James Rothman

Primary familial brain calcification (PFBC) is characterized by calcium deposition in the brain, causing progressive movement disorders, psychiatric symptoms, and cognitive decline. PFBC is a heterogeneous disorder currently linked to variants in six different genes, but most patients remain genetically undiagnosed. Here, we identify biallelic NAA60 variants in ten individuals from seven families with autosomal recessive PFBC. The NAA60 variants lead to loss-of-function with lack of protein N-terminal (Nt)-acetylation activity. We show that the phosphate importer SLC20A2 is a substrate of NAA60 in vitro. In cells, loss of NAA60 caused reduced surface levels of SLC20A2 and a reduction in extracellular phosphate uptake. This study establishes NAA60 as a causal gene for PFBC, provides a possible biochemical explanation of its disease-causing mechanisms and underscores NAA60-mediated Nt-acetylation of …

The intricate molecular environment of the native membrane profoundly influences every aspect of membrane protein (MP) biology. Despite this, the most prevalent method of studying MPs uses detergent- like molecules that disrupt and remove this vital local membrane context. This severely impedes our ability to quantitatively decipher the local molecular context and comprehend its regulatory role in the structure, function, and biogenesis of MPs. Using a library of membrane-active polymers we have developed a platform for the high-throughput analysis of the membrane proteome. The platform enables near-complete spatially resolved extraction of target MPs directly from their endogenous membranes into native nanodiscs that maintain the local membrane context. We accompany this advancement with an open-access quantitative database that provides the most efficient extraction conditions of 2065 unique mammalian MPs. Our method enables rapid and near-complete extraction and purification of target MPs directly from their endogenous organellar membranes at physiological expression levels while maintaining the nanoscale local membrane environment. Going beyond the plasma membrane proteome, our platform enables extraction from any target organellar membrane including the endoplasmic reticulum, mitochondria, lysosome, Golgi, and even transient organelles such as the autophagosome. To further validate this platform we took several independent MPs and demonstrated how our resource can enable rapid extraction and purification of target MPs from different organellar membranes with high efficiency and purity. Further …

Mapping the intricate spatial relationships between the many different molecules inside a cell is essential to understanding cellular functions in all their complexity. Super-resolution fluorescence microscopy offers the required spatial resolution but struggles to reveal more than four different targets simultaneously. Exchanging labels in subsequent imaging rounds for multiplexed imaging extends this number but is limited by its low throughput. Here, we present a method for rapid multiplexed super-resolution microscopy that can, in principle, be applied to a nearly unlimited number of molecular targets by leveraging fluorogenic labeling in conjunction with transient adapter-mediated switching for high-throughput DNA-PAINT (FLASH-PAINT). We demonstrate the versatility of FLASH-PAINT with four applications: mapping nine proteins in a single mammalian cell, elucidating the functional organization of primary cilia by …

1Department of Cell Biology, Yale University, New Haven, CT, USA, 2Department of Pharmacology, Yale University, New Haven, CT, USA. The local membrane environment plays a paramount role in regulating the biology of membrane proteins (MP). Standard detergent-based MP-solubilization strategies disrupt the native bilayer-environment, leading to a fundamental gap in our understanding of how nanoscale molecular organization of MPs regulates their ability to elicit cellular responses. Addressing this, we have developed a proteome-wide quantitative guide for high-throughput, spatiallyresolved extraction of MPs into tunable native nanodiscs of 8-20 nm diameter. This enables downstream structural/functional studies of target MPs directly from native membranes. Combining existing MAPs with in-house chaincontrolled polymers, we developed a high-throughput, fluorescence-based assay for rapid screening …

This year’s Albert Lasker Basic Medical Research Award recognizes the invention of AlphaFold, a revolutionary advance in the history of protein research which for the first time offers the practical ability to accurately predict the three-dimensional arrangement of amino acids in the vast majority of proteins on a genomic scale on the basis of sequence alone [J. Jumper et al., Nature 596, 583–589 (2021) and K. Tunyasuvunakool et al., Nature 596, 590–596 (2021)]. This extraordinary achievement by Demis Hassabis and John Jumper and their coworkers at Google’s DeepMind and other collaborators was built on decades of experimental protein structure determination (structural biology) as well as the gradual development of multiple strategies incorporating biologically inspired statistical approaches. But when Jumper and Hassabis added a brew of innovative neural network-based machine learning approaches to …

Golgins are long (50-400 nm) rod-like membrane proteins, enveloping the Golgi apparatus (GA). They initially capture cognate cargo vesicle, to facilitate Snare-mediated membrane fusion. Most of them can phase separate and this may result to internal organization of the GA. Golgi matrix protein 130 (GM130), is the most abundant Golgin located at the cis Golgi. In our study of recombinant GM130 on supported bilayer system, we observed that GM130 self-assembles and phase separates into two distinct phases: A Mesh phase and a Condensate phase. Here we will describe the precise nature of these phases and their hypothetical physiological roles in the GA.

The subject matter of the present disclosure generally relates to techniques for neuromodulation of a tissue that include applying energy (eg, ultrasound energy) into the tissue to cause altered activity at a synapse between a neuron and a non-neuronal cell.

Evidence from biochemistry, genetics, and electron microscopy strongly supports the idea that a ring of Synaptotagmin is central to the clamping and release of synaptic vesicles (SVs) for synchronous neurotransmission. Recent direct measurements in cell‐free systems suggest there are 12 SNAREpins in each ready‐release vesicle, consisting of six peripheral and six central SNAREpins. The six central SNAREpins are directly bound to the Synaptotagmin ring, are directly released by Ca++, and they initially open the fusion pore. The six peripheral SNAREpins are indirectly bound to the ring, each linked to a central SNAREpin by a bridging molecule of Complexin. We suggest that the primary role of peripheral SNAREpins is to provide additional force to ‘turbocharge’ neurotransmitter release, explaining how it can occur much faster than other forms of membrane fusion. The SV protein Synaptophysin forms …