SLDP and LIPA mediate lipid droplet-plasma membrane tethering in Arabidopsis thaliana

Membrane contact sites (MCS) are inter-organellar connections that allow for the direct exchange of molecules, such as lipids or Ca2+ between organelles, but can also serve to tether organelles at specific locations within cells. Here we identified and characterised three proteins that form a lipid droplet (LD)-plasma membrane (PM) tethering complex in plant cells, namely LD-localised SEED LD PROTEIN (SLDP) 1 and 2 and PM-localised LD-PLASMA MEMBRANE ADAPTOR (LIPA). Using proteomics and different protein-protein interaction assays, we show that both SLDPs associate with LIPA. Disruption of either SLDP1 and 2 expression, or that of LIPA, leads to an aberrant clustering of LDs in Arabidopsis seedlings. Ectopic co-expression of one of the SLDPs with LIPA on the other hand is sufficient to reconstitute LD-PM tethering in Nicotiana tabacum pollen tubes, a cell type characterised by dynamically moving LDs in the cytosolic streaming. Further, confocal laser scanning microscopy revealed both SLDP2.1 and LIPA to be enriched at LD-PM contact sites in seedlings. These and other results suggest that SLDP and LIPA interact to form a tethering complex that anchors a subset of LDs to the PM during post-germinative seedling growth in Arabidopsis thaliana.

The Role of Junctophilin Proteins in Cellular Function

Junctophilins (JPHs) comprise a family of structural proteins that connect the plasma membrane to intracellular organelles such as the endo/sarcoplasmic reticulum. Tethering of these membrane structures results in the formation of highly organized subcellular junctions that play important signaling roles in all excitable cell types. There are four JPH isoforms, expressed primarily in muscle and neuronal cell types. Each JPH protein consists of 6 'membrane occupation and recognition nexus' (MORN) motifs, a joining region connecting these to another set of 2 MORN motifs, a putative alpha-helical region, a divergent region exhibiting low homology between JPH isoforms, and a carboxy-terminal transmembrane region anchoring into the ER/SR membrane. JPH isoforms play essential roles in developing and maintaining subcellular membrane junctions. Conversely, inherited mutations in JPH2 cause hypertrophic or dilated cardiomyopathy, while trinucleotide expansions in the JPH3 gene cause Huntington Disease-Like 2. Loss of JPH1 protein levels can cause skeletal myopathy, while loss of cardiac JPH2 levels causes heart failure and atrial fibrillation, among other disease. This review will provide a comprehensive overview of the JPH gene family, phylogeny, and evolutionary analysis of JPH genes and other MORN domain proteins. JPH biogenesis, membrane tethering, and binding partners will be discussed, as well as functional roles of JPH isoforms in excitable cells. Finally, potential roles of JPH isoform deficits in human disease pathogenesis will be reviewed.

Weibel Palade Bodies: Unique Secretory Organelles of Endothelial Cells that Control Blood Vessel Homeostasis

Vascular endothelial cells produce and release compounds regulating vascular tone, blood vessel growth and differentiation, plasma composition, coagulation and fibrinolysis, and also engage in interactions with blood cells thereby controlling hemostasis and acute inflammatory reactions. These interactions have to be tightly regulated to guarantee smooth blood flow in normal physiology, but also allow specific and often local responses to blood vessel injury and infectious or inflammatory insults. To cope with these challenges, endothelial cells have the remarkable capability of rapidly changing their surface properties from non-adhesive (supporting unrestricted blood flow) to adhesive (capturing circulating blood cells). This is brought about by the evoked secretion of major adhesion receptors for platelets (von-Willebrand factor, VWF) and leukocytes (P-selectin) which are stored in a ready-to-be-used form in specialized secretory granules, the Weibel-Palade bodies (WPB). WPB are unique, lysosome related organelles that form at the trans-Golgi network and further mature by receiving material from the endolysosomal system. Failure to produce correctly matured VWF and release it through regulated WPB exocytosis results in pathologies, most importantly von-Willebrand disease, the most common inherited blood clotting disorder. The biogenesis of WPB, their intracellular motility and their fusion with the plasma membrane are regulated by a complex interplay of proteins and lipids, involving Rab proteins and their effectors, cytoskeletal components as well as membrane tethering and fusion machineries. This review will discuss aspects of WPB biogenesis, trafficking and exocytosis focussing on recent findings describing factors contributing to WPB maturation, WPB-actin interactions and WPB-plasma membrane tethering and fusion.

Programming cell surface signaling via phase separation-controlled compartmentalization

Cell surface signaling landscapes are formidably complex. Robust tools capable of manipulating the spatiotemporal distribution of cell surface proteins (CSPs) for dissecting signaling are in high demand. Some CSPs are regulated via multivalency-driven liquid-liquid phase separation (LLPS). Employing the robustness and versatility of LLPS, we decided to engineer LLPS-based tools for precisely manipulating CSPs. We generated membrane-tethering LLPS systems by fusing multivalent modular phase separation scaffold pairs with CSP binders. Phase separation of the scaffold pairs, concomitant compartmentalization of CSPs on membranes, and cluster-dependent signaling outputs of CSPs require membrane recruitment of one or both scaffolds. We also engineered orthogonal phase separation systems to segregate CSPs into mutually exclusive compartments. The engineered phase separation systems can robustly cluster individual CSPs, co-cluster two or more CSPs, or segregate different CSPs into distinct compartments on cell surfaces. These novel tools will enable the dissection of complicated cell signaling landscapes with unprecedented precision.

How to build a ribosome from RNA fragments in Chlamydomonas mitochondria

Mitochondria are the powerhouse of eukaryotic cells. They possess their own gene expression machineries where highly divergent and specialized ribosomes, named hereafter mitoribosomes, translate the few essential messenger RNAs still encoded by mitochondrial genomes. Here, we present a biochemical and structural characterization of the mitoribosome in the model green alga Chlamydomonas reinhardtii, as well as a functional study of some of its specific components. Single particle cryo-electron microscopy resolves how the Chlamydomonas mitoribosome is assembled from 13 rRNA fragments encoded by separate non-contiguous gene pieces. Additional proteins, mainly OPR, PPR and mTERF helical repeat proteins, are found in Chlamydomonas mitoribosome, revealing the structure of an OPR protein in complex with its RNA binding partner. Targeted amiRNA silencing indicates that these ribosomal proteins are required for mitoribosome integrity. Finally, we use cryo-electron tomography to show that Chlamydomonas mitoribosomes are attached to the inner mitochondrial membrane via two contact points mediated by Chlamydomonas-specific proteins. Our study expands our understanding of mitoribosome diversity and the various strategies these specialized molecular machines adopt for membrane tethering.

The endoplasmic reticulum-plasma membrane tethering protein TMEM24 is a regulator of cellular Ca2+ homeostasis

Endoplasmic reticulum (ER) - plasma membrane (PM) contacts are sites of lipid exchange and Ca2+ transport, and both lipid transport proteins and Ca2+ channels specifically accumulate at these locations. In pancreatic β-cells, both lipid- and Ca2+ signaling are essential for insulin secretion. The recently characterized lipid transfer protein TMEM24 dynamically localize to ER-PM contact sites and provide phosphatidylinositol, a precursor of PI(4)P and PI(4,5)P2, to the plasma membrane. β-cells lacking TMEM24 exhibit markedly suppressed glucose-induced Ca2+ oscillations and insulin secretion but the underlying mechanism is not known. We now show that TMEM24 only weakly interact with the PM, and dissociates in response to both diacylglycerol and nanomolar elevations of cytosolic Ca2+. Loss of TMEM24 results in hyper-accumulation of Ca2+ in the ER and in excess Ca2+ entry into mitochondria, with resulting impairment in glucose-stimulated ATP production.

Min waves without MinC can pattern FtsA-FtsZ filaments on model membranes

Although the essential proteins that drive bacterial cytokinesis have been identified and reconstituted in vitro, the precise mechanisms by which they dynamically interact to enable symmetrical division are largely unknown. In Escherichia coli, cell division begins with the formation of a proto-ring composed of FtsZ and its membrane-tethering proteins FtsA and ZipA. In the broadly proposed molecular scenario for ring positioning, Min waves composed of MinD and MinE distribute the FtsZ-polymerization inhibitor MinC away from mid-cell, where the Z-ring can form. Therefore, MinC is believed to be an essential element connecting the Min and FtsZ systems. Here, by using cell-free gene expression on planar lipid membranes, we demonstrate that MinDE drive the formation of dynamic, antiphase patterns of FtsZ-FtsA co-filaments even in the absence of MinC. This behavior is also observed when the proteins are compartmentalized inside microdroplets. These results suggest that Z-ring positioning may be achieved with a more minimal set of proteins than previously envisaged, providing a fresh perspective about the role of MinC. Moreover, we propose that MinDE oscillations may constitute the minimal localization mechanism of an FtsA-FtsZ constricting ring in a prospective synthetic cell.

Programmed cortical ER collapse drives selective ER degradation and inheritance in yeast meiosis

The endoplasmic reticulum (ER) carries out essential and conserved cellular functions, which depend on the maintenance of its structure and subcellular distribution. Here, we report developmentally regulated changes in ER morphology and composition during budding yeast meiosis, a conserved differentiation program that gives rise to gametes. A subset of the cortical ER collapses away from the plasma membrane at anaphase II, thus separating into a spatially distinct compartment. This programmed collapse depends on the transcription factor Ndt80, conserved ER membrane structuring proteins Lnp1 and reticulons, and the actin cytoskeleton. A subset of ER is retained at the mother cell plasma membrane and excluded from gamete cells via the action of ER–plasma membrane tethering proteins. ER remodeling is coupled to ER degradation by selective autophagy, which relies on ER collapse and is regulated by timed expression of the autophagy receptor Atg40. Thus, developmentally programmed changes in ER morphology determine the selective degradation or inheritance of ER subdomains by gametes.

A mechanism for exocyst-mediated tethering via Arf6 and PIP5K1C driven phosphoinositide conversion

Polarized trafficking is necessary for the development of eukaryotes and is regulated by a conserved molecular machinery. Late steps of cargo delivery are mediated by the exocyst complex, which integrates lipid and protein components to tether vesicles for plasma membrane fusion. However, the molecular mechanisms of this process are poorly defined. Here, we reconstitute functional octameric human exocyst, demonstrating the basis for holocomplex coalescence and biochemically stable subcomplexes. We determine that each subcomplex independently binds to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), which is minimally sufficient for membrane tethering. Through reconstitution and epithelial cell biology experiments, we show that Arf6-mediated recruitment of the lipid kinase PIP5K1C rapidly converts phosphatidylinositol 4-phosphate (PI(4)P) to PI(4,5)P2, driving exocyst recruitment and membrane tethering. These results provide a molecular mechanism of exocyst-mediated tethering and a unique functional requirement for phosphoinositide signaling on late-stage vesicles in the vicinity of the plasma membrane.