Just like the body contains organs that perform different jobs, the cells within the body contain organelles that carry out different tasks. The endoplasmic reticulum, for example, makes proteins that are sent to other organelles or to destinations outside the cell. Each organelle is typically sealed within a membrane made from a double layer of phospholipids—molecules that have a phosphate ‘head’ group at one end, and two fatty acid ‘tails’ at the other. Proteins are shuttled between the organelles inside membrane-bound packages called vesicles. There is, however, an exception to this rule. Lipid droplets are organelles that store fats and oils inside a single layer of phospholipids. This layer can include enzymes that break down the contents of the droplet, or make new fat molecules, depending on the needs of the cell and the organism. However, it is not clear how these enzymes get from the endoplasmic reticulum to the lipid droplet. Previous work had suggested that a protein complex called Arf1/COP—which is also involved in the movement of vesicles around the cell—might recruit the enzymes to the lipid droplets. However, none of the other proteins known to be involved in vesicle transport were needed to transport the enzymes to the droplets, which suggested that the Arf1/COPI complex was using a previously unknown mechanism to move the enzymes. Now Wilfling, Thiam et al. have shown that Arf1/COPI complexes trigger the establishment of membrane bridges between the endoplasmic reticulum and the droplets, which means that vesicles are not needed to get the enyzmes to the lipid droplets. It was also shown that the Arf1/COPI complexes could pinch off tiny droplets from full-size lipid droplets taken from living cells. Wilfling, Thiam et al. suggest that this ‘budding’ process changes the composition of the phospholipid layer around the larger droplet in a way that allows it to interact directly with the membrane of the endoplasmic reticulum. By providing new insights into the trafficking of proteins between organelles, the work of Wilfling, Thiam et al. reveals mechanisms that govern the composition of lipid droplets. In the future, these pathways could be manipulated to treat conditions that result from excessive storage of fat, such as obesity or cardiovascular diseases.

Arf1/COPI machinery acts directly on lipid droplets and enables their connection to the ER for protein targeting

Who's In and Who's Out-Compositional Control of Biomolecular Condensates.

Neutral lipid stores and lipase PNPLA5 contribute to autophagosome biogenesis

Our knowledge of inter-organellar communication has grown exponentially in recent years. This review focuses on the interactions that cytoplasmic lipid droplets have with other organelles. Twenty-five years ago droplets were considered simply particles of coalesced fat. Ten years ago there were hints from proteomics studies that droplets might interact with other structures to share lipids and proteins. Now it is clear that the droplets interact with many if not most cellular structures to maintain cellular homeostasis and to buffer against insults such as starvation. The evidence for this statement, as well as probes to understand the nature and results of droplet interactions, are presented.

/pdf/the-lipid-droplet-a-well-connected-organelle-4sypgjslsx.pdf

The lipid droplet—a well-connected organelle

Human actin-related protein 2/3 complex (Arp2/3), required for actin filament branching, has two ARPC1 component isoforms, with ARPC1B prominently expressed in blood cells. Here we show in a child with microthrombocytopenia, eosinophilia and inflammatory disease, a homozygous frameshift mutation in ARPC1B (p.Val91Trpfs*30). Platelet lysates reveal no ARPC1B protein and greatly reduced Arp2/3 complex. Missense ARPC1B mutations are identified in an unrelated patient with similar symptoms and ARPC1B deficiency. ARPC1B-deficient platelets are microthrombocytes similar to those seen in Wiskott-Aldrich syndrome that show aberrant spreading consistent with loss of Arp2/3 function. Knockout of ARPC1B in megakaryocytic cells results in decreased proplatelet formation, and as observed in platelets from patients, increased ARPC1A expression. Thus loss of ARPC1B produces a unique set of platelet abnormalities, and is associated with haematopoietic/immune symptoms affecting cell lineages where this isoform predominates. In agreement with recent experimental studies, our findings suggest that ARPC1 isoforms are not functionally interchangeable.

/pdf/loss-of-the-arp2-3-complex-component-arpc1b-causes-platelet-2burmalkho.pdf

Loss of the Arp2/3 complex component ARPC1B causes platelet abnormalities and predisposes to inflammatory disease

Actin assembly and inward flow in the plane of the immunological synapse (IS) drives the centralization of T cell receptor microclusters (TCR MCs) and the integrin leukocyte functional antigen 1 (LFA-1) Using structured-illumination microscopy (SIM), we show that actin arcs populating the medial, lamella-like region of the IS arise from linear actin filaments generated by one or more formins present at the IS distal edge After traversing the outer, Arp2/3-generated, lamellipodia-like region of the IS, these linear filaments are organized by myosin II into antiparallel concentric arcs Three-dimensional SIM shows that active LFA-1 often aligns with arcs, whereas TCR MCs commonly reside between arcs, and total internal reflection fluorescence SIM shows TCR MCs being swept inward by arcs Consistently, disrupting actin arc formation via formin inhibition results in less centralized TCR MCs, missegregated integrin clusters, decreased T–B cell adhesion, and diminished TCR signaling Together, our results define the origin, organization, and functional significance of a major actomyosin contractile structure at the IS that directly propels TCR MC transport

/pdf/formin-generated-actomyosin-arcs-propel-t-cell-receptor-1nbq5126md.pdf

Formin-generated actomyosin arcs propel T cell receptor microcluster movement at the immune synapse.

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.

https://www.pnas.org/content/pnas/114/32/E6585.full.pdf

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

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. Analyses of additional mutants in ATGL, PNPLA5, and Brummer Lipase, the Drosophila 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.

/pdf/identification-of-diverse-lipid-droplet-targeting-motifs-in-37kjvgvxxl.pdf

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

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.

/pdf/contractile-actomyosin-arcs-promote-the-activation-of-6plf1oxyzr.pdf

Sricharan Murugesan

Papers

Formin-generated actomyosin arcs propel T cell receptor microcluster movement at the immune synapse.

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

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

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

Conserved C‐terminal arginines are crucial for targeting PNPLA5/GS2‐like triglyceride lipase to lipid droplets