Vesicles: Looking inside the cell.

Advances in imaging techniques have shed new light on the structure of vesicles formed by COPI protein complexes

Dissection of GTPase activating proteins reveals functional asymmetry in the COPI coat of budding yeast.

The Arf GTPase controls formation of the COPI vesicle coat. Recent structural models of COPI revealed the positioning of two Arf1 molecules in contrasting molecular environments. Each of these pockets for Arf1 is expected to also accommodate an Arf GTPase-activating protein (ArfGAP). Structural evidence and protein interactions observed between isolated domains indirectly suggests that each niche may preferentially recruit one of the two ArfGAPs known to affect COPI, Gcs1/ArfGAP1 and Glo3/ArfGAP2/3, although only partial structures are available. The functional role of the unique non-catalytic domain of either ArfGAP has not been integrated into the current COPI structural model. Here, we delineate key differences in the consequences of triggering GTP hydrolysis via the activity of one versus the other ArfGAP. We demonstrate that Glo3/ArfGAP2/3 specifically triggers Arf1 GTP hydrolysis impinging on the stability of the COPI coat. We show that the yeast homologue of AMP kinase, Snf1, phosphorylates the region of Glo3 that is critical for this effect and thereby regulates its function in the COPI-vesicle cycle. Our results revise the model of ArfGAP function in the molecular context of COPI

The SND proteins constitute an alternative targeting route to the endoplasmic reticulum.

In eukaryotes, up to one-third of cellular proteins are targeted to the endoplasmic reticulum, where they undergo folding, processing, sorting and trafficking to subsequent endomembrane compartments(1). Targeting to the endoplasmic reticulum has been shown to occur co-translationally by the signal recognition particle (SRP) pathway(2) or post-translationally by the mammalian transmembrane recognition complex of 40 kDa (TRC40)(3,4) and homologous yeast guided entry of tail-anchored proteins (GET)(5,6) pathways. Despite the range of proteins that can be catered for by these two pathways, many proteins are still known to be independent of both SRP and GET, so there seems to be a critical need for an additional dedicated pathway for endoplasmic reticulum relay(7,8). We set out to uncover additional targeting proteins using unbiased high-content screening approaches. To this end, we performed a systematic visual screen using the yeast Saccharomyces cerevisiae(9,10), and uncovered three uncharacterized proteins whose loss affected targeting. We suggest that these proteins work together and demonstrate that they function in parallel with SRP and GET to target a broad range of substrates to the endoplasmic reticulum. The three proteins, which we name Snd1, Snd2 and Snd3 (for SRP-independent targeting), can synthetically compensate for the loss of both the SRP and GET pathways, and act as a backup targeting system. This explains why it has previously been difficult to demonstrate complete loss of targeting for some substrates. Our discovery thus puts in place an essential piece of the endoplasmic reticulum targeting puzzle, highlighting how the targeting apparatus of the eukaryotic cell is robust, interlinked and flexible

Complex consequences of Cantu syndrome SUR2 variant R1154Q in genetically modified mice

Cantu syndrome (CS) is caused by gain-of-function (GOF) mutations in pore-forming (Kir6.1, KCNJ8) and accessory (SUR2, ABCC9) ATP-sensitive potassium (KATP) channel subunits, the most common mutations being SUR2[R1154Q] and SUR2[R1154W], carried by approximately 30% of patients. We used CRISPR/Cas9 genome engineering to introduce the equivalent of the human SUR2[R1154Q] mutation into the mouse ABCC9 gene. Along with minimal CS disease features, R1154Q cardiomyocytes and vascular smooth muscle showed much lower KATP current density and pinacidil activation than WT cells. Almost complete loss of SUR2-dependent protein and KATP in homozygous R1154Q ventricles revealed underlying diazoxide-sensitive SUR1-dependent KATP channel activity. Surprisingly, sequencing of SUR2 cDNA revealed 2 distinct transcripts, one encoding full-length SUR2 protein; and the other with an in-frame deletion of 93 bases (corresponding to 31 amino acids encoded by exon 28) that was present in approximately 40% and approximately 90% of transcripts from hetero- and homozygous R1154Q tissues, respectively. Recombinant expression of SUR2A protein lacking exon 28 resulted in nonfunctional channels. CS tissue from SUR2[R1154Q] mice and human induced pluripotent stem cell-derived (hiPSC-derived) cardiomyocytes showed only full-length SUR2 transcripts, although further studies will be required in order to fully test whether SUR2[R1154Q] or other CS mutations might result in aberrant splicing and variable expressivity of disease features in human CS

Axial tubule junctions control rapid calcium signaling in atria.

The canonical atrial myocyte (AM) is characterized by sparse transverse tubule (TT) invaginations and slow intracellular Ca2+ propagation but exhibits rapid contractile activation that is susceptible to loss of function during hypertrophic remodeling. Here, we have identified a membrane structure and Ca2+-signaling complex that may enhance the speed of atrial contraction independently of phospholamban regulation. This axial couplon was observed in human and mouse atria and is composed of voluminous axial tubules (ATs) with extensive junctions to the sarcoplasmic reticulum (SR) that include ryanodine receptor 2 (RyR2) clusters. In mouse AM, AT structures triggered Ca2+ release from the SR approximately 2 times faster at the AM center than at the surface. Rapid Ca2+ release correlated with colocalization of highly phosphorylated RyR2 clusters at AT-SR junctions and earlier, more rapid shortening of central sarcomeres. In contrast, mice expressing phosphorylation-incompetent RyR2 displayed depressed AM sarcomere shortening and reduced in vivo atrial contractile function. Moreover, left atrial hypertrophy led to AT proliferation, with a marked increase in the highly phosphorylated RyR2-pS2808 cluster fraction, thereby maintaining cytosolic Ca2+ signaling despite decreases in RyR2 cluster density and RyR2 protein expression. AT couplon "super-hubs" thus underlie faster excitation-contraction coupling in health as well as hypertrophic compensatory adaptation and represent a structural and metabolic mechanism that may contribute to contractile dysfunction and arrhythmias

The essential role of multi-point measurements in investigations of turbulence, three-dimensional structure, and dynamics: the solar wind beyond single scale and the Taylor Hypothesis

Space plasmas are three-dimensional dynamic entities. Except under very special circumstances, their structure in space and their behavior in time are not related in any simple way. Therefore, single spacecraft in situ measurements cannot unambiguously unravel the full space-time structure of the heliospheric plasmas of interest in the inner heliosphere, in the Geospace environment, or the outer heliosphere. This shortcoming leaves numerous central questions incompletely answered. Deficiencies remain in at least two important subjects, Space Weather and fundamental plasma turbulence theory, due to a lack of a more complete understanding of the space-time structure of dynamic plasmas. Only with multispacecraft measurements over suitable spans of spatial separation and temporal duration can these ambiguities be resolved. We note that these characterizations apply to turbulence across a wide range of scales, and also equally well to shocks, flux ropes, magnetic clouds, current sheets, stream interactions, etc. In the following, we will describe the basic requirements for resolving space-time structure in general, using turbulence' as both an example and a principal target or study. Several types of missions are suggested to resolve space-time structure throughout the Heliosphere.Comment: White Paper submitted to: Decadal Survey for Solar and Space Physics (Heliophysics) 2024-2033. arXiv admin note: substantial text overlap with arXiv:1903.0689

Pore timing:the evolutionary origins of the nucleus and nuclear pore complex

The name “eukaryote” is derived from Greek, meaning “true kernel”, and describes the domain of organisms whose cells have a nucleus. The nucleus is thus the defining feature of eukaryotes and distinguishes them from prokaryotes (Archaea and Bacteria), whose cells lack nuclei. Despite this, we discuss the intriguing possibility that organisms on the path from the first eukaryotic common ancestor to the last common ancestor of all eukaryotes did not possess a nucleus at all—at least not in a form we would recognize today—and that the nucleus in fact arrived relatively late in the evolution of eukaryotes. The clues to this alternative evolutionary path lie, most of all, in recent discoveries concerning the structure of the nuclear pore complex. We discuss the evidence for such a possibility and how this impacts our views of eukaryote origins and how eukaryotes have diversified subsequent to their last common ancestor

Large-Eddy Simulations of Magnetohydrodynamic Turbulence in Heliophysics and Astrophysics

We live in an age in which high-performance computing is transforming the way we do science. Previously intractable problems are now becoming accessible by means of increasingly realistic numerical simulations. One of the most enduring and most challenging of these problems is turbulence. Yet, despite these advances, the extreme parameter regimes encountered in space physics and astrophysics (as in atmospheric and oceanic physics) still preclude direct numerical simulation. Numerical models must take a Large Eddy Simulation (LES) approach, explicitly computing only a fraction of the active dynamical scales. The success of such an approach hinges on how well the model can represent the subgrid-scales (SGS) that are not explicitly resolved. In addition to the parameter regime, heliophysical and astrophysical applications must also face an equally daunting challenge: magnetism. The presence of magnetic fields in a turbulent, electrically conducting fluid flow can dramatically alter the coupling between large and small scales, with potentially profound implications for LES/SGS modeling. In this review article, we summarize the state of the art in LES modeling of turbulent magnetohydrodynamic (MHD) ows. After discussing the nature of MHD turbulence and the small-scale processes that give rise to energy dissipation, plasma heating, and magnetic reconnection, we consider how these processes may best be captured within an LES/SGS framework. We then consider several special applications in heliophysics and astrophysics, assessing triumphs, challenges,and future directions

Formation of COPI-coated vesicles at a glance.

The coat protein complex I (COPI) allows the precise sorting of lipids and proteins between Golgi cisternae and retrieval from the Golgi to the ER. This essential role maintains the identity of the early secretory pathway and impinges on key cellular processes, such as protein quality control. In this Cell Science at a Glance and accompanying poster, we illustrate the different stages of COPI-coated vesicle formation and revisit decades of research in the context of recent advances in the elucidation of COPI coat structure. By calling attention to an array of questions that have remained unresolved, this review attempts to refocus the perspectives of the field