homology. Examination of the dpy-13 sequence fails to reveal an appropriately placed splice donor consensus, again suggesting that splicing with dpy- 13 is not involved. It remains a formal possibility that a collagen gene more 5' from dpy-13 is the source of the ama-l 5' 'exon'; a minimum size for the resultant 'intron' is 25.5 kb. A consequence of this scenario is that both dpy-13 and col-34 would lie within an ama-l intron. Observations by Miguel Estevez (Estevez and Riddle, pers. comm. ) support the notion of a complex formation of the ama-l 5'-end. He has found that the cosmid CB#C17D7, which spans from left of col-34 to the right of col-33, fails to rescue an ama1 lethal following microinjection.

Roka (Pun), Himal et al. (2021) International Worm Meeting "An RNAi Screen to Identify Factors that Enhance microRNA Activity After Dauer"

Roka (Pun), Himal, Karp, Xantha

[International Worm Meeting, 2021]

Proper execution of developmental events and their timing is crucial for animals to develop normally. Many animal species can pause their development by entering a stress-resistant and developmentally arrested diapause stage. This interruption can disrupt the timing of important biological processes such as cell fate specification and differentiation. Despite this disruption, wild-type animals develop normally after diapause. The mechanisms by which developmental pathways accommodate diapause can be studied using C. elegans dauer larvae. Dauer occurs after the second larval molt in response to unfavorable conditions. During dauer, progenitor cells such as seam cells pause their development. Seam cells divide in a particular pattern and sequence at each larval stage, called stage-specific cell fate, and differentiate at adulthood. After dauer, seam cells complete development normally. MicroRNAs act as a molecular switch to regulate seam cell fate by downregulating target genes that specify early cell fate. MicroRNAs regulate their targets as a part of the microRNA-Induced Silencing Complex (miRISC) that includes the core Argonaute proteins ALG-1 and ALG-2. In alg-1(0) mutants, stage-specific cell fates are reiterated. Interestingly, seam cell fates occur normally in alg-1(0) mutants that have experienced dauer diapause. This observation suggests that miRISC function is enhanced after dauer to allow cell fates to occur normally. Here, we are using RNAi to screen for factors that potentiate miRISC function in post-dauer animals. Specifically, we are screening for reiterative phenotypes in post-dauer alg-1(0) adults. We are focused on conserved kinases and RNA-binding proteins as factors that are most likely to regulate miRISC function. Thus far, we have screened 25% of the genes in our list. We have identified nekl-3 as a potential candidate gene to enhance microRNA function after dauer. nekl-3 encodes a kinase that promotes molting. We are currently investigating the mechanism underlying the nekl-3(RNAi) phenotype observed in our screen. Once complete, we expect our work to provide insight into the mechanisms by which microRNA pathways can be modulated to allow normal development after diapause. Because we are focusing on conserved genes, these findings may be relevant across animal species.

Ghosh R et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Quantitative Genetic Dissection Of A Sensorimotor Transformation In C.elegans"

Ryu W S, KRUGLYAK L, Ghosh R

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Individuals within and between species often exhibit heritable variation in sub-steps of a behavioral sequence. What genetic changes and architecture allow for such plasticity? To address this we transiently raised the local temperature around a worm by an IR laser and recorded several aspects of the resulting escape response. Typically upon sensation of a thermal pulse worms exhibit a behavioral sequence in which they enter a pause state followed by reversals, omega turn and resumption of forward movement. We found that these behavioral states were accompanied by a decrease in pre-stimulus speed followed by a rapid increase in speed, which decelerated back to the pre-stimulus speed. We quantitatively characterized several aspects of this sensorimotor transformation in the laboratory strain (N2) and several wild isolates of C. elegans including one from Hawaii (CB4856) for 0.3, 0.8, 3 or 7.5&deg;C rise in temperature and found responses to &Delta;T~0.3&deg;C exhibited the greatest variation. We measured several traits including but not limited to speeds at every 180 milliseconds during the assay, probability of worms responding by reversals, duration and number of reversals, probability of a reversal ending in omega turn in 149 recombinant inbred lines made from N2 and CB4856 parent strains for a thermal pulse of &Delta;T~0.3&deg;C. We have identified several unique and pleiotoropic quantitative trait loci (QTL) underlying different aspects of escape response e.g. reversal duration and number of body waves during reversals are modulated by a common locus on Chromosome V as well as distinct QTL on Chromosomes IV and X respectively implying partially independent molecular mechanisms underlying these two traits. We found that npr-1, previously shown to be a lab-derived major locus responsible for variation in speed between N2 and CB4856, contributes substantially to variation in pre-stimulus speed and deceleration to pause state. Interestingly, other aspects of speed was npr-1 independent e.g. QTL on Chromosome IV contributed to variation in acceleration to maximum speed after the thermal impulse. By phenotyping introgression strains we have narrowed several of these QTL to less than 200Kb. We will present the genetic architecture of the sensorimotor transformation that emerged from this study and our efforts to identify the genes underlying the QTL.