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1. HSP70 chaperones RNA-free TDP-43 into anisotropic intranuclear liquid spherical shells

   https://doi.org/0.1126/science.abb4309  

   Yu, Haiyang; Lu, Shan; Gasior, Kelsey; Singh, Digvijay; Vazquez-Sanchez, Sonia; Tapia, Olga; Toprani, Divek; Beccari, Melinda S; Yates, John R; Da Cruz, Sandrine; et al (December 2020, Science)

   null (Ed.)

   The RNA-binding protein TDP-43 forms intranuclear or cytoplasmic aggregates in age-related neurodegenerative diseases. Here we found that RNA-binding deficient TDP-43 (produced by neurodegeneration-causing mutations or post-translational acetylation in its RNA recognition motifs) drove TDP-43 de-mixing into intranuclear liquid spherical shells with liquid cores. We named these droplets anisosomes, whose shells exhibited birefringence, evidence of liquid crystal formation. Guided by mathematical modeling, we identified the major components of the liquid core to be HSP70 family chaperones, whose ATP-dependent activity maintained the liquidity of shells and cores. In vivo proteasome inhibition within neurons, to mimic aging-related reduction of proteasome activity, induced TDP-43-containing spherical shells. These structures converted into aggregates when ATP levels were reduced. Thus, acetylation, HSP70, and proteasome activities regulate TDP-43 phase separation and conversion into a gel/solid phase. 

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2. HSP70 chaperones RNA-free TDP-43 into anisotropic intranuclear liquid spherical shells

   https://doi.org/10.1126/science.abb4309  

   Yu, Haiyang; Lu, Shan; Gasior, Kelsey; Singh, Digvijay; Vazquez-Sanchez, Sonia; Tapia, Olga; Toprani, Divek; Beccari, Melinda S.; Yates, III, John R.; Da Cruz, Sandrine; et al (December 2020, Science)

   The RNA binding protein TDP-43 forms intranuclear or cytoplasmic aggregates in age-related neurodegenerative diseases. In this study, we found that RNA binding–deficient TDP-43 (produced by neurodegeneration-causing mutations or posttranslational acetylation in its RNA recognition motifs) drove TDP-43 demixing into intranuclear liquid spherical shells with liquid cores. These droplets, which we named “anisosomes”, have shells that exhibit birefringence, thus indicating liquid crystal formation. Guided by mathematical modeling, we identified the primary components of the liquid core to be HSP70 family chaperones, whose adenosine triphosphate (ATP)–dependent activity maintained the liquidity of shells and cores. In vivo proteasome inhibition within neurons, to mimic aging-related reduction of proteasome activity, induced TDP-43–containing anisosomes. These structures converted to aggregates when ATP levels were reduced. Thus, acetylation, HSP70, and proteasome activities regulate TDP-43 phase separation and conversion into a gel or solid phase. 

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3. Phe-Gly motifs drive fibrillization of TDP-43’s prion-like domain condensates

   https://doi.org/10.1371/journal.pbio.3001198  

   Pantoja-Uceda, David; Stuani, Cristiana; Laurents, Douglas V.; McDermott, Ann E.; Buratti, Emanuele; Mompeán, Miguel (April 2021, PLOS Biology)

   Walters, Kylie J. (Ed.)

   Transactive response DNA-binding Protein of 43 kDa (TDP-43) assembles various aggregate forms, including biomolecular condensates or functional and pathological amyloids, with roles in disparate scenarios (e.g., muscle regeneration versus neurodegeneration). The link between condensates and fibrils remains unclear, just as the factors controlling conformational transitions within these aggregate species: Salt- or RNA-induced droplets may evolve into fibrils or remain in the droplet form, suggesting distinct end point species of different aggregation pathways. Using microscopy and NMR methods, we unexpectedly observed in vitro droplet formation in the absence of salts or RNAs and provided visual evidence for fibrillization at the droplet surface/solvent interface but not the droplet interior. Our NMR analyses unambiguously uncovered a distinct amyloid conformation in which Phe-Gly motifs are key elements of the reconstituted fibril form, suggesting a pivotal role for these residues in creating the fibril core. This contrasts the minor participation of Phe-Gly motifs in initiation of the droplet form. Our results point to an intrinsic (i.e., non-induced) aggregation pathway that may exist over a broad range of conditions and illustrate structural features that distinguishes between aggregate forms. 

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4. Optimizing photoswitchable MEK

   https://doi.org/10.1073/pnas.1912320116  

   Patel, Aleena L.; Yeung, Eyan; McGuire, Sarah E.; Wu, Andrew Y.; Toettcher, Jared E.; Burdine, Rebecca D.; Shvartsman, Stanislav Y. (December 2019, Proceedings of the National Academy of Sciences)

   Optogenetic approaches are transforming quantitative studies of cell-signaling systems. A recently developed photoswitchable mitogen-activated protein kinase kinase 1 (MEK1) enzyme (psMEK) short-circuits the highly conserved Extracellular Signal-Regulated Kinase (ERK)-signaling cascade at the most proximal step of effector kinase activation. However, since this optogenetic tool relies on phosphorylation-mimicking substitutions in the activation loop of MEK, its catalytic activity is predicted to be substantially lower than that of wild-type MEK that has been phosphorylated at these residues. Here, we present evidence that psMEK indeed has suboptimal functionality in vivo and propose a strategy to circumvent this limitation by harnessing gain-of-function, destabilizing mutations in MEK. Specifically, we demonstrate that combining phosphomimetic mutations with additional mutations in MEK, chosen for their activating potential, restores maximal kinase activity in vitro. We establish that this modification can be tuned by the choice of the destabilizing mutation and does not interfere with reversible activation of psMEK in vivo in both Drosophila and zebrafish. To illustrate the types of perturbations enabled by optimized psMEK, we use it to deliver pulses of ERK activation during zebrafish embryogenesis, revealing rheostat-like responses of an ERK-dependent morphogenetic event. 

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5. Yeast Models of Amyotrophic Lateral Sclerosis Type 8 Mimic Phenotypes Seen in Mammalian Cells Expressing Mutant VAPBP56S

   https://doi.org/10.3390/biom13071147  

   Stump, AnnaMari L.; Rioux, Daniel J.; Albright, Richard; Melki, Guiliano L.; Prosser, Derek C. (July 2023, Biomolecules)

   Bryant, Nia; MacDonald, Chris (Ed.)

   Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disease that results in the loss of motor neurons and can occur sporadically or due to genetic mutations. Among the 30 genes linked to familial ALS, a P56S mutation in VAPB, an ER-resident protein that functions at membrane contact sites, causes ALS type 8. Mammalian cells expressing VAPBP56S have distinctive phenotypes, including ER collapse, protein and/or membrane-containing inclusions, and sensitivity to ER stress. VAPB is conserved through evolution and has two homologs in budding yeast, SCS2 and SCS22. Previously, a humanized version of SCS2 bearing disease-linked mutations was described, and it caused Scs2-containing inclusions when overexpressed in yeast. Here, we describe a yeast model for ALS8 in which the two SCS genes are deleted and replaced with a single chromosomal copy of either wild-type or mutant yeast SCS2 or human VAPB expressed from the SCS2 promoter. These cells display ER collapse, the formation of inclusion-like structures, and sensitivity to tunicamycin, an ER stress-inducing drug. Based on the phenotypic similarity to mammalian cells expressing VAPBP56S, we propose that these models can be used to study the molecular basis of cell death or dysfunction in ALS8. Moreover, other conserved ALS-linked genes may create opportunities for the generation of yeast models of disease. 

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