Cytosolic phospholipase A2ε drives recycling through the clathrin-independent endocytic route.

Previous studies have demonstrated that membrane tubule-mediated transport events in biosynthetic and endocytic routes require phospholipase A2 (PLA2) activity. Here, we show that cytosolic phospholipase A2ε (cPLA2ε, also known as PLA2G4E) is targeted to the membrane compartments of the clathrin-independent endocytic route through a C-terminal stretch of positively charged amino acids, which allows the enzyme to interact with phosphoinositide lipids [especially PI(4,5)P2] that are enriched in clathrin-independent endosomes. Ablation of cPLA2ε suppressed the formation of tubular elements that carry internalized clathrin-independent cargoes, such as MHC-I, CD147 and CD55, back to the cell surface and, therefore, caused their intracellular retention. The ability of cPLA2ε to support recycling through tubule formation relies on the catalytic activity of the enzyme, because the inactive cPLA2ε(S420A) mutant was not able to recover either tubule growth or transport from clathrin-independent endosomes. Taken together, our findings indicate that cPLA2ε is a new important regulator of trafficking processes within the clathrin-independent endocytic and recycling route. The affinity of cPLA2ε for this pathway supports a new hypothesis that different PLA2 enzymes use selective targeting mechanisms to regulate tubule formation locally during specific trafficking steps in the secretory and/or endocytic systems.

Polarised apical-like intracellular sorting and trafficking regulates invadopodia formation and degradation of the extracellular matrix in cancer cells.

Invadopodia are proteolytically active protrusions formed by invasive tumoral cells when grown on an extracellular matrix (ECM) substratum. A current challenge is to understand how proteolytic activity is so precisely localised at discrete sites of the plasma membrane to produce focalised ECM degradation at invadopodia. Indeed, a number of components including metalloproteases need to be directed to invadopodia to ensure proper segregation of proteolytic activities. We recently found invadopodia to feature the properties of cholesterol-rich membrane domains (a.k.a. lipid drafts) and that ECM degradation depends on the tight control of cholesterol homeostasis. Since apically directed polarised sorting and transport in epithelial cells relies on segregation of proteins into lipid rafts at the Golgi complex, we hypothesised that invadopodia-dependent ECM degradation might also rely on lipid raft-dependent polarised transport routes. To investigate this issue we undertook a three-pronged approach. First, we found that microtubule depolymerisation, which is known to disrupt polarised transport in polarised cells, strongly inhibited invadopodia formation, while not affecting overall protein transport. In the second approach we found that glycosylphosphatidylinositol-anchored green fluorescent protein (an apical model protein), but not vesicular stomatitis virus G-protein or influenza virus hemagglutinin (both model basolateral model cargoes), was transported to sites of ECM degradation. Finally, RNAi-mediated knock-down of proteins known to specifically regulate polarised apical or basolateral transport in epithelial cells, such as caveolin 1 and annexin XIIIB or clathrin, respectively, demonstrated that the selective inhibition of the apical, but not the basolateral, transport route impairs invadopodia formation and ECM degradation. Taken together, our findings suggest that invadopodia are apical-like membrane domains, where signal transduction and local membrane remodelling events might be temporally and spatially confined via selective raft-dependent apical transport routes.

Shaping tubular carriers for intracellular membrane transport.

The particular compositions of the intracellular membrane organelles rely on the proteins and lipids received frequently through membrane trafficking. The delivery of these molecules is driven by the membrane-bound organelles known as transport carriers (TCs). Advanced microscopy approaches have revealed that TC morphology ranges from small vesicles to complex tubular membrane structures. These tubular TCs (TTCs) support effectively both sorting and transport events within the biosynthetic and endocytic pathways, while a coherent picture of the processes that define the formation and further fate of TTCs is still missing. Here, we present an overview of the mechanisms operating during the TTC life cycle, as well as of the emerging role of tubular carriers in different intracellular transport routes.

Group IV phospholipase A(2)alpha controls the formation of inter-cisternal continuities involved in intra-Golgi transport.

The organization of intra-Golgi trafficking and the nature of the transport intermediates involved (e.g., vesicles, tubules, or tubular continuities) remain incompletely understood. It was recently shown that successive cisternae in the Golgi stack are interconnected by membrane tubules that form during the arrival of transport carriers from the endoplasmic reticulum. Here, we examine the mechanisms of generation and the function of these tubules. In principle, tubule formation might depend on several protein- and/or lipid-based mechanisms. Among the latter, we have studied the phospholipase A(2) (PLA(2))-mediated generation of wedge-shaped lysolipids, with the resulting local positive membrane curvature. We show that the arrival of cargo at the Golgi complex induces the recruitment of Group IVA Ca(2+)-dependent, cytosolic PLA(2) (cPLA(2)alpha) onto the Golgi complex itself, and that this cPLA(2)alpha is required for the formation of the traffic-dependent intercisternal tubules and for intra-Golgi transport. In contrast, silencing of cPLA(2)alpha has no inhibitory effects on peri-Golgi vesicles. These findings identify cPLA(2)alpha as the first component of the machinery that is responsible for the formation of intercisternal tubular continuities and support a role for these continuities in transport through the Golgi complex.

Faciogenital dysplasia protein (FGD1) regulates export of cargo proteins from the golgi complex via Cdc42 activation.

Mutations in the FGD1 gene are responsible for the X-linked disorder known as faciogenital dysplasia (FGDY). FGD1 encodes a guanine nucleotide exchange factor that specifically activates the GTPase Cdc42. In turn, Cdc42 is an important regulator of membrane trafficking, although little is known about FGD1 involvement in this process. During development, FGD1 is highly expressed during bone growth and mineralization, and therefore a lack of the functional protein leads to a severe phenotype. Whether the secretion of proteins, which is a process essential for bone formation, is altered by mutations in FGD1 is of great interest. We initially show here that FGD1 is preferentially associated with the trans-Golgi network (TGN), suggesting its involvement in export of proteins from the Golgi. Indeed, expression of a dominant-negative FGD1 mutant and RNA interference of FGD1 both resulted in a reduction in post-Golgi transport of various cargoes (including bone-specific proteins in osteoblasts). Live-cell imaging reveals that formation of post-Golgi transport intermediates directed to the cell surface is inhibited in FGD1-deficient cells, apparently due to an impairment of TGN membrane extension along microtubules. These effects depend on FGD1 regulation of Cdc42 activation and its association with the Golgi membranes, and they may contribute to FGDY pathogenesis.

A traffic-activated Golgi-based signalling circuit coordinates the secretory pathway.

As with other complex cellular functions, intracellular membrane transport involves the coordinated engagement of a series of organelles and machineries; however, the molecular basis of this coordination is unknown. Here we describe a Golgi-based signalling system that is activated by traffic and is involved in monitoring and balancing trafficking rates into and out of the Golgi complex. We provide evidence that the traffic signal is due to protein chaperones that leave the endoplasmic reticulum and reach the Golgi complex where they bind to the KDEL receptor. This initiates a signalling reaction that includes the activation of a Golgi pool of Src kinases and a phosphorylation cascade that in turn activates intra-Golgi trafficking, thereby maintaining the dynamic equilibrium of the Golgi complex. The concepts emerging from this study should help to understand the control circuits that coordinate high-order cellular functions.