Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - SYSMET (Systems Biology of Membrane Trafficking)

Teaser

Membrane trafficking is fundamental for homeostasis of the internal membrane system and transport to and from the extracellular medium. Although we have gained detailed knowledge on the molecular organization of membrane trafficking machineries a global view of its function...

Summary

Membrane trafficking is fundamental for homeostasis of the internal membrane system and transport to and from the extracellular medium. Although we have gained detailed knowledge on the molecular organization of membrane trafficking machineries a global view of its function and regulation is lacking. To date membrane trafficking is often regarded as a constitutive process with a high degree of functional redundancy. However, the fact that mutations of single trafficking genes with ubiquitous expression give rise to tissue-specific human diseases and discrete sets of trafficking genes have differential effects on tissue development challenge this view.
Here, using a combination of state-of the-art technologies, we will apply a systems biology approach in specialized cell types to establish a physiological and functional spatiotemporal map of membrane trafficking genes and proteins (membrane trafficking modules; MTMs). To this end we have curated a list of 1,187 genes representing ER, Golgi, Endosomes and Lysosomes (EGEL) around which we develop independent but interconnected approaches: (i) RNA-seq and antibody microarrays to identify co-regulated MTMs; (ii) high-content siRNA screening to define functional MTMs; (iii) epistatic functional analysis between EGEL genes and five membrane trafficking disease genes (TRAPPC2 in chondrocytes, Sec23A in osteoblasts, OCRL and CLCN5 in proximal tubular epithelial kidney cells, and VAPB in neuronal cells); and (iv) studies of protein-protein interactions to generate functional and physical networks centered on the disease genes.
SYSMET will generate a unique resource by defining the impact and interplay of the different regulatory layers of the entire membrane trafficking system with important implications for human health.

The overall objectives of the project are:
Objective 1: Identifying gene and protein regulatory networks in membrane trafficking
Objective 2: Identifying functional membrane trafficking modules
Objective 3: Assessing the impact of membrane trafficking modules in human disease

Work performed

Objective 1: Identifying gene and protein regulatory networks in membrane trafficking
Using data from the Genotype-Tissue Expression project (2,897 samples from 25 tissues) we constructed a co-expression network and identified 23 consensus Membrane Trafficking Modules (MTMs) significantly preserved across the analyzed tissues. We checked whether the co-expression got from spatial criteria could also occur according to temporal criteria. Thus we evaluated the behavior of MTMs during the process of reprogramming/differentiation and found that for instance, MTM8 had a trend overlapping with that of mesenchymal genes with some of the overlapping genes having a known role in collagen secretion, thus indicating that the entire module may correspond to a collagen-dedicated module that is switched off during reprogramming and switched on during mesodermal differentiation. We are preparing a manuscript reporting the identification of MTMs (an important resource for the community of cell biologists) and the functional validation of some of them.

Objective 2: Identifying functional membrane trafficking modules
We pursued the experimental validation of the functional relevance of the MTMs assessing their involvement in conventional membrane trafficking pathways and in the non-vesicular communication between organelles that occurs at the level of membrane contact sites (CS). CS between the ER and the TransGolgiNetwork (ERTGoCs) have been poorly characterized mainly due to methodological limits. We therefore developed an innovative FLIM-FRET–based approach to study ERTGoCS. We showed that ERTGoCS control PI4P at the TGN by providing a spatial setting suitable for the ER localized Sac1 phosphatase to dephosphorylate PI4P in trans at the TGN with the help of FAPP1.
Having identified FAPP1 as a key regulator of PI4P levels, we wanted to expand the knowledge of its interplay with other membrane trafficking components, so we interrogated our MTM database. FAPP1 belongs to MTM14 where it shows a high degree of co-expression with GOLPH3, a PI4P effector and an oncogene. We have obtained results indicating that FAPP1 controls GOLPH3 activity in promoting cell migration and invasion by regulating PI4P at the Golgi complex thus showing that the definition of the membrane trafficking modules was highly informative and instrumental in identifying biologically relevant processes.

Objective 3: Assessing the impact of membrane trafficking modules in human disease
We obtained evidence that: 1. OCRL (mutated in OculoCerebroRenal Lowe syndrome) interacts with synaptic vesicle components and controls synaptic vesicle recycling in hyppocampal neurons; 2. TRAPPC2 (mutated in spondyloepiphyseal dysplasia tarda) plays a pivotal role the biogenesis of stress granules the membrane less organelles that assemble in response to a variety of stress conditions; and 3. VAPB (mutated in Amyotrophic Lateral Sclerosis 8) establishes a bidirectional functional relationship with mitochondria. This was possible thanks to the novel yeast model we generated to mimic the autosomal dominant nature of ALS8.

Final results

We have achieved the first demonstration that membrane trafficking genes are organized in modules including components that are highly and significantly co-expressed, we have obtained initial demonstration that these co-expression modules have a functional correlate. In the following we will expand the functional analysis of the modules and investigate their relevance for human disease involving membrane trafficking genes.