Selected Projects Overview
Luminal Ca2+, the Ca2+ Sensor ALG-2, and Sec31 Regulate pre-Golgi Membrane Trafficking
Morphological evidence suggests that the first fusion event in the secretory pathway may represent homotypic fusion of COPII-derived vesicles to form vesicular tubular clusters (VTCs). The VTCs are then speculated to carry cargo from the cell periphery toward the Golgi region where a second membrane fusion delivers the cargo to the cis-Golgi. We developed in vitro assay systems which isolate and recapitulate this first fusion event in cargo transport, leading to the first direct demonstration of the COPII vesicle homotpyic fusion model for VTC generation (see Xu et al. 2004). Figure 1 shows our current working model of the steps in ER-to-Golgi vesicle transport.
Figure 1. Model of VTC formation & function. red, COPII coat and anterograde cargo; yellow, COPI coat and retrograde cargo. Blue intensity = luminal Ca2+ concentration.
We have accumulated substantial evidence that luminal Ca2+ regulates early steps in VTC formation/maturation. To elucidate a Ca2+ effector mechanism, we examined isolated COPII fusion products and discovered that Ca2+ chelation caused a selective loss of the COPII subunit sec31, and that the calcium sensor ALG-2 stabilized sec31 on COPII vesicles (see Bentley et al. 2010). Significantly, we demonstrated that ALG-2 "placed" sec31 at a critical functional site on the membrane. Thisrepresents the first evidence that the targeting of sc31 at a functional site is regulated by the Ca2+ sensor ALG-2, and have led to a model in which ALG-2 stabilizes sec31 at the membrane upon efflux of luminal Ca2+ through simultaneously binding sec31 and an unknown membrane-associated protein (see Figure 2). The ALG-2/sec31 interaction has several important potential consequences: 1) it could facilitate budding; 2) post-budding, it could restrict the fusogenicity of COPII derived membranes, regulate the precise coordination of VTC formation, and prevent COPII vesicle back-fusion with the ER; 3) by contributing to the immobilization of sec23/24, it could create anterograde cargo micro-domains on the VTC surface that could facilitate cargo sorting. Any of these mechanisms could provide explanations for the regulation of ER/Golgi transport we have documented for luminal Ca2+.
Figure 2. Model for action of luminal Ca2+, ALG-2, and COPII coat components. "Component M" represents an unidentified, hypothetical ALG-2 effector present on COPII vesicles.
Mechanism of Alpha-Synuclein Intoxication of ER/Golgi Transport
Alpha-synuclein is a cytosolic membrane-associated neuronal protein whose over-expression or mutation in humans causes Parkinson’s Disease (PD) involving the selective death of dopaminergic neurons. Investigations of the mechanisms underlying alpha-synuclein toxicity have gone in several directions. A central problem in interpreting studies of alpha-synuclein toxicity is the unknown degree of directness of the observed effects in cellular studies. Starting in 2006, a flurry of genomic studies reported that the most potent suppressors of alpha-synuclein-induced killing of yeast cells or neurodegeneration in worms involved overexpression of ER/Golgi transport proteins, arguing that ER/Golgi transport is a proximal target of alpha-synuclein toxicity [Cooper et al., 2006. Science 313:324; Humamachi et al., 2008. Proc. Natl. Acad. Sci. 105:728]. In the meantime, we developed a quantitative morphological ER-to-Golgi transport assay employing intact normal rat kidney (NRK) cells. Remarkably we found that overexpression of alpha-synuclein A53T, which causes PD in humans, retards ER-to Golgi transport of GFP-VSVG, a model secretory cargo membrane protein (Thayanidhi et al. 2010). Our work was the first to demonstrate that alpha-synuclein is a potent inhibitor of ER-to-Golgi transport in mammalian cells, even at moderate expression levels and in the absence of indirect effects (see Figure 3). The potentially important role that ER/Golgi trafficking machinery plays in PD is a major paradigm shift in the neurodegeneration field. Current work on this project in the Hay lab is focused on elucidating the mechanism of alpha-synuclein toxicity and understanding how the neuronal secretory pathway copes with the stress caused by its mutation or over-expression.
Figure 3. Collage summary of our findings on alpha-synuclein and ER/Golgi transport. The image shows immunofluoresence in normal rat kidney cells transfected with a GFP-tagged secretory cargo protein (VSV-G-GFP, green) that has been allowed to progress from the ER toward the Golgi for 12 minutes. In the single cell that is also transfected with the PD-causing mutant a-synuclein A53T (red), ER-to-Golgi transport is severely slowed and most of the cargo remains in the ER and pre-Golgi intermediates scattered throughout the cytoplasm. In surrounding cells lacking the a-synuclein A53T construct, the cargo has by this time already concentrated in the Golgi, visible as a series of bright juxtanuclear spots and ribbons. The red punctate structures in the inhibited cell are autophagolysosomes participating in the concentration and degradation of a-synuclein A53T. The key role of a-synuclein in neuronal toxicity and PD led to its representation as an unstructured serpentine polymer of 140 rat brains. The A53T mutation is depicted as a disordered brain in the polymer.