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1.
bioRxiv ; 2023 Feb 14.
Article in English | MEDLINE | ID: mdl-36824970

ABSTRACT

Aggregation of mutant Huntingtin protein (mHtt) leads to neuronal cell death and human disease. We investigated the effect of inclusion formation on yeast cells. Previous work indicates that mHtt protein moves both in and out of inclusions, potentially undergoing refolding in the inclusion. However, the sustained influx of unfolded protein into an inclusion leads to a dramatic change from a phase-separated body to an irregular, less soluble form at a threshold inclusion size. Altered morphology was associated with a prion-like seeding that accelerated inclusion growth despite loss of soluble cytoplasmic protein. The structural change abolished exchange of material between the inclusion and the cytosol and resulted in early cell death. Affected cells continued to divide occasionally, giving rise to daughters with a similar phenotype. Most newly born cells were able to reverse the prion-like aggregation, restoring both soluble cytoplasmic protein and a normal inclusion structure.

2.
Commun Biol ; 4(1): 971, 2021 08 16.
Article in English | MEDLINE | ID: mdl-34400761

ABSTRACT

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.


Subject(s)
Huntingtin Protein/metabolism , Inclusion Bodies/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism
3.
Life Sci Alliance ; 2(5)2019 10.
Article in English | MEDLINE | ID: mdl-31527136

ABSTRACT

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.


Subject(s)
Heat-Shock Proteins/metabolism , Huntingtin Protein/genetics , Huntingtin Protein/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Green Fluorescent Proteins/metabolism , Humans , Inclusion Bodies/metabolism , Microscopy, Electron, Transmission , Mutation , Optical Imaging , Protein Aggregates , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/metabolism
4.
J Neurosci ; 32(46): 16285-95, 2012 Nov 14.
Article in English | MEDLINE | ID: mdl-23152612

ABSTRACT

To identify molecular mechanisms that function in G-protein signaling, we have performed molecular genetic studies of a simple behavior of the nematode Caenorhabditis elegans, egg laying, which is driven by a pair of serotonergic neurons, the hermaphrodite-specific neurons (HSNs). The activity of the HSNs is regulated by the G(o)-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 ChannelRhodopsin-2-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 G(o) 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 via a dedicated pool of G(o) not involved in other G(o)-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 G-protein-coupled receptor to its effectors can be determined by factors other than its associated G-proteins.


Subject(s)
Caenorhabditis elegans/physiology , GTP-Binding Protein alpha Subunits, Gi-Go/physiology , Neuropeptides/pharmacology , Potassium Channels, Inwardly Rectifying/physiology , Serotonergic Neurons/physiology , Alleles , Amino Acid Sequence , Animals , Animals, Genetically Modified , Behavior, Animal/physiology , Genome , Ion Channel Gating/physiology , Molecular Sequence Data , Oocytes , Polymerase Chain Reaction , Potassium Channels, Inwardly Rectifying/genetics , Sexual Behavior, Animal/physiology , Signal Transduction/physiology , Xenopus laevis
5.
Proc Natl Acad Sci U S A ; 108(10): 3982-7, 2011 Mar 08.
Article in English | MEDLINE | ID: mdl-21368137

ABSTRACT

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 ß-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.


Subject(s)
Caenorhabditis elegans/metabolism , Gene Expression , Microtubules/metabolism , Neurons/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Caenorhabditis elegans/enzymology , Caenorhabditis elegans/genetics , Colchicine/pharmacology , Mutation , Neurons/drug effects , Neurons/enzymology
6.
PLoS One ; 4(2): e4613, 2009.
Article in English | MEDLINE | ID: mdl-19242552

ABSTRACT

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.


Subject(s)
Behavior, Animal , Caenorhabditis elegans Proteins/physiology , Cell Adhesion Molecules, Neuronal/physiology , Guanylate Kinases/physiology , Receptors, AMPA/metabolism , Animals , Caenorhabditis elegans/physiology , Nerve Tissue Proteins , Neuronal Plasticity , Receptors, AMPA/physiology
7.
Neuron ; 44(5): 795-807, 2004 Dec 02.
Article in English | MEDLINE | ID: mdl-15572111

ABSTRACT

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.


Subject(s)
Caenorhabditis elegans Proteins/physiology , Caenorhabditis elegans/physiology , Extracellular Matrix Proteins/physiology , Mechanoreceptors/physiology , Touch/physiology , Alleles , Amino Acid Sequence , Animals , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Collagen/metabolism , Epidermal Growth Factor/genetics , Extracellular Matrix Proteins/genetics , Membrane Proteins/metabolism , Molecular Sequence Data , Protein Structure, Tertiary , Tissue Distribution
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