Specialized extracellular matrix (ECM) is associated with virtually every mechanosensory system studied. C. elegans touch receptor neurons have specialized ECM and attach to the surrounding epidermis. The mec-1 gene encodes an ECM protein with multiple EGF and Kunitz domains. MEC-1 is needed for the accumulation of the collagen MEC-5 and other ECM components, attachment, and, separately, for touch sensitivity. MEC-1 and MEC-5 bind to touch processes uniformly and in puncta. These puncta colocalize with and localize the mechanosensory channel complex in the touch neurons. In turn, the production of the MEC-1 and MEC-5 puncta appears to rely on interactions with the neighboring epidermal tissue. These and other observations lead us to propose that extracellular, but not cytoskeletal, tethering of the degenerin channel is needed for mechanosensory transduction. Additionally, our experiments demonstrate an important role of the ECM in organizing the placement of the channel complex.

Microtubules are integral to neuronal development and function. They endow cells with polarity, shape, and structure, and their extensive surface area provides substrates for intracellular trafficking and scaffolds for signaling molecules. Consequently, microtubule polymerization dynamics affect not only structural features of the cell but also the subcellular localization of proteins that can trigger intracellular signaling events. In the nematode Caenorhabditis elegans , the processes of touch receptor neurons are filled with a bundle of specialized large-diameter microtubules. We find that conditions that disrupt these microtubules (loss of either the MEC-7 β-tubulin or MEC-12 α-tubulin or growth in 1 mM colchicine) cause a general reduction in touch receptor neuron (TRN) protein levels. This reduction requires a p38 MAPK pathway (DLK-1, MKK-4, and PMK-3) and the transcription factor CEBP-1. Cells may use this feedback pathway that couples microtubule state and MAPK activation to regulate cellular functions.

Inclusions of disordered protein are a characteristic feature of most neurodegenerative diseases, including Huntington’s disease. Huntington’s disease is caused by expansion of a polyglutamine tract in the huntingtin protein; mutant huntingtin protein (mHtt) is unstable and accumulates in large intracellular inclusions both in affected individuals and when expressed in eukaryotic cells. Using mHtt-GFP expressed in Saccharomyces cerevisiae, we find that mHtt-GFP inclusions are dynamic, mobile, gel-like structures that concentrate mHtt together with the disaggregase Hsp104. Although inclusions may associate with the vacuolar membrane, the association is reversible and we find that inclusions of mHtt in S. cerevisiae are not taken up by the vacuole or other organelles. Instead, a pulse-chase study using photoconverted mHtt-mEos2 revealed that mHtt is directly and continuously removed from the inclusion body. In addition to mobile inclusions, we also imaged and tracked the movements of small particles of mHtt-GFP and determine that they move randomly. These observations suggest that inclusions may grow through the collision and coalescence of small aggregative particles.

To identify molecular mechanisms that function in G protein signaling, we have performed molecular genetic studies of a simple behavior of the nematode C. elegans, egg laying, which is driven by a pair of serotonergic neurons, the HSNs. The activity of the HSNs is regulated by the Go-coupled receptor EGL-6, which mediates inhibition of the HSNs by neuropeptides. We report here that this inhibition requires one of three inwardly rectifying K+ channels encoded by the C. elegans genome: IRK-1. Using ChannelRhodopsin2-mediated stimulation of HSNs, we observed roles for egl-6 and irk-1 in regulating the excitability of HSNs. Although irk 1 is required for inhibition of HSNs by EGL-6 signaling, we found that other Go signaling pathways that inhibit HSNs involve irk-1 little or not at all. These findings suggest that the neuropeptide receptor EGL-6 regulates the potassium channel IRK 1 is via a dedicated pool of Go not involved in other Go-mediated signaling. We conclude that G protein-coupled receptors that signal through the same G protein in the same cell might activate distinct effectors and that specific coupling of a GPCR to its effectors can be determined by factors other than its associated G proteins.

It is well established that the efficacy of synaptic connections can be rapidly modified by neural activity, yet how the environment and prior experience modulate such synaptic and behavioral plasticity is only beginning to be understood. Here we show in C. elegans that the broadly conserved scaffolding molecule MAGI-1 is required for the plasticity observed in a glutamatergic circuit. This mechanosensory circuit mediates reversals in locomotion in response to touch stimulation, and the AMPA-type receptor (AMPAR) subunits GLR-1 and GLR-2, which are required for reversal behavior, are localized to ventral cord synapses in this circuit. We find that animals modulate GLR-1 and GLR-2 localization in response to prior mechanosensory stimulation; a specific isoform of MAGI-1 (MAGI-1L) is critical for this modulation. We show that MAGI-1L interacts with AMPARs through the intracellular domain of the GLR-2 subunit, which is required for the modulation of AMPAR synaptic localization by mechanical stimulation. In addition, mutations that prevent the ubiquitination of GLR-1 prevent the decrease in AMPAR localization observed in previously stimulated magi-1 mutants. Finally, we find that previously-stimulated animals later habituate to subsequent mechanostimulation more rapidly compared to animals initially reared without mechanical stimulation; MAGI-1L, GLR-1, and GLR-2 are required for this change in habituation kinetics. Our findings demonstrate that prior experience can cause long-term alterations in both behavioral plasticity and AMPAR localization at synapses in an intact animal, and indicate a new, direct role for MAGI/S-SCAM proteins in modulating AMPAR localization and function in the wake of variable sensory experience.

The processes underlying formation and growth of unfolded protein inclusions are relevant to neurodegenerative diseases but poorly characterized in living cells. In S. cerevisiae, inclusions formed by mutant huntingtin (mHtt) have some characteristics of biomolecular condensates but the physical nature and growth mechanisms of inclusion bodies remain unclear. We have probed the relationship between concentration and inclusion growth in vivo and find that growth of mHtt inclusions in living cells is triggered at a cytoplasmic threshold concentration, while reduction in cytoplasmic mHtt causes inclusions to shrink. The growth rate is consistent with incorporation of new material through collision and coalescence. A small remnant of the inclusion is relatively long-lasting, suggesting that it contains a core that is structurally distinct, and which may serve to nucleate it. These observations support a model in which aggregative particles are incorporated by random collision into a phase-separated condensate composed of a particle-rich mixture.

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The processes underlying formation and growth of unfolded protein inclusions are relevant to neurodegenerative diseases. In S. cerevisiae, inclusion bodies formed by mutant huntingtin have characteristics of phase-separated compartments: they are mobile, ovoid, and the contents are diffusible. We have used molecular genetics and quantitative confocal microscopy to probe the relationship between concentration and inclusion growth in vivo. Our analysis and modeling of the growth of mutant huntingtin inclusion bodies (mHtt IBs) suggests that there is a cytoplasmic threshold concentration that triggers the formation of an IB, regardless of proteasome capacity, and that reduction in cytoplasmic mHtt causes IBs to shrink. These findings confirm that the IB is a phase-separated compartment that continuously exchanges material with the cytoplasm. The growth rate of the IB is most consistent with a model in which material is incorporated through collision with the IB. A small remnant of the IB is relatively long-lasting, suggesting that the IB contains a core that is structurally distinct, and which may serve to nucleate it.