John G. Lock

E-cadherin transport from the trans-Golgi network in tubulovesicular carriers is selectively regulated by Golgin-97

E‐cadherin is a cell–cell adhesion protein that is trafficked and delivered to the basolateral cell surface. Membrane‐bound carriers for the post‐Golgi exocytosis of E‐cadherin have not been characterized. Green fluorescent protein (GFP)‐tagged E‐cadherin (Ecad‐GFP) is transported from the trans‐Golgi network (TGN) to the recycling endosome on its way to the cell surface in tubulovesicular carriers that resemble TGN tubules labeled by members of the golgin family of tethering proteins. Here, we examine the association of golgins with tubular carriers containing E‐cadherin as cargo. Fluorescent GRIP domains from golgin proteins replicate the membrane binding of the full‐length proteins and were coexpressed with Ecad‐GFP. The GRIP domains of p230/golgin‐245 and golgin‐97 had overlapping but nonidentical distributions on the TGN; both domains were on TGN‐derived tubules but only the golgin‐97 GRIP domain coincided with Ecad‐GFP tubules in live cells. When the Arl1‐binding endogenous golgins, p230/golgin‐245 and golgin‐97 were displaced from Golgi membranes by overexpression of the p230 GRIP domain, trafficking of Ecad‐GFP was inhibited. siRNA knockdown of golgin‐97 also inhibited trafficking of Ecad‐GFP. Thus, the GRIP domains of p230/golgin‐245 and golgin‐97 bind discriminately to distinct membrane subdomains of the TGN. Golgin‐97 is identified as a selective and essential component of the tubulovesicular carriers transporting E‐cadherin out of the TGN.

Domains of the TGN: Coats, Tethers and G Proteins

The trans‐Golgi network is the major sorting compartment of the secretory pathway for protein, lipid and membrane traffic. There is a constant flow of membrane and cargo to and from this compartment. Evidence is emerging that the trans‐Golgi network has multiple biochemically and functionally distinct subdomains, each of which contributes to the combined sorting and transport requirements of this dynamic compartment. The recruitment of distinct arrays of protein complexes to trans‐Golgi network membranes is likely to produce the diversity of structure and biochemistry observed amongst subdomains that serve to generate different carriers or maintain resident trans‐Golgi network components. This review discusses how these subdomains may be formed and examines the molecular players involved, including G proteins, clathrin adaptors and golgin tethers. Diversity within these protein families is highlighted and shown to be critical for the functionality of the trans‐Golgi network, as a mediator of protein sorting and membrane transport, and for the maintenance of Golgi structure.

Single fluorescent protein-based Ca2+ sensors with increased dynamic range

BackgroundGenetically encoded sensors developed on the basis of green fluorescent protein (GFP)-like proteins are becoming more and more popular instruments for monitoring cellular analytes and enzyme activities in living cells and transgenic organisms. In particular, a number of Ca2+ sensors have been developed, either based on FRET (Fluorescence Resonance Energy Transfer) changes between two GFP-mutants or on the change in fluorescence intensity of a single circularly permuted fluorescent protein (cpFP).ResultsHere we report significant progress on the development of the latter type of Ca2+ sensors. Derived from the knowledge of previously reported cpFP-based sensors, we generated a set of cpFP-based indicators with different spectral properties and fluorescent responses to changes in Ca2+ concentration. Two variants, named Case12 and Case16, were characterized by particular high brightness and superior dynamic range, up to 12-fold and 16.5-fold increase in green fluorescence between Ca2+-free and Ca2+-saturated forms. We demonstrated the high potential of these sensors on various examples, including monitoring of Ca2+ response to a prolonged glutamate treatment in cortical neurons.ConclusionWe believe that expanded dynamic range, high brightness and relatively high pH-stability should make Case12 and Case16 popular research tools both in scientific studies and high throughput screening assays.

A trans-Golgi network golgin is required for the regulated secretion of TNF in activated macrophages in vivo.

The transmembrane precursor of tumor necrosis factor-α (TNF) exits the trans-Golgi network (TGN) in tubular carriers for subsequent trafficking and delivery to the cell surface; however, the molecular machinery responsible for Golgi export is unknown. We previously reported that members of the TGN golgin family are associated with subdomains and tubules of the TGN. Here, we show that the TGN golgin, p230/golgin-245 (p230), is essential for intracellular trafficking and cell surface delivery of TNF in transfected HeLa cells and activated macrophages. Live-cell imaging revealed that TNF transport from the TGN is mediated selectively by tubules and carriers marked by p230. Significantly, LPS activation of macrophages resulted in a dramatic increase of p230-labeled tubules and carriers emerging from the TGN, indicating that macrophages up-regulate the transport pathway for TNF export. Depletion of p230 in LPS-stimulated macrophages reduced cell surface delivery of TNF by >10-fold compared with control cells. To determine whether p230 depletion blocked TNF secretion in vivo, we generated retrogenic mice expressing a microRNA-vector to silence p230. Bone-marrow stem cells were transduced with recombinant retrovirus containing microRNA constructs and transplanted into irradiated recipients. LPS-activated peritoneal macrophages from p230 miRNA retrogenic mice were depleted of p230 and had dramatically reduced levels of cell surface TNF. Overall, these studies have identified p230 as a key regulator of TNF secretion and have shown that LPS activation of macrophages results in increased Golgi carriers for export. Also, we have demonstrated a previously undescribed approach to control cytokine secretion by the specific silencing of trafficking machinery.

TLR activation regulates damage‐associated molecular pattern isoforms released during pyroptosis

Infection of macrophages by bacterial pathogens can trigger Toll‐like receptor (TLR) activation as well as Nod‐like receptors (NLRs) leading to inflammasome formation and cell death dependent on caspase‐1 (pyroptosis). Complicating the study of inflammasome activation is priming. Here, we develop a priming‐free NLRC4 inflammasome activation system to address the necessity and role of priming in pyroptotic cell death and damage‐associated molecular pattern (DAMP) release. We find pyroptosis is not dependent on priming and when priming is re‐introduced pyroptosis is unaffected. Cells undergoing unprimed pyroptosis appear to be independent of mitochondrial involvement and do not produce inflammatory cytokines, nitrous oxide (NO), or reactive oxygen species (ROS). Nevertheless, they undergo an explosive cell death releasing a chemotactic isoform of the DAMP high mobility group protein box 1 (HMGB1). Importantly, priming through surface TLRs but not endosomal TLRs during pyroptosis leads to the release of a new TLR4‐agonist cysteine redox isoform of HMGB1. These results show that pyroptosis is dominant to priming signals and indicates that metabolic changes triggered by priming can affect how cell death is perceived by the immune system.

The GRIP domain is a specific targeting sequence for a population of trans-Golgi network derived tubulo-vesicular carriers.

Vesicular carriers for intracellular transport associate with unique sets of accessory molecules that dictate budding and docking on specific membrane domains. Although many of these accessory molecules are peripheral membrane proteins, in most cases the targeting sequences responsible for their membrane recruitment have yet to be identified. We have previously defined a novel Golgi targeting domain (GRIP) shared by a family of coiled‐coil peripheral membrane Golgi proteins implicated in membrane trafficking. We show here that the docking site for the GRIP motif of p230 is a specific domain of Golgi membranes. By immuno‐electron microscopy of HeLa cells stably expressing a green fluorescent protein (GFP)‐p230GRIP fusion protein, we show binding specifically to a subset of membranes of the trans‐Golgi network (TGN). Real‐time imaging of live HeLa cells revealed that the GFP‐p230GRIP was associated with highly dynamic tubular extensions of the TGN, which have the appearance and behaviour of transport carriers. To further define the nature of the GRIP membrane binding site, in vitro budding assays were performed using purified rat liver Golgi membranes and cytosol from GFP‐p230GRIP‐transfected cells. Analysis of Golgi‐derived vesicles by sucrose gradient fractionation demonstrated that GFP‐p230GRIP binds to a specific population of vesicles distinct from those labelled for β‐COP or γ‐adaptin. The GFP‐p230GRIP fusion protein is recruited to the same vesicle population as full‐length p230, demonstrating that the GRIP domain is solely proficient as a targeting signal for membrane binding of the native molecule. Therefore, p230 GRIP is a targeting signal for recruitment to a highly selective membrane attachment site on a specific population of trans‐Golgi network tubulo‐vesicular carriers.

PtdIns(3,4,5)P3 is a regulator of myosin-X localization and filopodia formation

Phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] is a key regulator of cell signaling that acts by recruiting proteins to the cell membrane, such as at the leading edge during cell migration. Here, we show that PtdIns (3,4,5)P3 plays a central role in filopodia formation via the binding of myosin-X (Myo10), a potent promoter of filopodia. We found that the second pleckstrin homology domain (Myo10-PH2) of Myo10 specifically binds to PtdIns(3,4,5)P3, and that disruption of this binding led to impairment of filopodia and partial re-localization of Myo10 to microtubule-associated Rab7-positive endosomal vesicles. Given that the localization of Myo10 was dynamically restored to filopodia upon reinstatement of PtdIns(3,4,5)P3-binding, our results indicate that PtdIns(3,4,5)P3 binding to the Myo10-PH2 domain is involved in Myo10 trafficking and regulation of filopodia dynamics.

Kindlin-2 is expressed in malignant mesothelioma and is required for tumor cell adhesion and migration

Kindlin‐2 is a novel integrin‐interacting focal adhesion protein that belongs to the Kindlin family. Focal adhesion proteins control cytoskeleton dynamics and promote cancer cell growth, survival, migration and metastasis. Little is known, however, about expression of Kindlin‐2 in association with human cancer. We now reveal high Kindlin‐2 expression in malignant mesothelioma (MM) cell lines using an affinity‐purified anti‐Kindlin‐2 antibody. Furthermore, we show by immunohistochemistry that Kindlin‐2 is highly expressed in 92 of 102 (90%) MMs with epitheliod; sarcomatoid, biphasic and poorly differentiated morphologies. In addition, Kindlin‐2 expression correlates to cell proliferation, suggesting a role for Kindlin‐2 in tumor growth. We also detect increased expression of Kindlin‐2 at the invasion front of tumors concurrent with increased expression of integrin‐linked kinase, a Kindlin‐binding protein. Besides the high expression of Kindlin‐2 in pleural MMs, pleural metastases of lung adenocarcinoma also express large amounts of Kindlin‐2, but not Kindlin‐1. Notably, in vitro, when endogenous Kindlin‐2 was knocked down with RNAi in MM cells, this impaired cell spreading, adhesion and migration. Overall, our study suggests that heightened expression of Kindlin‐2 might contribute to tumor progression in MM.

Integrin-mediated cell attachment induces a PAK4-dependent feedback loop regulating cell adhesion through modified integrin alpha v beta 5 clustering and turnover.

This article presents a novel mechanism deployed by cells to tune cell adhesion levels through the autoinhibitory regulation of integrin adhesion involving the activation of PAK4.

GAIP Participates in Budding of Membrane Carriers at the Trans‐Golgi Network

Galpha interacting protein (GAIP) is a regulator of G protein signaling protein that associates dynamically with vesicles and has been implicated in membrane trafficking, although its specific role is not yet known. Using an in vitro budding assay, we show that GAIP is recruited to a specific population of trans‐Golgi network‐derived vesicles and that these are distinct from coatomer or clathrin‐coated vesicles. A truncation mutant (NT‐GAIP) encoding only the N‐terminal half of GAIP is recruited to trans‐Golgi network membranes during the formation of vesicle carriers. Overexpression of NT‐GAIP induces the formation of long, coated tubules, which are stabilized by microtubules. Results from the budding assay and from imaging in live cells show that these tubules remain attached to the Golgi stack rather than being released as carrier vesicles. NT‐GAIP expression blocks membrane budding and results in the accumulation of tubular carrier intermediates. NT‐GAIP‐decorated tubules are competent to load vesicular stomatitis virus protein G‐green fluorescent protein as post‐Golgi, exocytic cargo and in cells expressing NT‐GAIP there is reduced surface delivery of vesicular stomatitis virus protein G‐green fluorescent protein. We conclude that GAIP functions as an essential part of the membrane budding machinery for a subset of post‐Golgi exocytic carriers derived from the trans‐Golgi network.