Titration of syntaxin1 in Mammalian synapses reveals multiple roles in vesicle docking, priming, and release probability.

Synaptic vesicles undergo sequential steps in preparation for neurotransmitter release. Individual SNARE proteins and the SNARE complex itself have been implicated in these processes. However, discrete effects of SNARE proteins on synaptic function have been difficult to assess using complete loss-of-function approaches. We therefore used a genetic titration technique in cultured mouse hippocampal neurons to evaluate the contribution of the neuronal SNARE protein Syntaxin1 (Stx1) in vesicle docking, priming, and release probability. We generated graded reductions of total Stx1 levels by combining two approaches, namely, endogenous hypomorphic expression of the isoform Stx1B and RNAi-mediated knockdown. Proximity of synaptic vesicles to the active zone was not strongly affected. However, overall release efficiency of affected neurons was severely impaired, as demonstrated by a smaller readily releasable pool size, slower refilling rate of primed vesicles, and lower release probability. Interestingly, dose-response fitting of Stx1 levels against readily releasable pool size and vesicular release probability showed similar Kd (dissociation constant) values at 18% and 19% of wild-type Stx1, with cooperativity estimates of 3.4 and 2.5, respectively. This strongly suggests that priming and vesicle fusion share the same molecular stoichiometry, and are governed by highly related mechanisms

Lack of Laminar Shear Stress Facilitates the Endothelial Uptake of Very Small Superparamagnetic Iron Oxide Nanoparticles by Modulating the Endothelial Surface Layer

Shailey Gale Twamley,1– 3 Niclas Gimber,4 Héctor Eduardo Sánchez-Ibarra,1,2 Tobias Christaller,1,2 Victoria Isakzai,1,2 Harald Kratz,5 Ronodeep Mitra,6 Lena Kampen,1– 3 Anke Stach,1,2 Heike Heilmann,7 Berit Söhl-Kielczynski,8 Eno Essien Ebong,6,9,10 Jan Schmoranzer,4 Agnieszka Münster-Wandowski,7,* Antje Ludwig1– 3,* 1Department of Cardiology, Angiology and Intensive Care Medicine, Deutsches Herzzentrum der Charité, Berlin, Germany; 2Department of Cardiology, Angiology and Intensive Care Medicine, Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität Zu Berlin, Berlin, Germany; 3DZHK (German Centre for Cardiovascular Research), Partner Site Berlin, Berlin, Germany; 4Advanced Medical Bioimaging Core Facility (AMBIO), Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany; 5Department of Radiology, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany; 6Department of Chemical Engineering, Northeastern University, Boston, MA, USA; 7Institute of Integrative Neuroanatomy, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany; 8Institute for Integrative Neurophysiology, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität Zu Berlin, and Berlin Institute of Health, Berlin, Germany; 9Department of Bioengineering, Northeastern University, Boston, MA, USA; 10Department of Neuroscience, Albert Einstein College of Medicine, New York, NY, USA*These authors contributed equally to this workCorrespondence: Shailey Gale Twamley; Antje Ludwig, Tel +49-30-450-528455 ; +49-30-450-513196, Fax +49-30-450-528922 ; +49-30-450941, Email [email protected]; [email protected]: To study whether the absence of laminar shear stress (LSS) enables the uptake of very small superparamagnetic iron oxide nanoparticles (VSOP) in endothelial cells by altering the composition, size, and barrier function of the endothelial surface layer (ESL).Methods and Results: A quantitative particle exclusion assay with living human umbilical endothelial cells using spinning disc confocal microscopy revealed that the dimension of the ESL was reduced in cells cultivated in the absence of LSS. By combining gene expression analysis, flow cytometry, high pressure freezing/freeze substitution immuno-transmission electron microscopy, and confocal laser scanning microscopy, we investigated changes in ESL composition. We found that increased expression of the hyaluronan receptor CD44 by absence of shear stress did not affect the uptake rate of VSOPs. We identified collagen as a previously neglected component of ESL that contributes to its barrier function. Experiments with inhibitor halofuginone and small interfering RNA (siRNA) demonstrated that suppression of collagen expression facilitates VSOP uptake in endothelial cells grown under LSS.Conclusion: The absence of laminar shear stress disturbs the barrier function of the ESL, facilitating membrane accessibility and endocytic uptake of VSOP. Collagen, a previously neglected component of ESL, contributes to its barrier function. Keywords: citrate coated nanoparticles, atherosclerosis, blood flow, endothelial barrier, permeability, internalizatio

Timed global reorganization of protein synthesis during neocortex neurogenesis at codon resolution

Translation modulates the timing and amplification of gene expression after transcription. Development of the brain’s neocortex requires precisely timed and spatially targeted gene expression, but the relationship between mRNA vs. protein synthesis throughout the genome is unknown. We perform a comprehensive analysis of the reactants, synthesis, and products of mRNA translation spanning mouse neocortex neurogenesis. Ribosome number in the cortical plate decreases sharply at mid-neurogenesis during a transition in neuronal subtype specification, shifting the fundamental kinetics of protein synthesis, with mRNA and protein levels frequently divergent. Satb2, which drives an essential neuronal subtype-specific program, is a highly dynamically translated mRNA with surprisingly broad transcription across diverse neuronal lineages. Satb2 protein achieves its neuronal subtype expression through timed regulation by the RNA-binding protein Pumilio2. Thus, the refinement of transcriptional programs by protein synthesis is a widespread feature of neuronal specification. Developmental neocortex translatome data are provided in an open-source resource: https://shiny.mdc-berlin.de/cortexomics/

The architecture of protein synthesis in the developing neocortex at near-atomic resolution reveals Ebp1-mediated neuronal proteostasis at the 60S tunnel exit

Protein synthesis must be finely tuned in the nervous system, as it represents an essential feature of neurodevelopmental gene expression, and dominant pathology in neurological disease. However, the architecture of ribosomal complexes in the developing mammalian brain has not been analyzed at high resolution. This study investigates the architecture of ribosomes ex vivo from the embryonic and perinatal mouse neocortex, revealing Ebp1 as a 60S peptide tunnel exit binding factor at near-atomic resolution by multiparticle cryo-electron microscopy. The impact of Ebp1 on the neuronal proteome was analyzed by pSILAC and BONCAT coupled mass spectrometry, implicating Ebp1 in neurite outgrowth proteostasis, with in vivo embryonic Ebp1 knockdown resulting in dysregulation of neurite outgrowth. Our findings reveal Ebp1 as a central component of neocortical protein synthesis, and the 60S peptide tunnel exit as a focal point of gene expression control in the molecular specification of neuronal morphology

A critical period of translational control during brain development at codon resolution

Translation modulates the timing and amplification of gene expression after transcription. Brain development requires uniquely complex gene expression patterns, but large-scale measurements of translation directly in the prenatal brain are lacking. We measure the reactants, synthesis and products of mRNA translation spanning mouse neocortex neurogenesis, and discover a transient window of dynamic regulation at mid-gestation. Timed translation upregulation of chromatin-binding proteins like Satb2, which is essential for neuronal subtype differentiation, restricts protein expression in neuronal lineages despite broad transcriptional priming in progenitors. In contrast, translation downregulation of ribosomal proteins sharply decreases ribosome biogenesis, coinciding with a major shift in protein synthesis dynamics at mid-gestation. Changing activity of eIF4EBP1, a direct inhibitor of ribosome biogenesis, is concurrent with ribosome downregulation and affects neurogenesis of the Satb2 lineage. Thus, the molecular logic of brain development includes the refinement of transcriptional programs by translation. Modeling of the developmental neocortex translatome is provided as an open-source searchable resource at https://shiny.mdc-berlin.de/cortexomics

Protein synthesis in the developing neocortex at near-atomic resolution reveals Ebp1-mediated neuronal proteostasis at the 60S tunnel exit

Protein synthesis must be finely tuned in the developing nervous system as the final essential step of gene expression. This study investigates the architecture of ribosomes from the neocortex during neurogenesis, revealing Ebp1 as a high-occupancy 60S peptide tunnel exit (TE) factor during protein synthesis at near-atomic resolution by cryoelectron microscopy (cryo-EM). Ribosome profiling demonstrated Ebp1-60S binding is highest during start codon initiation and N-terminal peptide elongation, regulating ribosome occupancy of these codons. Membrane-targeting domains emerging from the 60S tunnel, which recruit SRP/Sec61 to the shared binding site, displace Ebp1. Ebp1 is particularly abundant in the early-born neural stem cell (NSC) lineage and regulates neuronal morphology. Ebp1 especially impacts the synthesis of membrane-targeted cell adhesion molecules (CAMs), measured by pulsed stable isotope labeling by amino acids in cell culture (pSILAC)/bioorthogonal noncanonical amino acid tagging (BONCAT) mass spectrometry (MS). Therefore, Ebp1 is a central component of protein synthesis, and the ribosome TE is a focal point of gene expression control in the molecular specification of neuronal morphology during development