Cortical actin recovery at the immunological synapse leads to termination of lytic granule secretion in cytotoxic T lymphocytes.

CD8+ cytotoxic T lymphocytes (CTLs) eliminate virally infected cells through directed secretion of specialized lytic granules. Because a single CTL can kill multiple targets, degranulation must be tightly regulated. However, how CTLs regulate the termination of granule secretion remains unclear. Previous work demonstrated that centralized actin reduction at the immune synapse precedes degranulation. Using a combination of live confocal, total internal reflection fluorescence, and superresolution microscopy, we now show that, after granule fusion, actin recovers at the synapse and no further secretion is observed. Depolymerization of actin led to resumed granule secretion, suggesting that recovered actin acts as a barrier preventing sustained degranulation. Furthermore, RAB27a-deficient CTLs, which do not secrete cytotoxic granules, failed to recover actin at the synapse, suggesting that RAB27a-mediated granule secretion is required for actin recovery. Finally, we show that both actin clearance and recovery correlated with synaptic phosphatidylinositol 4,5-bisphosphate (PIP2) and that alterations in PIP2 at the immunological synapse regulate cortical actin in CTLs, providing a potential mechanism through which CTLs control cortical actin density. Our work provides insight into actin-related mechanisms regulating CTL secretion that may facilitate serial killing during immune responses

Contractile actomyosin arcs promote the activation of primary mouse T cells in a ligand-dependent manner.

Mechano-transduction is an emerging but still poorly understood component of T cell activation. Here we investigated the ligand-dependent contribution made by contractile actomyosin arcs populating the peripheral supramolecular activation cluster (pSMAC) region of the immunological synapse (IS) to T cell receptor (TCR) microcluster transport and proximal signaling in primary mouse T cells. Using super resolution microscopy, OT1-CD8+ mouse T cells, and two ovalbumin (OVA) peptides with different affinities for the TCR, we show that the generation of organized actomyosin arcs depends on ligand potency and the ability of myosin 2 to contract actin filaments. While weak ligands induce disorganized actomyosin arcs, strong ligands result in organized actomyosin arcs that correlate well with tension-sensitive CasL phosphorylation and the accumulation of ligands at the IS center. Blocking myosin 2 contractility greatly reduces the difference in the extent of Src and LAT phosphorylation observed between the strong and the weak ligand, arguing that myosin 2-dependent force generation within actin arcs contributes to ligand discrimination. Together, our data are consistent with the idea that actomyosin arcs in the pSMAC region of the IS promote a mechano-chemical feedback mechanism that amplifies the accumulation of critical signaling molecules at the IS

A deep cascade of ensemble of dual domain networks with gradient-based T1 assistance and perceptual refinement for fast MRI reconstruction

Deep learning networks have shown promising results in fast magnetic resonance imaging (MRI) reconstruction. In our work, we develop deep networks to further improve the quantitative and the perceptual quality of reconstruction. To begin with, we propose reconsynergynet (RSN), a network that combines the complementary benefits of independently operating on both the image and the Fourier domain. For a single-coil acquisition, we introduce deep cascade RSN (DC-RSN), a cascade of RSN blocks interleaved with data fidelity (DF) units. Secondly, we improve the structure recovery of DC-RSN for T2 weighted Imaging (T2WI) through assistance of T1 weighted imaging (T1WI), a sequence with short acquisition time. T1 assistance is provided to DC-RSN through a gradient of log feature (GOLF) fusion. Furthermore, we propose perceptual refinement network (PRN) to refine the reconstructions for better visual information fidelity (VIF), a metric highly correlated to radiologists opinion on the image quality. Lastly, for multi-coil acquisition, we propose variable splitting RSN (VS-RSN), a deep cascade of blocks, each block containing RSN, multi-coil DF unit, and a weighted average module. We extensively validate our models DC-RSN and VS-RSN for single-coil and multi-coil acquisitions and report the state-of-the-art performance. We obtain a SSIM of 0.768, 0.923, 0.878 for knee single-coil-4x, multi-coil-4x, and multi-coil-8x in fastMRI. We also conduct experiments to demonstrate the efficacy of GOLF based T1 assistance and PRN.Comment: Accepted in CMIG 202

Identification of Diverse Lipid Droplet Targeting Motifs in the PNPLA Family of Triglyceride Lipases

<div><p>Members of the Patatin-like Phospholipase Domain containing Protein A (PNPLA) family play key roles in triglyceride hydrolysis, energy metabolism, and lipid droplet (LD) homoeostasis. Here we report the identification of two distinct LD targeting motifs (LTM) for PNPLA family members. Transient transfection of truncated versions of human adipose triglyceride lipase (ATGL, also known as PNPLA2), PNPLA3/adiponutrin, or PNPLA5 (GS2-like) fused to GFP revealed that the C-terminal third of these proteins contains sequences that are sufficient for targeting to LDs. Furthermore, fusing the C-termini of PNPLA3 or PNPLA5 confers LD localization to PNPLA4, which is otherwise cytoplasmic<b>.</b> Analyses of additional mutants in ATGL, PNPLA5, and Brummer Lipase, the <i>Drosophila</i> homolog of mammalian ATGL, identified two different types of LTMs. The first type, in PNPLA5 and Brummer lipase, is a set of loosely conserved basic residues, while the second type, in ATGL, is contained within a stretch of hydrophobic residues. These results show that even closely related members of the PNPLA family employ different molecular motifs to associate with LDs.</p></div

Actomyosin arcs promote tension at the IS in a ligand-dependent manner.

<p>(A and B) Representative 3D-SIM images of OT1 T cells that had been pretreated with either DMSO or pnBB in DMSO, allowed to attach to the activating surface for 7 min, and then fixed and stained with phalloidin (red) and anti-pCasL antibody (green). The merged images are shown in the bottom row. The cells were activated on glass surfaces coated with either OVA:H-2K^b plus CD80 (A) or G4:H-2K^b plus CD80 (B). Scale bar, 5 μm. (C) Mean intensities of pCasL at the IS from 3D-SIM images of OT1 cells pretreated with either DMSO or pnBB in DMSO and activated on OVA- or G4-coated surfaces. Mean ± SEM. An unequal variance T-test (Welch's T-test) was used. * and ** indicate p < 0.05 and < 0.01, respectively.</p

The ATPase activity of myosin 2 promotes actomyosin arc formation in a ligand-dependent manner.

<p>(A and B) Representative 3D-SIM images of OT1 T cells that had been pretreated with either DMSO or pnBB in DMSO, allowed to attach to the activating surface for 7 min, and then fixed and stained with phalloidin (red) and anti-myosin 2A antibody (green). The merged images are shown in the bottom row. The cells were activated on glass surfaces coated with either OVA:H-2K^b plus CD80 (A) or G4:H-2K^b plus CD80 (B). The pSMAC regions are labeled with yellow or white brackets. Scale bar, 5 μm. (C) Frequency of arc morphologies in OT1 cells pretreated with either DMSO or pnBB in DMSO and activated on OVA- or G4-coated surfaces. Arcs were scored as either Organized (i.e. present and concentric), Disorganized (i.e. present but either pointing inwards or entangled), or No arcs (i.e. not present). (D) Average anisotropies of actin arcs in the pSMAC region of OT1 cells pretreated with either DMSO or pnBB in DMSO and activated on OVA- or G4-coated surfaces. (E) Histogram of total anisotropies from all ROIs measured on OVA-coated surfaces with or without pnBB treatment (p < 0.0001). (F) Same as (E) but using G4-coated surfaces (p = 0.44). Mean ± SEM. **** indicates p < 0.0001.</p

Myosin 2 facilitates proximal TCR signaling in a ligand-dependent manner.

<p>(A) Representative example of AMNIS Imagestream images used to quantitate signaling molecule phosphorylation at the IS of OT1 T cells conjugated with EL4 target cells by employing a combination masking strategy (see Methods for details). Shown from left to right are the bright-field (BF) image with the portion of the combination mask that corresponds to the IS (blue), the signal for GFP-F-Tractin (green) in the OT1 T cell, the signal for farnesylated RFP (pseudocolored orange) in the EL4 target cell, the signals for the nucleus (purple) in both cells, and the portion of the combination mask that corresponds to the IS (blue) overlaid with the signal for pLAT at the IS (red). Scale bar, 10 μm. (B, C, and D) MFIs for pSrc (B), pZap70 (C), and pLAT (D) at the IS of OT1 T cells that had been pretreated with either DMSO or pnBB in DMSO and allowed to form conjugates for 10 min with EL4 target cells loaded with either OVA:H-2K^b or G4:H-2K^b prior to fixation and staining. Each circle represents a single OT1: EL4 conjugate. (E, F, G) Normalized MFIs from three or more experiments performed exactly as in (B), (C) and (D), except that these normalized MFIs were background corrected by subtracting the MFI for the null peptide control. (H, I, and J) Shown are the differences in mean MFI values presented in (H), (I) and (J) between OVA:H-2K^b-engaged and G4:H-2K^b-engaged T cells. The value obtained, referred to as “ΔMFI OVA vs G4” on the y axis, represents a measure of the contribution made by myosin 2 contractility to ligand discrimination. Mean ± SEM (N ≥ 3). *, **, *** indicate p < 0.05, 0.01, 0.001.</p

Actomyosin arcs facilitate the accumulation of TCR MCs at the IS center in a ligand-dependent and contractility-dependent manner.

<p>(A and B) Representative 3D-SIM images of OT1 T cells that had been pretreated with either DMSO or pnBB in DMSO, allowed to attach for 7 min to planar lipid bilayers containing ICAM-1 and either OVA:H-2K^b (A) or G4:H-2K^b (B) bound via fluorescent SA (to indirectly report the position of TCR MCs in the T cell), and then fixed and stained with phalloidin (red). The distribution of SA (green) and the merged images are shown in the middle and bottom rows, respectively. Scale bar, 5 μm. (C) Averaged, normalized radial integral intensity profiles of fluorescent SA in bilayers containing OVA:H-2K^b (red) or G4:H-2K^b (blue) (p <0.001). (D) Averaged, normalized radial integral intensity profiles of fluorescent SA in bilayers containing OVA:H-2K^b using OT1 T cells pretreated with either DMSO (red) or pnBB in DMSO (black) (p < 0.0001). (E) Averaged, normalized radial integral intensity profiles of fluorescent SA in bilayers containing G4:H-2K^b using OT1 T cells pretreated with either DMSO (blue) or pnBB in DMSO (black) (p < 0.0001). For (C), (D) and (E), the mean value is represented by the bold line, while the SEM is represented by the surrounding shaded area.</p