Function of the Cytoplasmic FMRP Interacting Protein 1 (CyFIP1) in mouse

Abstract

CyFIP (Cytoplasmic Fragile X mental retardation protein Interacting Protein) is a large scaffolding protein that was shown to be a central regulatory component of the WAVE complex. The pentameric WAVE complex is a nucleation promoting factor (NPF) regulated by phosphoinositides and phosphorylation, which translates signals from the small GTPase Rac1 by exposing the VCA domain in the WAVE subunit, thus activating the Arp2/3 actin nucleation complex and initiating actin polymerization. Actin reorganization is important for cell motility, migration, locomotion, proliferation and more. Except for HSPC300/Brick, each subunit is represented by a gene family - CyFIP1 and CyFIP2 for CyFIP, Nap1/Hem2 and Hem1 for Nap, WAVE1, WAVE2, and WAVE3 for WAVE, and Abi1, Abi2, and Abi3 for Abi. In principle this allows to build many different WAVE complexes, a few of which have also been crystalized and their structure determined. Despite the extensive sequence similarity, the expression pattern of CyFIP1 and CyFIP2 in mouse tissues suggests distinct physiological roles. CyFIP1 is ubiquitously expressed, while CyFIP2 has a more specialized function in the nervous system, where it is ten-fold more abundant than its homologue. CyFIP1 has been proposed to function as a tumor suppressor gene and is a known interaction partner of FMRP in the brain (Fragile X Mental Retardation Protein), hence having a distinct role in translational control. CyFIP1 has also been linked to autism spectrum disorder phenotypes in Prader-Willi, Angelman and Fragile X Syndromes.<br /> In this work the role of CyFIP1 during mouse embryonic development, ES cells in vitro differentiation and female fertility was analyzed, as well as the composition of the WAVE complex. The results showed that CyFIP1 has an essential role in early embryonic development and CyFIP1-/- embryos die around embryonic day 6.5. To obtain better insight into the function of CyFIP1 in development two strategies were used: 1. analysis of the embryos and 2. Generation and differentiation of ES cells. Generated CyFIP1-/- ES cells showed disturbed cell proliferation, differentiation and adhesion. Differentiation studies of CyFIP1-/- ES cells into embryoid bodies (EB) indicated that lineage determination is altered. EBs showed an increase in endodermal tissue, a decrease in meso-endodermal tissue and higher apoptosis. Importantly ES cells showed alterations in the levels of several WAVE complex components. Microarray analysis of in vitro differentiated ES cells revealed down-stream effectors and pathways affected by CyFIP1 depletion.<br /> CyFIP1+/- females showed an infertility disorder similar to women with Fragile X-dependent POI (primary ovarian insufficiency) Syndrome. Investigations in the ovaries and follicles of wild type and CyFIP1+/- females showed some important differences. Altered follicle distribution in the ovaries of CyFIP1+/- females was observed. Antral and preovulatory follicles of CyFIP1+/- females appeared significantly smaller than wt. Studies on the ligands and composition of the WAVE complex indicated that the WAVE complex is not an irreversibly stable pentameric complex. Biochemical analyses under different ionic conditions were made and results are in agreement with the predictions from the structure determined by the Rosen lab, showing how CyFIP/Nap forms a core complex around which the other subunits assemble. Variable complexes were recognized in different tissues. Novel Cyfip1/2 ligand candidates were identified by mass spectrometry after pull-down of the complex in brain, such as Cofilin1, CapZ, Myosin1 and Myosin4. One surprising novel Cyfip1/2 ligand was POF1 (Premature ovarian failure 1B), an actin binding protein mutated in human premature ovarian failure. These latest findings expand the current view of the WAVE complex.