Synaptotagmin-Mediated Bending of the Target Membrane Is a Critical Step in Ca2+-Regulated Fusion

SummaryDecades ago it was proposed that exocytosis involves invagination of the target membrane, resulting in a highly localized site of contact between the bilayers destined to fuse. The vesicle protein synaptotagmin-I (syt) bends membranes in response to Ca2+, but whether this drives localized invagination of the target membrane to accelerate fusion has not been determined. Previous studies relied on reconstituted vesicles that were already highly curved and used mutations in syt that were not selective for membrane-bending activity. Here, we directly address this question by utilizing vesicles with different degrees of curvature. A tubulation-defective syt mutant was able to promote fusion between highly curved SNARE-bearing liposomes but exhibited a marked loss of activity when the membranes were relatively flat. Moreover, bending of flat membranes by adding an N-BAR domain rescued the function of the tubulation-deficient syt mutant. Hence, syt-mediated membrane bending is a critical step in membrane fusion

Ca2+ and synaptotagmin VII–dependent delivery of lysosomal membrane to nascent phagosomes

Synaptotagmin (Syt) VII is a ubiquitously expressed member of the Syt family of Ca2+ sensors. It is present on lysosomes in several cell types, where it regulates Ca2+-dependent exocytosis. Because [Ca2+]i and exocytosis have been associated with phagocytosis, we investigated the phagocytic ability of macrophages from Syt VII−/− mice. Syt VII−/− macrophages phagocytose normally at low particle/cell ratios but show a progressive inhibition in particle uptake under high load conditions. Complementation with Syt VII rescues this phenotype, but only when functional Ca2+-binding sites are retained. Reinforcing a role for Syt VII in Ca2+-dependent phagocytosis, particle uptake in Syt VII−/− macrophages is significantly less dependent on [Ca2+]i. Syt VII is concentrated on peripheral domains of lysosomal compartments, from where it is recruited to nascent phagosomes. Syt VII recruitment is rapidly followed by the delivery of Lamp1 to phagosomes, a process that is inhibited in Syt VII−/− macrophages. Thus, Syt VII regulates the Ca2+-dependent mobilization of lysosomes as a supplemental source of membrane during phagocytosis

Phase separation of signaling molecules promotes T cell receptor signal transduction

Author Posting. © The Author(s), 2016. This is the author's version of the work. It is posted here by permission of American Association for the Advancement of Science for personal use, not for redistribution. The definitive version was published in Science 352 (2016): 595-599, doi:10.1126/science.aad9964.Activation of various cell surface receptors triggers the reorganization of downstream signaling molecules into micron- or submicron-sized clusters. However, the functional consequences of such clustering has been unclear. We biochemically reconstituted a 12-component signaling pathway on model membranes, beginning with T cell receptor (TCR) activation and ending with actin assembly. When TCR phoshophorylation was triggered, downstream signaling proteins spontaneously separated into liquid-like clusters that promoted signaling outputs both in vitro and in human Jurkat T cells. Reconstituted clusters were enriched in kinases but excluded phosphatases, and enhanced actin filament assembly by recruiting and organizing actin regulators. These results demonstrate that protein phase separation can create a distinct physical and biochemical compartment that facilitates signaling.This work was supported by the HCIA program of HHMI, the NIH (R01-GM56322 to M.K.R.) and Welch Foundation (I–1544 to M.K.R.). X.S. was supported by CRI Irvington postdoctoral fellowship. J.A.D. was supported by NRSA F32 award 5-F32-DK101188. E.H. was supported as a fellow of the Leukemia and Lymphoma Society. J.O. was supported by funds from Tobacco-Related Disease Research Program of the University of California (19FT-0090).2016-10-0

Transmission Roles of Symptomatic and Asymptomatic COVID-19 Cases: A Modelling Study

Coronavirus disease 2019 (COVID-19) asymptomatic cases are hard to identify, impeding transmissibility estimation. The value of COVID-19 transmissibility is worth further elucidation for key assumptions in further modelling studies. Through a population-based surveillance network, we collected data on 1342 confirmed cases with a 90-days follow-up for all asymptomatic cases. An age-stratified compartmental model containing contact information was built to estimate the transmissibility of symptomatic and asymptomatic COVID-19 cases. The difference in transmissibility of a symptomatic and asymptomatic case depended on age and was most distinct for the middle-age groups. The asymptomatic cases had a 66.7% lower transmissibility rate than symptomatic cases, and 74.1% (95% CI 65.9–80.7) of all asymptomatic cases were missed in detection. The average proportion of asymptomatic cases was 28.2% (95% CI 23.0–34.6). Simulation demonstrated that the burden of asymptomatic transmission increased as the epidemic continued and could potentially dominate total transmission. The transmissibility of asymptomatic COVID-19 cases is high and asymptomatic COVID-19 cases play a significant role in outbreaks

In vitro membrane reconstitution of the T-cell receptor proximal signaling network.

T-cell receptor (TCR) phosphorylation is controlled by a complex network that includes Lck, a Src family kinase (SFK), the tyrosine phosphatase CD45 and the Lck-inhibitory kinase Csk. How these competing phosphorylation and dephosphorylation reactions are modulated to produce T-cell triggering is not fully understood. Here we reconstituted this signaling network using purified enzymes on liposomes, recapitulating the membrane environment in which they normally interact. We demonstrate that Lcks enzymatic activity can be regulated over an ~10-fold range by controlling its phosphorylation state. By varying kinase and phosphatase concentrations, we constructed phase diagrams that reveal ultrasensitivity in the transition from the quiescent to the phosphorylated state and demonstrate that co-clustering TCR and Lck or detaching Csk from the membrane can trigger TCR phosphorylation. Our results provide insight into the mechanism of TCR signaling as well as other signaling pathways involving SFKs

Molecular features underlying differential SHP1/SHP2 binding of immune checkpoint receptors

A large number of inhibitory receptors recruit SHP1 and/or SHP2, tandem-SH2-containing phosphatases through phosphotyrosine-based motifs immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM). Despite the similarity, these receptors exhibit differential effector binding specificities, as exemplified by the immune checkpoint receptors PD-1 and BTLA, which preferentially recruit SHP2 and SHP1, respectively. The molecular basis by which structurally similar receptors discriminate SHP1 and SHP2 is unclear. Here, we provide evidence that human PD-1 and BTLA optimally bind to SHP1 and SHP2 via a bivalent, parallel mode that involves both SH2 domains of SHP1 or SHP2. PD-1 mainly uses its ITSM to prefer SHP2 over SHP1 via their C-terminal SH2 domains (cSH2): swapping SHP1-cSH2 with SHP2-cSH2 enabled PD-1:SHP1 association in T cells. In contrast, BTLA primarily utilizes its ITIM to prefer SHP1 over SHP2 via their N-terminal SH2 domains (nSH2). The ITIM of PD-1, however, appeared to be de-emphasized due to a glycine at pY+1 position. Substitution of this glycine with alanine, a residue conserved in BTLA and several SHP1-recruiting receptors, was sufficient to induce PD-1:SHP1 interaction in T cells. Finally, structural simulation and mutagenesis screening showed that SHP1 recruitment activity exhibits a bell-shaped dependence on the molecular volume of the pY+1 residue of ITIM. Collectively, we provide a molecular interpretation of the SHP1/SHP2-binding specificities of PD-1 and BTLA, with implications for the mechanisms of a large family of therapeutically relevant receptors