Proteomics Of Trans-acing Factors And Identification Of Filamin A-Binding Partner In Mechanotransduction

The biological processes that cells sense and response to the mechanical forces(such as gravity,stress and pressure)are called mechanotransduction.Mechanotransduction is important for mechano-sensitive cell growth,differentiation,and survival.For example,mesenchymal stem cells can differentiate into different type of cells depending on substrate stiffness.However,molecular mechanism of how cells sense and transmit the signal to regulate gene expression is not well known.Here,this thesis concerns to identify mechano-sensitive molecules using two different approaches.In Part I,I attempted to develop a new method to identify trans-acting factors using SILAC(Stable Isotope Labeling with Amino Acids in Cell Culture)followed by the mass spectrometric analysis.Since YAP,co-transcription factor,is known to shuttle upon mechanical stimulation,we used YAP as an internal marker.First,we tested commercial kits to isolate nucleus to detect YAP in the isolated nucleus.However,none of the commercial kits successfully fractionated YAP in the nuclear fraction even though YAP was mainly localized in nucleus in growing cells.Instead most of YAP was fractionated in soluble cytoplasmic fraction.Therefore,we attempted to isolate the nucleus using hypotonic buffer with detergent like NP-40 and Triton X-100.We found that addition of mannitol can keep YAP in nucleus during organelle fractionation.We also found that DNase I treatment can extract YAP from the nuclear fraction presumably by cleaving DNase I hypersensitive sites(DHSs),where trans-acting factors associate.WBP2(WW domain-binding protein 2)was identified as a preliminary mechanosensitive shuttling molecule upon the stimulation of blebbistatin.In the meantime,I have established manufacturing elastic hydrogels which will be used to study the mechanism of stem cells differentiation on substrates with different stiffness.Mouse embryonic fibroblast(MEF)cells and rat mesenchymal stem cells can attach to the hydrogels well and the YAP translocation was detected as expected.In part II,we found a novel filamin A mechanobinding partner.Filamin A(FLNA),an actin cross-linking protein,acts as a mechanosensor and mechanotransducer by exposing the cryptic binding site on repeat 21(R21)to interact with integrin.Here,we investigated if any other biological molecules interact with the cryptic binding site on R21.Using proteomics and screening for a FLNA-binding motif in silico,we identified and characterized a protein termed fimbacin(filamin mechanobinding actin crosslinking protein),encoded in the LUZP1 gene,as a novel FLNA-binding partner.Fimbacin does not interact with canonical full-length FLNA,but the exposure of a cryptic integrin-binding site of FLNA R21 enables fimbacin to interact.We have identified two FLNA binding sites on fimbacin and determined critical amino acid residues for the interaction.We also found that fimbacin itself is a new actin-crosslinking protein and mapped the actin-binding site on amino acid residues 400-500.Fimbacin oligomerizes(estimated as an octamer on size-exclusion chromatography)through the amino-terminal domain that is predicted to be a coiled-coil to cross-link actin filaments.When expressed,fimbacin localized to actin stress fibers in tissue culture cells.Although the interaction with FLNA is not necessary for fimbacin to colocalize with F-actin,fluorescent recovery after photobleaching(FRAP)revealed that their interaction stabilizes fimbacin on the actin cytoskeleton and inhibition of Rho-kinase,an upstream activator of myosin II,also decreases the interaction presumably due to loss of internal mechanical stress.Taken together,these data identify fimbacin as a new actin-crosslinking protein that interacts with the FLNA mechanosensing domain R21.