Periodic Report Summary - MEMBRANE DYNAMICS (The role of membrane trafficking in immune cell function)
Project objective and goal of the study
The general theme of the study is to investigate the roles of receptor endocytosis and the trafficking/sorting machinery in immune cell signalling/function. Specifically, the major research objective is to analyse the biological function of two regulators of small GTPases, RIN3 and ACAP1, which are involved in membrane trafficking and the regulation of the actin cytoskeleton. In addition to studying the biological function of these molecules, the project also focuses on their potential role in the development of allergy/asthma and autoimmunity, which will help to better understand the underlying mechanisms leading to disease.
Work performed and main results achieved so far
In initial studies, my laboratory focused on the analysis of the gene expression profile of RIN3 and ACAP1 in immune cells. Furthermore, we have established a comprehensive experimental system with relevant genetic tools - a lentiviral based siRNA gene knock-down system, and the cloning of RIN3 and ACAP1 cDNA for overexpression studies to examine the biological function of RIN3 and ACAP1 in in vitro systems.
Our detailed gene expression studies on purified mouse immune cell subpopulations revealed a particularly high expression of RIN3 in mast cells and dendritic cells, while RIN3 was hardly detectable in other immune cells, including T and B lymphocytes. In contrast, ACAP1 gene expression was highest in T and B-lymphocytes. ACAP1 was also expressed in macrophages and dendritic cells, albeit at lower levels.
Based on the high expression levels of RIN3 in mast cells, we have initiated a project aiming at the characterisation of the potential role of RIN3 in mast cell function. Our on-going studies have revealed that specific knockdown of RIN3 in mast cells results in an increased sensitivity to IgE-induced mast cell degranulation. Moreover, in activated mast cells, RIN3 knockdown leads to an enhanced release of cytokines. These data demonstrate an inhibitory function of RIN3 on mast cell degranulation and identify RIN3 as a novel negative regulator of mast cell function. The mechanism by which RIN3 affects granule trafficking and its physiological function/relevance is currently under investigation.
As an experimental complementation for the RIN3 gene knock-down approach, we have cloned murine RIN3 from primary mast cells into expression vectors to be utilised in overexpression studies. These experiments will further define the cellular function of RIN3.
Furthermore, to characterise the role of RIN3 in immune cell function in an in vivo setting, we are in the process of generating RIN3 deficient mice. The current status is that we now have identified targeted embryonic stem (ES) cell clones carrying a RIN3 knockout allele. The completed RIN3 knockout mouse is expected to be available in early 2010. In addition, we have also generated a polyclonal antibody specific for murine RIN3, which is an essential tool for protein expression and localisation studies.
The expected final results and their potential impact
Mast cells play a pivotal role in human allergic disease and intensively require specifically regulated vesicle transport and membrane fusion processes. However, the molecular mechanisms responsible for the intracellular trafficking of mast cell granular vesicles and regulated fusion of mast cell granules with the plasma membrane are still largely unknown. Therefore our novel finding on the negative regulatory role of RIN3 in mast cell function adds important new information to our understanding of mast cell biology. In further experiments we will investigate the molecular mechanisms underlying RIN3 function in mast cells, which will significantly advance our knowledge on the molecular processes that regulate membrane trafficking during mast cell degranulation and allergic responses. Moreover, the RIN3 deficient mice also open up new prospects of generating mouse disease models to study allergy/asthma.
The general theme of the study is to investigate the roles of receptor endocytosis and the trafficking/sorting machinery in immune cell signalling/function. Specifically, the major research objective is to analyse the biological function of two regulators of small GTPases, RIN3 and ACAP1, which are involved in membrane trafficking and the regulation of the actin cytoskeleton. In addition to studying the biological function of these molecules, the project also focuses on their potential role in the development of allergy/asthma and autoimmunity, which will help to better understand the underlying mechanisms leading to disease.
Work performed and main results achieved so far
In initial studies, my laboratory focused on the analysis of the gene expression profile of RIN3 and ACAP1 in immune cells. Furthermore, we have established a comprehensive experimental system with relevant genetic tools - a lentiviral based siRNA gene knock-down system, and the cloning of RIN3 and ACAP1 cDNA for overexpression studies to examine the biological function of RIN3 and ACAP1 in in vitro systems.
Our detailed gene expression studies on purified mouse immune cell subpopulations revealed a particularly high expression of RIN3 in mast cells and dendritic cells, while RIN3 was hardly detectable in other immune cells, including T and B lymphocytes. In contrast, ACAP1 gene expression was highest in T and B-lymphocytes. ACAP1 was also expressed in macrophages and dendritic cells, albeit at lower levels.
Based on the high expression levels of RIN3 in mast cells, we have initiated a project aiming at the characterisation of the potential role of RIN3 in mast cell function. Our on-going studies have revealed that specific knockdown of RIN3 in mast cells results in an increased sensitivity to IgE-induced mast cell degranulation. Moreover, in activated mast cells, RIN3 knockdown leads to an enhanced release of cytokines. These data demonstrate an inhibitory function of RIN3 on mast cell degranulation and identify RIN3 as a novel negative regulator of mast cell function. The mechanism by which RIN3 affects granule trafficking and its physiological function/relevance is currently under investigation.
As an experimental complementation for the RIN3 gene knock-down approach, we have cloned murine RIN3 from primary mast cells into expression vectors to be utilised in overexpression studies. These experiments will further define the cellular function of RIN3.
Furthermore, to characterise the role of RIN3 in immune cell function in an in vivo setting, we are in the process of generating RIN3 deficient mice. The current status is that we now have identified targeted embryonic stem (ES) cell clones carrying a RIN3 knockout allele. The completed RIN3 knockout mouse is expected to be available in early 2010. In addition, we have also generated a polyclonal antibody specific for murine RIN3, which is an essential tool for protein expression and localisation studies.
The expected final results and their potential impact
Mast cells play a pivotal role in human allergic disease and intensively require specifically regulated vesicle transport and membrane fusion processes. However, the molecular mechanisms responsible for the intracellular trafficking of mast cell granular vesicles and regulated fusion of mast cell granules with the plasma membrane are still largely unknown. Therefore our novel finding on the negative regulatory role of RIN3 in mast cell function adds important new information to our understanding of mast cell biology. In further experiments we will investigate the molecular mechanisms underlying RIN3 function in mast cells, which will significantly advance our knowledge on the molecular processes that regulate membrane trafficking during mast cell degranulation and allergic responses. Moreover, the RIN3 deficient mice also open up new prospects of generating mouse disease models to study allergy/asthma.