Tatiana Khromykh

Syntaxin 6 and Vti1b form a novel SNARE complex, which is up-regulated in activated macrophages to facilitate exocytosis of tumor necrosis factor-alpha

A key function of activated macrophages is to secrete proinflammatory cytokines such as TNFα; however, the intracellular pathway and machinery responsible for cytokine trafficking and secretion is largely undefined. Here we show that individual SNARE proteins involved in vesicle docking and fusion are regulated at both gene and protein expression upon stimulation with the bacterial cell wall component lipopolysaccharide. Focusing on two intracellular SNARE proteins, Vti1b and syntaxin 6 (Stx6), we show that they are up-regulated in conjunction with increasing cytokine secretion in activated macrophages and that their levels are selectively titrated to accommodate the volume and timing of post-Golgi cytokine trafficking. In macrophages, Vti1b and syntaxin 6 are localized on intracellular membranes and are present on isolated Golgi membranes and on Golgi-derived TNFα vesicles budded in vitro. By immunoprecipitation, we find that Vti1b and syntaxin 6 interact to form a novel intracellular Q-SNARE complex. Functional studies using overexpression of full-length and truncated proteins show that both Vti1b and syntaxin 6 function and have rate-limiting roles in TNFα trafficking and secretion. This study shows how macrophages have uniquely adapted a novel Golgi-associated SNARE complex to accommodate their requirement for increased cytokine secretion.

Syntaxin 6 and Vti1b form a novel SNARE complex which is upregulated in activated macrophages to facilitate exocytosis of TNFα

A key function of activated macrophages is to secrete proinflammatory cytokines such as TNFα; however, the intracellular pathway and machinery responsible for cytokine trafficking and secretion is largely undefined. Here we show that individual SNARE proteins involved in vesicle docking and fusion are regulated at both gene and protein expression upon stimulation with the bacterial cell wall component lipopolysaccharide. Focusing on two intracellular SNARE proteins, Vti1b and syntaxin 6 (Stx6), we show that they are up-regulated in conjunction with increasing cytokine secretion in activated macrophages and that their levels are selectively titrated to accommodate the volume and timing of post-Golgi cytokine trafficking. In macrophages, Vti1b and syntaxin 6 are localized on intracellular membranes and are present on isolated Golgi membranes and on Golgi-derived TNFα vesicles budded in vitro. By immunoprecipitation, we find that Vti1b and syntaxin 6 interact to form a novel intracellular Q-SNARE complex. Functional studies using overexpression of full-length and truncated proteins show that both Vti1b and syntaxin 6 function and have rate-limiting roles in TNFα trafficking and secretion. This study shows how macrophages have uniquely adapted a novel Golgi-associated SNARE complex to accommodate their requirement for increased cytokine secretion.

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.

Recycling endosome-dependent and -independent mechanisms for IL-10 secretion in LPS-activated macrophages

IL‐10 is a key anti‐inflammatory cytokine secreted by activated macrophages as a feedback control mechanism to prevent excessive inflammatory responses. Here, we define multiple intracellular trafficking pathways involved in the secretion of newly synthesized IL‐10 from macrophages following TLR4 activation with LPS, and show how this relates to the previously defined trafficking pathways for IL‐6 and TNF in macrophages simultaneously producing these proinflammatory cytokines. IL‐10 exits the Golgi in multiple tubular carriers, including those dependent on p230GRIP. Some of the IL‐10 is then delivered to recycling endosomes, where cytokine sorting may occur prior to its release. Another portion of the IL‐10 is delivered to the cell surface in distinct vesicles colabeled for apoE. Thus, we show at least two post‐Golgi pathways via which IL‐10 is trafficked, ensuring its secretion from activated macrophages under different physiological conditions.

The Rho GTPase Rac1 is required for recycling endosome-mediated secretion of TNF in macrophages

Rho GTPases are required for many cellular events such as adhesion, motility, and membrane trafficking. Here we show that in macrophages, the Rho GTPases Rac1 and Cdc42 are involved in lamellipodia and filopodia formation, respectively, and that both of these Rho GTPases are essential for the efficient surface delivery of tumor necrosis factor (TNF) to the plasma membrane following TLR4 stimulation. We have previously demonstrated intracellular trafficking of TNF via recycling endosomes in lipopolysaccharide (LPS)‐activated macrophages. Here, we further define a specific role for Rac1 in intracellular TNF trafficking, demonstrating impairment in TNF release following TLR4 stimulation in the presence of a Rac inhibitor, in cells expressing a dominant negative (DN) form of Rac1, and following small interfering RNA (siRNA) knockdown of Rac1. Rac1 activity was required for TNF trafficking but not for TLR4 signaling following LPS stimulation. Reduced TNF secretion was due to a defect in Rac1 activity, but not of the closely related Rho GTPase Rac2, demonstrated by the additional use of macrophages derived from Rac2‐deficient mice. Labeling recycling endosomes by the uptake of fluorescent transferrin enabled us to show that Rac1 was required for the final stages of TNF trafficking and delivery from recycling endosomes to the plasma membrane. Thus, actin remodeling by the Rho GTPase Rac1 is required for TNF cell surface delivery and release from macrophages.

Development of SH2 probes and pull-down assays to detect pathogen-induced, site-specific tyrosine phosphorylation of the TLR adaptor SCIMP

Protein tyrosine phosphorylation guides many molecular interactions for cellular functions. SCIMP is a transmembrane adaptor protein (TRAP) family member that mediates selective proinflammatory cytokine responses generated by pathogen‐activated Toll‐like receptor (TLR) pathways in macrophages. TLR activation triggers SCIMP phosphorylation and selective phosphorylation of distinct tyrosine residues on this adaptor offers the potential for regulating or biasing inflammatory responses. To analyze site‐specific phosphorylation events, we developed three probes based on the SH2 domains of known SCIMP effectors, and used them for pull‐downs from macrophage extracts. CRISPR‐mediated SCIMP‐deficient RAW264.7 macrophage‐like cells were reconstituted with various phosphorylation‐deficient (Y58F, Y96F, Y120F) SCIMPs, and used to demonstrate the specificity of LPS/TLR4‐induced, site‐specific phosphorylation of SCIMP for the temporal recruitment of the effectors Grb2, Csk and SLP65. Our findings reveal potential for differential SCIMP phosphorylation and specific effectors to influence TLR signaling and inflammatory programs. Furthermore, the use of Csk‐SH2 pull‐downs to identify additional known and new Csk targets in LPS‐activated macrophages reveals the wider utility of our SH2 probes.