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Signal Transduction Proteomics
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Research area
Mutations in MYO5b cause microvillus inclusion disease. Autosomal recessive microvillus inclusion disease (MVID) is characterized by an intractable diarrhea starting within the first few weeks of life. The hallmarks of MVID are a lack of microvilli on the surface of villous enterocytes, occurrence of intracellular vacuoles lined by microvilli (microvillus inclusions), and the cytoplasmic accumulation of periodic acid-Schiff (PAS)-positive vesicles in enterocytes. Recently, we together with our collaborators from the Department of Pediatrics II, MUI and the Division of Histology and Embryology, MUI, were the first to identify mutations in MYO5B, encoding the unconventional type Vb myosin motor protein, in a first cohort of nine MVID patients (Mueller et al., Nature Genetics 2008). In a follow up study, we identified 15 novel nonsense and missense mutations in MYO5B in 11 unrelated MVID patients. Fluorescence microscopy, Western blotting, and electron microscopy were applied to analyze the effects of MYO5B siRNA knock-down in polarized, brush border possessing CaCo-2 cells.
Cell surface phenotype of CaCo2 cells following MYO5B-si-RNA knock-down treatment
Loss of surface microvilli, increased formation of microvillus inclusions, and subapical enrichment of PAS-positive endomembrane compartments were induced in polarized, filter-grown CaCo-2 cells, following MYO5B knock-down (see Figure 1). Our data indicate that MYO5B mutations are a major cause of microvillus inclusion disease and that MYO5B knock-down recapitulates most of the cellular phenotype in vitro, thus independently showing loss of MYO5B function as the cause of microvillus inclusion disease (Ruemmele et al., Human Mutation 2010). Meanwhile we have embarked on generation a MYO5b knockout mouse model.
Regulation of focal adhesions dynamics and cell migration through p14/MP1/MAP kinase signaling endosomes
A novel role for late endosomes carrying the p14/MP1 MAPK scaffold complex in signaling compartmentalization and cell migration. In control cells, IQGAP1 is localized to the plasma membrane and to focal complexes (FC) and supports Rac1 activation and formation of new FC. Upon maturation of FC into focal adhesions (FA), IQGAP1 dissociates from FA and recycles back to the plasma membrane. The process of IQGAP1 dissociation from the FA requires endosomal MEK/ERK signaling and the binding of IQGAP1 to the p14/MP1 complex. When p14/MP1 is absent from the late endosome, disassociation of IQGAP1 does not take place efficiently and IQGAP1 accumulates at FA. As a result, Rac1 is no longer activated at the plasma membrane and new FC are not formed. The absence of endosomal MEK/ERK activation on the other hand results in impaired FA dynamics. Disbalance between FC and subpopulations of FA in cells where p14/MP1 is no longer present on late endosomes, results in a phenotype where new FC are not efficiently formed (due to impaired Rac1)
Focal adhesions govern cell motility. Asymmetric distribution and dynamics of focal adhesions rely on a variety of signaling cascades and require polarized cellular distribution of molecular components. Within the SFB021 and in close collaboration with the Fässler group (MPI, Munich) we could recently show that late endosomes, carrying the p14/MP1 MAPK scaffold complex, can move to the cell periphery where they specifically target focal adhesions. This was the first presentation of MAP kinase signaling complexes that move along on late endosomes in order to target specific structures in the cell periphery for signal propagation. In the absence of the p14/MP1 signaling complexes on late endosomes we observed strongly impaired cell migration and a defect in focal adhesion remodeling. Our data showed further that binding of the signaling complex through MP1 to the IQ domain of IQGAP1 is required for IQGAP1 localization to the plasma membrane, activation of Rac1 at the leading edge and proper focal adhesion remodeling. We propose a novel role for late endosomes carrying the p14/MP1 MAPK scaffold complex in signaling compartmentalization and cell migration; see Figure 2.
International cooperations
- Max Planck Institute of Biochemistry, Department of Molecular Medicine (Reinhard Fässler)
- Harvard Medical School Brigham and Women`s Hospita, Boston, Mass. (David B. Sacks)