Biology subjects

Miller, E. A.

Publications and source records attributed to Miller, E. A..

25 records · Page 2Linked to original sources

bioRxiv2020

Cargo crowding drives sorting stringency in COPII vesicles

Accurate maintenance of organelle identity in the secretory pathway relies on retention and retrieval of resident proteins. In the endoplasmic reticulum (ER), secretory proteins are packaged into COPII vesicles that largely exclude ER residents and misfolded proteins by mechanisms that remain unresolved. Here we combined biochemistry and genetics with correlative light and electron microscopy (CLEM) to explore how selectivity is achieved. Our data suggest that vesicle occupancy dictates ER retention: in the absence of abundant cargo, non-specific bulk flow increases. We demonstrate that ER leakage is influenced by vesicle size and cargo occupancy: overexpressing an inert cargo protein, or reducing vesicle size restores sorting stringency. We propose that cargo recruitment into vesicles creates lumenal steric pressure that drives selectivity. Sorting stringency is thus an emergent property of the biophysical process of cargo enrichment into a constrained spherical membrane-bound carrier. SummaryCombining correlative light and electron microscopy with yeast genetics and biochemistry, Gomez-Navarro, Melero and colleagues show that cargo recruitment into a constrained COPII vesicle restricts bulk flow, thereby contributing to sorting stringency and ER quality control.

cell biology↗

bioRxiv2020

An in vitro vesicle formation assay to analyze protein sorting in the secretory transport pathway

The fidelity of protein transport in the secretory transport pathway relies on the accurate sorting of proteins to their correct destination. To deepen our understanding of the underlying molecular mechanisms, it is important to develop a robust approach to systematically reveal cargo proteins that depend on a specific cargo sorting machinery to be efficiently packaged into vesicles. Here, we used an in vitro assay that reconstitutes packaging of human cargo proteins into vesicles to quantify cargo capture. Quantitative mass spectrometry analyses of the isolated vesicles revealed novel cytosolic proteins that are associated with vesicle membranes in a GTP-dependent manner or that interact with GTP-bound Sar1A on vesicle membranes. Functional analysis indicates that two of them, FAM84B and PRRC1, regulate anterograde trafficking. Comparing control cells with cells depleted of the cargo receptors, SURF4 or ERGIC53, we revealed specific clients of each of these two export adaptors. Moreover, our results indicate that vesicles enriched with a specific cargo protein contain specific transmembrane cargo and SNARE proteins. A SNARE protein, Vti1B, is identified to be in vesicles enriched with a planar cell polarity protein, Frizzled6, and promotes vesicular release of Frizzled6. Our results indicate that the vesicle formation assay in combination with quantitative mass spectrometric analysis is a robust and powerful tool to reveal novel cytosolic and transmembrane proteins that regulate trafficking of a specific cargo protein.

cell biology↗

bioRxiv2020

Gatekeeper helix activates Golgi SM protein Sly1 and directly mediates close-range vesicle tethering

The essential Golgi protein Sly1 is a member of the SM (Sec1/mammalian Unc-18) family of SNARE chaperones. Sly1 was originally identified through gain-of-function alleles that bypass requirements for diverse vesicle tethering factors. Employing genetic analyses and chemically defined reconstitutions of ER-Golgi fusion, we discovered that a loop conserved among Sly1 family members is not only autoinhibitory, but also acts as a positive effector. An amphipathic helix within the loop directly binds high-curvature membranes; membrane binding is required for relief of Sly1 autoinhibition and allows Sly1 to directly tether incoming vesicles to the Qa-SNARE on the target organelle. The SLY1-20 allele bypasses requirements for diverse tethering factors but loses this functionality if Sly1 membrane binding is impaired. We propose that long-range tethers, including Golgins and multisubunit tethering complexes, hand off vesicles to Sly1, which then tethers at close range to activate SNARE assembly and fusion in the early secretory pathway.

cell biology↗

bioRxiv2019

Emergence of a novel Salmonella enterica serotype Reading clone is linked to its expansion in commercial turkey production, resulting in unanticipated human illness in North America

Concurrent separate human outbreaks of Salmonella enterica serotype Reading occurred in 2017-2019 in the United States and Canada, which were both linked to the consumption of raw turkey products. In this study, a comprehensive genomic investigation was conducted to reconstruct the evolutionary history of S. Reading from turkeys, and to determine the genomic context of outbreaks involving this rarely isolated Salmonella serotype. A total of 988 isolates of U.S. origin were examined using whole genome-based approaches, including current and historical isolates from humans, meat, and live food animals. Broadly, isolates clustered into three major clades, with one apparently highly adapted turkey clade. Within the turkey clade isolates clustered into three subclades, including an "emergent" clade that only contained isolates dated 2016 or later, including many of the isolates from these outbreaks. Genomic differences were identified between emergent and other turkey subclades suggesting that the apparent success of currently circulating subclades clade is, in part, attributable to plasmid acquisitions conferring antimicrobial resistance, gain of phage-like sequences with cargo virulence factors, and mutations in systems that may be involved in beta-glucuronidase activity and resistance towards colicins. U.S. and Canadian outbreak isolates were found interspersed throughout the emergent subclade and the other circulating subclade. The emergence of a novel S. Reading turkey subclade, coinciding temporally with expansion in commercial turkey production and with U.S. and Canadian human outbreaks, indicates that emergent strains with higher potential for niche success were likely vertically transferred and rapidly disseminated from a common source. ImportanceIncreasingly, outbreak investigations involving foodborne pathogens are confounded by the inter-connectedness of food animal production and distribution, necessitating high-resolution genomic investigations to determine their basis. Fortunately, surveillance and whole genome sequencing, combined with the public availability of these data, enable comprehensive queries to determine underlying causes of such outbreaks. Utilizing this pipeline, it was determined that a novel clone of Salmonella Reading has emerged that coincides with increased abundance in raw turkey products and two outbreaks of human illness in North America. The rapid dissemination of this highly adapted and conserved clone indicates that it was likely obtained from a common source and rapidly disseminated across turkey production. Key genomic changes may have contributed to its apparent continued success in the barn environment, and ability to cause illness in humans.

microbiology↗

bioRxiv2019

Dynamic preferences account for inter-animal variability during the continual learning of a cognitive task

Individual animals perform tasks in different ways, yet the nature and origin of that variability is poorly understood. In the context of spatial memory tasks, variability is often interpreted as resulting from differences in memory ability, but the validity of this interpretation is seldom tested since we lack a systematic approach for identifying and understanding factors that make one animals behavior different than another. Here we identify such factors in the context of spatial alternation in rats, a task often described as relying solely on memory of past choices. We combine hypothesis-driven behavioral design and reinforcement learning modeling to identify spatial preferences that, when combined with memory, support learning of a spatial alternation task. Identifying these preferences allows us to capture differences among animals, including differences in overall learning ability. Our results show that to understand the complexity of behavior requires quantitative accounts of the preferences of each animal.

neuroscience↗

bioRxiv2019

Robust and replicable measurement for prepulse inhibition of the acoustic startle response

Measuring animal behavior in the context of experimental manipulation is critical for modeling and understanding neuro-psychiatric disease. Prepulse inhibition of the acoustic startle response (PPI) is a behavioral paradigm used extensively for this purpose, but the results of PPI studies are often inconsistent. As a result, the utility of this metric remains uncertain. Here we deconstruct the phenomenon of PPI. We first confirm several limitations of the traditional PPI metric, including that the underlying startle response has a non-Gaussian distribution and that the traditional PPI metric changes with different stimulus condition. We then develop a novel model that reveals PPI to be a combination of the previously appreciated scaling of the startle response, as well as a scaling of sound perception. Using our model, we find no evidence for differences in PPI in a rat model of Fragile-X Syndrome (FXS) compared to wild-type controls. These results in the rat provide a reliable methodology that could be used to clarify inconsistent PPI results in mice and humans. In addition, we find robust differences between wild-type male and female rats. Our model allows us to understand the nature of these differences, and we find that both the startle-scaling and sound-scaling components of PPI are a function of the baseline startle response. Males and females differ specifically in the startle-scaling, but not the sound-scaling, component of PPI. These findings establish a robust experimental and analytical approach that has the potential to provide a consistent biomarker of brain function.

animal behavior and cognition↗

bioRxiv2019

Rod-shaped tricalbins contribute to PM asymmetry at curved ER-PM contact sites

Lipid flow between cellular organelles occurs via membrane contact sites that form dynamic conduits. Extended-synaptotagmins, known as tricalbins in yeast, mediate lipid transfer between the endoplasmic reticulum (ER) and plasma membrane (PM). How these proteins regulate the membrane architecture to transport lipids across the aqueous space between bilayers remains unknown. Using correlative microscopy, electron cryo-tomography and high-throughput genetics we address this interplay of architecture and function in budding yeast. We find that ER-PM contacts are diverse in protein composition and membrane morphology, not in intermembrane distance. In situ cryo-EM of tricalbins reveals their molecular organisation that suggests an unexpected structural framework for lipid transfer. Genetic analysis identifies functional redundancies, both for tricalbin domains and cellular lipid routes, and points to tricalbin function in maintenance of PM asymmetry. These results uncover a modularity of molecular and structural functions of tricalbins, and of their roles within the network of cellular lipid fluxes.

cell biology↗