Rubén Fernández-Busnadiego

Quantitative analysis of the native presynaptic cytomatrix by cryoelectron tomography

The filamentous structures that tether exocytic vesicles to the plasma membrane in the active zone are rearranged in response to synaptic stimulation.

Three-dimensional architecture of extended synaptotagmin-mediated endoplasmic reticulum–plasma membrane contact sites

Significance Membrane contact sites, the sites of close physical proximity between intracellular membranes, are emerging as key players in multiple cellular processes. In particular, endoplasmic reticulum (ER)–plasma membrane (PM) contact sites play important roles in calcium homeostasis, signaling, and lipid regulation/exchange. However, the architecture of these contact sites remains poorly understood. Here we study the 3D architecture of ER–PM contact sites at molecular resolution using cryo-electron tomography. We define the structural signature of the ER–PM tethers mediated by the extended synaptotagmins (E-Syts) and show that E-Syts regulate ER–PM distance in a cytosolic Ca2+-dependent manner. These findings provide an important foundation towards elucidating E-Syt function and more generally the mechanisms of cross-talk between the ER and the PM. The close apposition between the endoplasmic reticulum (ER) and the plasma membrane (PM) plays important roles in Ca2+ homeostasis, signaling, and lipid metabolism. The extended synaptotagmins (E-Syts; tricalbins in yeast) are ER-anchored proteins that mediate the tethering of the ER to the PM and are thought to mediate lipid transfer between the two membranes. E-Syt cytoplasmic domains comprise a synaptotagmin-like mitochondrial-lipid–binding protein (SMP) domain followed by five C2 domains in E-Syt1 and three C2 domains in E-Syt2/3. Here, we used cryo-electron tomography to study the 3D architecture of E-Syt–mediated ER–PM contacts at molecular resolution. In vitrified frozen-hydrated mammalian cells overexpressing individual E-Syts, in which E-Syt–dependent contacts were by far the predominant contacts, ER–PM distance (19–22 nm) correlated with the amino acid length of the cytosolic region of E-Syts (i.e., the number of C2 domains). Elevation of cytosolic Ca2+ shortened the ER–PM distance at E-Syt1–dependent contacts sites. E-Syt–mediated contacts displayed a characteristic electron-dense layer between the ER and the PM. These features were strikingly different from those observed in cells exposed to conditions that induce contacts mediated by the stromal interaction molecule 1 (STIM1) and the Ca2+ channel Orai1 as well as store operated Ca2+ entry. In these cells the gap between the ER and the PM was spanned by filamentous structures perpendicular to the membranes. Our results define specific ultrastructural features of E-Syt–dependent ER–PM contacts and reveal their structural plasticity, which may impact on the cross-talk between the ER and the PM and the functions of E-Syts in lipid transport between the two bilayers.

Cryo–electron tomography reveals a critical role of RIM1α in synaptic vesicle tethering

RIM1α-deficient synapses show structural defects in presynaptic vesicle distribution and tethering to the active zone that can be reversed by proteasome inhibition.

Cryo-electron tomography: methodology, developments and biological applications

Cryo‐electron tomography allows three‐dimensional visualization of frozen‐hydrated, vitrified biological material at molecular resolution. Here, we summarize the most important sample preparation methods and technical aspects relevant for cryo‐electron tomography, as well as its recent biological applications from isolated macromolecular complexes to entire cells and tissues.

Epsin deficiency impairs endocytosis by stalling the actin-dependent invagination of endocytic clathrin-coated pits

Epsin is an evolutionarily conserved endocytic clathrin adaptor whose most critical function(s) in clathrin coat dynamics remain(s) elusive. To elucidate such function(s), we generated embryonic fibroblasts from conditional epsin triple KO mice. Triple KO cells displayed a dramatic cell division defect. Additionally, a robust impairment in clathrin-mediated endocytosis was observed, with an accumulation of early and U-shaped pits. This defect correlated with a perturbation of the coupling between the clathrin coat and the actin cytoskeleton, which we confirmed in a cell-free assay of endocytosis. Our results indicate that a key evolutionary conserved function of epsin, in addition to other roles that include, as we show here, a low affinity interaction with SNAREs, is to help generate the force that leads to invagination and then fission of clathrin-coated pits. DOI: http://dx.doi.org/10.7554/eLife.03311.001

Insights into the molecular organization of the neuron by cryo-electron tomography

Despite great progress in the identification and characterization of the key molecular players in neuronal function, remarkably little is known about their supramolecular organization. Cryo-electron tomography (cryo-ET), providing three-dimensional views of the molecular components of the cell in their native, fully hydrated environment, is uniquely positioned to elucidate the native architecture of the molecular machinery of the neuron. In our laboratory, we employ cryo-ET to study neuronal morphology in a variety of experimental systems and develop methods to extract quantitative and functional information from tomographic data. This approach has allowed us to shed light onto the intricate organization of the molecules of the synaptic cleft and the presynaptic cytomatrix, providing evidence for their functional roles. Also, cryo-ET of cultured neurons is beginning to open new perspectives on neuronal ultrastructure and the architecture of synaptic complexes in situ. Here, we will review these findings and discuss future directions towards the elucidation of the molecular landscape of the neuron.

Synucleins Have Multiple Effects on Presynaptic Architecture

Synucleins (α, β, γ-synuclein) are a family of abundant presynaptic proteins. α-Synuclein is causally linked to the pathogenesis of Parkinsons disease (PD). In an effort to define their physiological and pathological function or functions, we investigated the effects of deleting synucleins and overexpressing α-synuclein PD mutations, in mice, on synapse architecture using electron microscopy (EM) and cryoelectron tomography (cryo-ET). We show that synucleins are regulators of presynapse size and synaptic vesicle (SV) pool organization. Using cryo-ET, we observed that deletion of synucleins increases SV tethering to the active zone but decreases the inter-linking of SVs by short connectors. These ultrastructural changes were correlated with discrete protein phosphorylation changes in αβγ-synuclein-/- neurons. We also determined that α-synuclein PD mutants (PARK1/hA30P and PARK4/hα-syn) primarily affected presynaptic cytomatrix proximal to the active zone, congruent with previous findings that these PD mutations decrease neurotransmission. Collectively, our results suggest that synucleins are important orchestrators of presynaptic terminal topography.

The cryo-electron microscopy structure of huntingtin.

Huntingtin (HTT) is a large (348 kDa) protein that is essential for embryonic development and is involved in diverse cellular activities such as vesicular transport, endocytosis, autophagy and the regulation of transcription. Although an integrative understanding of the biological functions of HTT is lacking, the large number of identified HTT interactors suggests that it serves as a protein–protein interaction hub. Furthermore, Huntington’s disease is caused by a mutation in the HTT gene, resulting in a pathogenic expansion of a polyglutamine repeat at the amino terminus of HTT. However, only limited structural information regarding HTT is currently available. Here we use cryo-electron microscopy to determine the structure of full-length human HTT in a complex with HTT-associated protein 40 (HAP40; encoded by three F8A genes in humans) to an overall resolution of 4 Å. HTT is largely α-helical and consists of three major domains. The amino- and carboxy-terminal domains contain multiple HEAT (huntingtin, elongation factor 3, protein phosphatase 2A and lipid kinase TOR) repeats arranged in a solenoid fashion. These domains are connected by a smaller bridge domain containing different types of tandem repeats. HAP40 is also largely α-helical and has a tetratricopeptide repeat-like organization. HAP40 binds in a cleft and contacts the three HTT domains by hydrophobic and electrostatic interactions, thereby stabilizing the conformation of HTT. These data rationalize previous biochemical results and pave the way for improved understanding of the diverse cellular functions of HTT.

Supramolecular architecture of endoplasmic reticulum-plasma membrane contact sites.

The endoplasmic reticulum (ER) forms membrane contact sites (MCS) with most other cellular organelles and the plasma membrane (PM). These ER-PM MCS, where the membranes of the ER and PM are closely apposed, were discovered in the early days of electron microscopy (EM), but only recently are we starting to understand their functional and structural diversity. ER-PM MCS are nowadays known to mediate excitation-contraction coupling (ECC) in striated muscle cells and to play crucial roles in Ca(2+)and lipid homoeostasis in all metazoan cells. A common feature across ER-PM MCS specialized in different functions is the preponderance of cooperative phenomena that result in the formation of large supramolecular assemblies. Therefore, characterizing the supramolecular architecture of ER-PM MCS is critical to understand their mechanisms of function. Cryo-electron tomography (cryo-ET) is a powerful EM technique uniquely positioned to address this issue, as it allows 3D imaging of fully hydrated, unstained cellular structures at molecular resolution. In this review I summarize our current structural knowledge on the molecular organization of ER-PM MCS and its functional implications, with special emphasis on the emerging contributions of cryo-ET.

Expression of DNAJB12 or DNAJB14 Causes Coordinate Invasion of the Nucleus by Membranes Associated with a Novel Nuclear Pore Structure

DNAJB12 and DNAJB14 are transmembrane proteins in the endoplasmic reticulum (ER) that serve as co-chaperones for Hsc70/Hsp70 heat shock proteins. We demonstrate that over-expression of DNAJB12 or DNAJB14 causes the formation of elaborate membranous structures within cell nuclei, which we designate DJANGOS for DNAJ-associated nuclear globular structures. DJANGOS contain DNAJB12, DNAJB14, Hsc70 and markers of the ER lumen and ER and nuclear membranes. Strikingly, they are evenly distributed underneath the nuclear envelope and are of uniform size in any one nucleus. DJANGOS are composed primarily of single-walled membrane tubes and sheets that connect to the nuclear envelope via a unique configuration of membranes, in which the nuclear pore complex appears anchored exclusively to the outer nuclear membrane, allowing both the inner and outer nuclear membranes to flow past the circumference of the nuclear pore complex into the nucleus. DJANGOS break down rapidly during cell division and reform synchronously in the daughter cell nuclei, demonstrating that they are dynamic structures that undergo coordinate formation and dissolution. Genetic studies showed that the chaperone activity of DNAJ/Hsc70 is required for the formation of DJANGOS. Further analysis of these structures will provide insight into nuclear pore formation and function, activities of molecular chaperones, and mechanisms that maintain membrane identity.