The main research question that we study is: how are proteins sorted to the appropriate location in a cell? We use a combination of biochemical and cell biological techniques to study this fundamental process. Our work has also taken on a human health-related aspect as described below.
MEMBRANE TRAFFIC. A eukaryotic cell is composed of many different compartments (eg. nucleus, mitochondria, Golgi apparatus, vacuole, endoplasmic reticulum). Each of these organelles is surrounded by a membrane, thus separating the inside of the organelle from the cytoplasm. Furthermore, each compartment contains its own unique protein and lipid composition. Material can be transferred between specific organelles by small vesicles that bud from one compartment and fuse with another compartment. The central question in this aspect of our research is: how is the specificity in vesicle targeting ensured?
The TRAPP complexes. While there are numerous factors that help to ensure the proper targeting of transport vesicles, our work has focused on a large protein complex called TRAPP. In humans, there are in fact two related complexes called TRAPP II and III. They are composed of the same core of proteins but each has distinct polypeptides that influence the location and function of the complexes. These complexes function in the endomembrane system and TRAPP III has been implicated in autophagy. Our group has identified novel subunits of these complexes (TRAPPC2L, TRAPPC11, TRAPPC12) and in collaboration with clinicians and geneticists have reported individuals with mutations in some of the genes encoding these TRAPP proteins. Our work has contributed to the first reports of individuals with variants in TRAPPC12, TRAPPC11, TRAPPC4 and TRAPPC2L.
We perform studies on standard cell lines (e.g. HeLa) as well as on cells derived from individuals with mutations in TRAPP genes to better understand the functions of the TRAPP complexes. Our work expands to proteins that influence the function of TRAPP or whose function is influenced by the complexes including Rab GTPases and their effectors, as well as autophagy proteins as examples. We employ techniques such as live-cell imaging, confocal microscopy, liquid chromatography, basic molecular biological techniques and recombinant protein expression/purification and western analysis to name a few.
A. TRAPPC11 mutations result in a complex phenotype including muscular dystrophy
A number of individuals with mutations in the gene encoding TRAPPC11 have been described. One common feature in these individuals is that they all suffer from some form of muscular dystrophy (e.g. limb girdle muscular dystrophy, congenital muscular dystrophy). Some also have fatty liver, similar to the main phenotype seen in zebrafish with a trappc11 mutation. Interestingly, depletion of TRAPPC11 in HeLa cells affects some aspect of protein glycosylation. Since protein glycosylation is linked to muscular dystrophy, we are pursuing this avenue of research. Glycosylation defects may also relate to the fatty liver involvement in these individuals since defective glycosylation induces endoplasmic reticulum stress which can lead to increased lipid synthesis. The functions of the TRAPPC11 protein are a main focus of the work in our group
B. TRAPPC12 mutations affect brain development
The first reported individuals with mutations in TRAPPC12 were recently reported by our group. These individuals suffer from brain degeneration (encephalopathy). It is not understood why the mutations affect only brain. Our group made the serendipitous finding that TRAPPC12 is involved in mitosis, and depletion of cells of the TRAPPC12 protein results in a chromosome congression defect where the cells cannot progress beyond metaphase. We are investigating the functions of TRAPPC12 to better understand its role in brain development.
TANGO2. The TANGO2 gene product was described in 2006 as a protein required for some unidentified aspect of Golgi organization. Nearly a decade later the first individuals with TANGO2 variants were described. While the phenotype of such individuals is complex, there is partial overlap with the phenotype seen for individuals with TRAPPC2L variants. Very little is known about the basic biology of TANGO2 and a portion of the work in the laboratory is devoted to understanding its localization and function(s). We are employing amongst other methodologies "big biology" approaches such as BioID, lipidomics and RNAseq to understand the pathways in which the protein functions. Recently, we were the first to show that vitamin B5 supplementation rescued phenotypes associated with TANGO2 dysfunction in both fruit flies and human cells, a treatment that is now recommended for all individuals suffering from TANGO2 deficiency disease. We now seek to understand the mechanism of this treatment.