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Full-Text Articles in Life Sciences
Multi-Scale 3d Cryo-Correlative Microscopy For Vitrified Cells, Gong-Her Wu, Patrick G. Mitchell, Jesus G. Galaz-Montoya, Corey W. Hecksel, Emily M. Sontag, Vimal Gangadharan, Jeffrey Marshman, David Mankus, Margaret E. Bisher, Abigail K.R. Lytton-Jean, Judith Frydman, Kirk Czymmek, Wah Chiu
Multi-Scale 3d Cryo-Correlative Microscopy For Vitrified Cells, Gong-Her Wu, Patrick G. Mitchell, Jesus G. Galaz-Montoya, Corey W. Hecksel, Emily M. Sontag, Vimal Gangadharan, Jeffrey Marshman, David Mankus, Margaret E. Bisher, Abigail K.R. Lytton-Jean, Judith Frydman, Kirk Czymmek, Wah Chiu
Biological Sciences Faculty Research and Publications
Three-dimensional (3D) visualization of vitrified cells can uncover structures of subcellular complexes without chemical fixation or staining. Here, we present a pipeline integrating three imaging modalities to visualize the same specimen at cryogenic temperature at different scales: cryo-fluorescence confocal microscopy, volume cryo-focused ion beam scanning electron microscopy, and transmission cryo-electron tomography. Our proof-of-concept benchmark revealed the 3D distribution of organelles and subcellular structures in whole heat-shocked yeast cells, including the ultrastructure of protein inclusions that recruit fluorescently-labeled chaperone Hsp104. Since our workflow efficiently integrates imaging at three different scales and can be applied to other types of cells, it could …
Spatial Sequestration And Oligomer Remodeling During De Novo [Psi+] Formation, Douglas Lyke, Anita L. Manogaran
Spatial Sequestration And Oligomer Remodeling During De Novo [Psi+] Formation, Douglas Lyke, Anita L. Manogaran
Biological Sciences Faculty Research and Publications
Prions are misfolded, aggregated, infectious proteins found in a range of organisms from mammals to bacteria. In mammals, prion formation is difficult to study because misfolding and aggregation take place prior to symptom presentation. The study of the yeast prion [PSI+], which is the misfolded infectious form of Sup35p, provides a tractable system to monitor prion formation in real time. Recently, we showed that the de novo formation of prion aggregates begins with the appearance of highly mobile cytoplasmic foci, called early foci, which assemble into larger ring or dot structures. We also observed SDS-resistant oligomers during …
Hsp70 And Hsp40 Functionally Interact With Soluble Mutant Huntingtin Oligomers In A Classic Atp-Dependent Reaction Cycle, Gregor P. Lotz, Justin Legleiter, Rebecca Aron, Emily M. Sontag, Shao-Yi Huang, Cheping Ng, Charles G. Glabe, Leslie M. Thompson, Paul J. Muchowski
Hsp70 And Hsp40 Functionally Interact With Soluble Mutant Huntingtin Oligomers In A Classic Atp-Dependent Reaction Cycle, Gregor P. Lotz, Justin Legleiter, Rebecca Aron, Emily M. Sontag, Shao-Yi Huang, Cheping Ng, Charles G. Glabe, Leslie M. Thompson, Paul J. Muchowski
Biological Sciences Faculty Research and Publications
Inclusion bodies of aggregated mutant huntingtin (htt) fragments are a neuropathological hallmark of Huntington disease (HD). The molecular chaperones Hsp70 and Hsp40 colocalize to inclusion bodies and are neuroprotective in HD animal models. How these chaperones suppress mutant htt toxicity is unclear but might involve direct effects on mutant htt misfolding and aggregation. Using size exclusion chromatography and atomic force microscopy, we found that mutant htt fragments assemble into soluble oligomeric species with a broad size distribution, some of which reacted with the conformation-specific antibody A11. Hsp70 associated with A11-reactive oligomers in an Hsp40- and ATP-dependent manner and inhibited their …
Mutant Huntingtin Fragments Form Oligomers In A Polyglutamine Length-Dependent Manner In Vitro And In Vivo, Justin Legleiter, Emily M. Sontag, Gregor P. Lotz, Ellen Sapp, Cheping Ng, Marian Difiglia, Leslie M. Thompson, Paul J. Muchowski
Mutant Huntingtin Fragments Form Oligomers In A Polyglutamine Length-Dependent Manner In Vitro And In Vivo, Justin Legleiter, Emily M. Sontag, Gregor P. Lotz, Ellen Sapp, Cheping Ng, Marian Difiglia, Leslie M. Thompson, Paul J. Muchowski
Biological Sciences Faculty Research and Publications
Huntington disease (HD) is caused by an expansion of more than 35–40 polyglutamine (polyQ) repeats in the huntingtin (htt) protein, resulting in accumulation of inclusion bodies containing fibrillar deposits of mutant htt fragments. Intriguingly, polyQ length is directly proportional to the propensity for htt to form fibrils and the severity of HD and is inversely correlated with age of onset. Although the structural basis for htt toxicity is unclear, the formation, abundance, and/or persistence of toxic conformers mediating neuronal dysfunction and degeneration in HD must also depend on polyQ length. Here we used atomic force microscopy to demonstrate mutant htt …