Refilins: A link between perinuclear actin bundle dynamics and mechanosensing signaling

Abstract

Actin cytoskeleton dynamics lie at the heart of cell mechanosensing signaling. In fibroblast cells, two perinuclear acto-myosin structures, the actin cap and the transmembrane actin-associated nuclear (TAN) line, are components of a physical pathway transducing extracellular physical signals to changes in nuclear shape and movements. We recently demonstrated the existence of a previously uncharacterized third apical perinuclear actin organization in epithelial cells that forms during epithelial-mesenchymal transition (EMT) mediated by TGFβ (TGFβ). A common regulatory mechanism for these different perinuclear actin architectures has emerged with the identification of a novel family of actin bundling proteins, the Refilins. Here we provide updates on some characteristics of Refilin proteins, and we discuss potential function of the Refilins in cell mechanosensing signaling.

Figure 1. Sequence analysis of the N-terminus of rat RefilinA and rat RefilinB. (A) Sequence alignment of the N-terminus of rat RefilinA (residues 1–99) and RefilinB (residues 1–112) proteins shows conserved regions with homologous (purple) or similar (blue) residues. A 15 amino-acid N-terminal sequence, harbouring a DSG(X)_2–4S motif is fully conserved between the two proteins (blue rectangle), whereas a specific adjacent sequence is only found in RefilinB (red rectangle). (B) Sequence comparison of the DSG(X)_2–4S motif within β-catenin, IκBα, EMI1, Snail, ATF4, NFκB1/p105, Refilin and Cdc25A proteins.

Figure 2. Localization of the Refilin/FLNA complex in U373 MG and U373A cells. U373MG cells and their tumorogenic U373A countepart were infected with recombinant adenovirus expressing RefilinB-GFP. Cells were fixed in paraformaldehyde and immunostained with antibodies against FLNA from USBiological (red). Bar = 20 µm.

Figure 3. RefilinB localizes on apical actin cytoskeleton in confluent RPE1 cells. Confluente RPE1 cells were fixed with methanol and double immunostained with guinea pig anti RefilinB antibody (Red) and mouse anti-FLNA antibody (green). Bar = 20 µm.

Figure 4. Parameters regulating formation of actin perinuclear strucuture by Refilins. (A) U373A cells plated on fibronectin (left column) or collagen 1 (right column) substrates were infected with RefilinB-GFP expressing adenoviral particles, fixed in 4% paraformaldehyde and immunostained with a mouse antibody against FLNA protein. Bar = 20 µm. (B) Single U373A cells plated on fibronectin micropatterns of different shapes (upper right of top image) were infected with RefilinB-GFP expressing adenoviral particles, fixed in 4% paraformaldehyde and immunostained with a mouse antibody against FLNA protein. RefilinB-GFP (top) and FLNA (bottom) co-localize on basal fibers but lack perinuclear staining. Similar results were obtained with microprinted fibronectin islands with the sizes of 700 µm², 1100 µm² and 1600 µm².

Figure 5. RefilinB and FLNA localize on apical actin cytoskeleton in NMuMG cells during epithelial-mesenchymal transition. NMuMG epithelial cells were cultured in the presence of TGFβ (2 ng/ml) for 12 h. Cells were then fixed with methanol and double immunostained with guinea pig anti RefilinB antibody (red) and mouse anti FilaminA antibody (green). Low (upper; Bar = 20 µm) and high (lower; Bar = 5 µm) magnification confocal microscopy images are shown.