Biology subjects

Sanders, D. W.

Publications and source records attributed to Sanders, D. W..

5 recordsLinked to original sources

bioRxiv2023

DnaJC7 specifically regulates tau seeding

Neurodegenerative tauopathies are caused by accumulation of toxic tau protein assemblies. This appears to involve template-based seeding events, whereby tau monomer changes conformation and is recruited to a growing aggregate. Several large families of chaperone proteins, including Hsp70s and J domain proteins (JDPs) cooperate to regulate the folding of intracellular proteins such as tau, but the factors that coordinate this activity are not well known. The JDP DnaJC7 binds tau and reduces its intracellular aggregation. However, it is unknown whether this is specific to DnaJC7 or if other JDPs might be similarly involved. We used proteomics within a cell model to determine that DnaJC7 co-purified with insoluble tau and colocalized with intracellular aggregates. We individually knocked out every possible JDP and tested the effect on intracellular aggregation and seeding. DnaJC7 knockout decreased aggregate clearance and increased intracellular tau seeding. This depended on the ability of the J domain (JD) of DnaJC7 to bind to Hsp70, as JD mutations that block binding to Hsp70 abrogated the protective activity. Disease-associated mutations in the JD and substrate binding site of DnaJC7 also abrogated its protective activity. DnaJC7 thus specifically regulates tau aggregation in cooperation with Hsp70.

molecular biology↗

bioRxiv2022

A rich get richer effect governs intracellular condensate size distributions

Phase separation of biomolecules into condensates has emerged as a ubiquitous mechanism for intracellular organization and impacts many intracellular processes, including reaction pathways through clustering of enzymes and their intermediates. Precise and rapid spatiotemporal control of reactions by condensates requires tuning of their sizes. However, the physical processes that govern the distribution of condensate sizes remain unclear. Here, we utilize a combination of synthetic and native condensates to probe the underlying physical mechanisms determining condensate size. We find that both native nuclear speckles and FUS condensates formed with the synthetic Corelet system obey an exponential size distribution, which can be recapitulated in Monte Carlo simulations of fast nucleation followed by coalescence. By contrast, pathological aggregation of cytoplasmic Huntingtin polyQ protein exhibits a power-law size distribution, with an exponent of -1.41 {+/-} 0.02. These distinct behaviors reflect the relative importance of nucleation and coalescence kinetics: introducing continuous condensate nucleation into the Monte Carlo coarsening simulations gives rise to polyQ-like power-law behavior. We demonstrate that the emergence of power-law distributions under continuous nucleation reflects a "rich get richer" effect, whose extent may play a general role in the determination of condensate size distributions.

biophysics↗

bioRxiv2022

The AAA+ chaperone VCP disaggregates Tau fibrils and generates aggregate seeds

Amyloid-like aggregates of the microtubule-associated protein Tau are associated with several neurodegenerative disorders including Alzheimers disease. The existence of cellular machinery for the removal of such aggregates has remained unclear, as specialized disaggregase chaperones are thought to be absent in mammalian cells. Here we show in cell culture and in neurons that the AAA+ chaperone VCP is recruited to ubiquitylated Tau fibrils, resulting in their efficient disaggregation. Aggregate clearance depends on the functional cooperation of VCP with Hsp70 and the ubiquitin-proteasome machinery. Inhibition of VCP activity stabilizes large Tau aggregates, and is accompanied by a reduction in the amount of Tau species competent of prion- like aggregate seeding in recipient cells. Thus, disaggregation by VCP generates seeding-active Tau as byproduct. These findings identify VCP as a core component of the machinery for the removal of neurodegenerative disease aggregates and suggest that its activity can be associated with enhanced aggregate spreading in tauopathies.

cell biology↗

bioRxiv2020

SARS-CoV-2 Requires Cholesterol for Viral Entry and Pathological Syncytia Formation

Many enveloped viruses induce multinucleated cells (syncytia), reflective of membrane fusion events caused by the same machinery that underlies viral entry. These syncytia are thought to facilitate replication and evasion of the host immune response. Here, we report that co-culture of human cells expressing the receptor ACE2 with cells expressing SARS-CoV-2 spike, results in synapse-like intercellular contacts that initiate cell-cell fusion, producing syncytia resembling those we identify in lungs of COVID-19 patients. To assess the mechanism of spike/ACE2-driven membrane fusion, we developed a microscopy-based, cell-cell fusion assay to screen [~]6000 drugs and >30 spike variants. Together with cell biological and biophysical approaches, the screen reveals an essential role for membrane cholesterol in spike-mediated fusion, which extends to replication-competent SARS-CoV-2 isolates. Our findings provide a molecular basis for positive outcomes reported in COVID-19 patients taking statins, and suggest new strategies for therapeutics targeting the membrane of SARS-CoV-2 and other fusogenic viruses. HighlightsO_LICell-cell fusion at ACE2-spike clusters cause pathological syncytia in COVID-19 C_LIO_LIDrug screen reveals critical role for membrane lipid composition in fusion C_LIO_LISpikes unusual membrane-proximal cysteines and aromatics are essential for fusion C_LIO_LICholesterol tunes relative infectivity of SARS-CoV-2 viral particles C_LI

cell biology↗

bioRxiv2019

Composition dependent phase separation underlies directional flux through the nucleolus

Intracellular bodies such as nucleoli, Cajal bodies, and various signaling assemblies, represent membraneless organelles, or condensates, that form via liquid-liquid phase separation (LLPS)1,2. Biomolecular interactions, particularly homotypic interactions mediated by self-associating intrinsically disordered protein regions (IDRs), are thought to underlie the thermodynamic driving forces for LLPS, forming condensates that can facilitate the assembly and processing of biochemically active complexes, such as ribosomal subunits within the nucleolus. Simplified model systems3-6 have led to the concept that a single fixed saturation concentration (Csat) is a defining feature of endogenous LLPS7-9, and has been suggested as a mechanism for intracellular concentration buffering2,7,8,10. However, the assumption of a fixed Csat remains largely untested within living cells, where the richly multicomponent nature of condensates could complicate this simple picture. Here we show that heterotypic multicomponent interactions dominate endogenous LLPS, and give rise to nucleoli and other condensates that do not exhibit a fixed Csat. As the concentration of individual components is varied, their partition coefficients change, in a manner that can be used to extract thermodynamic interaction energies, that we interpret within a framework we term polyphasic interaction thermodynamic analysis (PITA). We find that heterotypic interactions between protein and RNA components stabilize a variety of archetypal intracellular condensates, including the nucleolus, Cajal bodies, stress granules, and P bodies. These findings imply that the composition of condensates is finely tuned by the thermodynamics of the underlying biomolecular interaction network. In the context of RNA processing condensates such as the nucleolus, this stoichiometric self-tuning manifests in selective exclusion of fully-assembled RNP complexes, providing a thermodynamic basis for vectorial ribosomal RNA (rRNA) flux out of the nucleolus. The PITA methodology is conceptually straightforward and readily implemented, and it can be broadly utilized to extract thermodynamic parameters from microscopy images. These approaches pave the way for a deep understanding of the thermodynamics of multi-component intracellular phase behavior and its interplay with nonequilibrium activity characteristic of endogenous condensates.

biophysics↗