To study the dynamics of stress fiber components in cultured fibroblasts, we expressed α-actinin and the myosin II regulatory myosin light chain (MLC) as fusion proteins with green fluorescent protein. Myosin activation was stimulated by treatment with calyculin A, a serine/threonine phosphatase inhibitor that elevates MLC phosphorylation, or with LPA, another agent that ultimately stimulates phosphorylation of MLC via a RhoA-mediated pathway. The resulting contraction caused stress fiber shortening and allowed observation of changes in the spacing of stress fiber components. We have observed that stress fibers, unlike muscle myofibrils, do not contract uniformly along their lengths. Although peripheral regions shortened, more central regions stretched. We detected higher levels of MLC and phosphorylated MLC in the peripheral region of stress fibers. Fluorescence recovery after photobleaching revealed more rapid exchange of myosin and α-actinin in the middle of stress fibers, compared with the periphery. Surprisingly, the widths of the myosin and α-actinin bands in stress fibers also varied in different regions. In the periphery, the banding patterns for both proteins were shorter, whereas in central regions, where stretching occurred, the bands were wider.

Talins are essential for integrin activation, focal adhesion formation and mesenchymal cell migration. The E3 ubiquitin ligase Smurf1 regulates talin head degradation and Cdk5-mediated phosphorylation of the head prevents Smurf1 action on talin. Cell migration is a dynamic process that requires temporal and spatial regulation of integrin activation and focal adhesion assembly/disassembly1. Talin, an actin and β-integrin tail-binding protein, is essential for integrin activation and focal adhesion formation2,3. Calpain-mediated cleavage of talin has a key role in focal adhesion turnover3; however, the talin head domain, one of the two cleavage products, stimulates integrin activation, localizes to focal adhesions and maintains cell edge protrusions2,4,5, suggesting that other steps, downstream of talin proteolysis, are required for focal adhesion disassembly. Here we show that talin head binds Smurf1, an E3 ubiquitin ligase involved in cell polarity and migration6,7, more tightly than full-length talin does and that this interaction leads to talin head ubiquitylation and degradation. We found that talin head is a substrate for Cdk5, a cyclin-dependent protein kinase that is essential for cell migration, synaptic transmission and cancer metastasis8,9,10,11. Cdk5 phosphorylated talin head at Ser 425, inhibiting its binding to Smurf1, thus preventing talin head ubiquitylation and degradation. Expression of the mutant talS425A, which resists Cdk5 phosphorylation thereby increasing its susceptibility to Smurf1-mediated ubiqitylation, resulted in extensive focal adhesion turnover and inhibited cell migration. Thus, talin head produced by calpain-induced cleavage of talin is degraded through Smurf1-mediated ubiquitylation; moreover, phosphorylation by Cdk5 regulates the binding of Smurf1 to talin head, controlling talin head turnover, adhesion stability and ultimately, cell migration.

The first steps of human coronavirus NL63 (HCoV-NL63) infection were previously described. The virus binds to target cells by use of heparan sulfate proteoglycans and interacts with the ACE2 protein. Subsequent events, including virus internalization and trafficking, remain to be elucidated. In this study, we mapped the process of HCoV-NL63 entry into the LLC-Mk2 cell line and ex vivo three-dimensional (3D) tracheobronchial tissue. Using a variety of techniques, we have shown that HCoV-NL63 virions require endocytosis for successful entry into the LLC-MK2 cells, and interaction between the virus and the ACE2 molecule triggers recruitment of clathrin. Subsequent vesicle scission by dynamin results in virus internalization, and the newly formed vesicle passes the actin cortex, which requires active cytoskeleton rearrangement. Finally, acidification of the endosomal microenvironment is required for successful fusion and release of the viral genome into the cytoplasm. For 3D tracheobronchial tissue cultures, we also observed that the virus enters the cell by clathrin-mediated endocytosis, but we obtained results suggesting that this pathway may be bypassed. IMPORTANCE Available data on coronavirus entry frequently originate from studies employing immortalized cell lines or undifferentiated cells. Here, using the most advanced 3D tissue culture system mimicking the epithelium of conductive airways, we systematically mapped HCoV-NL63 entry into susceptible cells. The data obtained allow for a better understanding of the infection process and may support development of novel treatment strategies.

Focal adhesion kinase (FAK) is an essential kinase that regulates developmental processes and functions in the pathology of human disease. An intramolecular autoinhibitory interaction between the FERM and catalytic domains is a major mechanism of regulation. Based upon structural studies, a fluorescence resonance energy transfer (FRET)-based FAK biosensor that discriminates between autoinhibited and active conformations of the kinase was developed. This biosensor was used to probe FAK conformational change in live cells and the mechanism of regulation. The biosensor demonstrates directly that FAK undergoes conformational change in vivo in response to activating stimuli. A conserved FERM domain basic patch is required for this conformational change and for interaction with a novel ligand for FAK, acidic phospholipids. Binding to phosphatidylinositol 4,5-bisphosphate (PIP2)-containing phospholipid vesicles activated and induced conformational change in FAK in vitro, and alteration of PIP2 levels in vivo changed the level of activation of the conformational biosensor. These findings provide direct evidence of conformational regulation of FAK in living cells and novel insight into the mechanism regulating FAK conformation.