SUMMARY1. A study has been made of the tension responses and sarcomere length changes produced by servo-controlled stretches applied to isometrically contracting frog muscle. Sarcomere lengths were monitored by cine-photography of diffraction spectra obtained by illuminating a small area of muscle with a laser.2. The tension increment produced by a ramp-and-hold stretch of approximately 1 mm (ca. 4 % of the muscle length) comprises three phases whose limits are defined by two points, S, and S2, where the slope of the response decreases abruptly. S1 and S2 correspond to extensions of 0 13 and 1-2% of the muscle length.3. Movements of the first order spectra relative to the zero order recorded during stretch reveal that S2 coincides with an abrupt elongation of the sarcomeres. This is termed sarcomere 'give' and it occurs when the filaments are displaced by 11-12 nm from their steady-state (isometric) position.4. The stiffness of the sarcomeres, E8, up to S2 decreases with increasing sarcomere length. The maximum force sustained by the muscle at S2, Ps,, also shows an inverse dependence on sarcomere length. Both E8 and Ps, fall to zero at an extrapolated sarcomere spacing of 3*6-3-7 /tm, coinciding with the length at which the actin and myosin filaments no longer overlap.5. The ratio Ps,/PO (where P0 = maximum isometric tension) varies with temperature and speed of stretch. It increases with increasing speeds of stretch until a certain critical velocity, VY, is reached, beyond which it is almost independent of any further increase. V, has a positive temperature coefficient, increasing 5-6 in the range 0-30 TC (Q10 = 1.8). There is a positive correlation between the maximum speed of isotonic shortening (Vmax.) and Vc in different muscles.6. Sarcomere 'give' during stretch is considered to be due to forcible detachment of cross-bridges between the actin and myosin filaments. This results in recoil of the extended series elastic elements in the muscle at the expense of the sarcomeres. The amount of filament displacement required to induce sarcomere 'give' (11-12 nm) is thought to represent the range of movement over which a cross-bridge can remain attached to actin during a stretch.

Abstract. The properties of keratin intermediate filaments (IFs) have been studied after transfection with green fluorescent protein (GFP)-tagged K18 and/or K8 (type I/II IF proteins). GFP-K8 and -K18 become incorporated into tonofibrils, which are comprised of bundles of keratin IFs. These tonofibrils exhibit a remarkably wide range of motile and dynamic activities. Fluorescence recovery after photobleaching (FRAP) analyses show that they recover their fluorescence slowly with a recovery t 1/2 of ‫ف‬ 100 min. The movements of bleach zones during recovery show that closely spaced tonofibrils ( Ͻ 1 m apart) often move at different rates and in different directions. Individual tonofibrils frequently change their shapes, and in some cases these changes appear as propagated waveforms along their long axes. In addition, short fibrils, termed keratin squiggles, are seen at the cell periphery where they move mainly towards the cell center. The motile properties of keratin IFs are also compared with those of type III IFs (vimentin) in PtK2 cells. Intriguingly, the dynamic properties of keratin tonofibrils and squiggles are dramatically different from those of vimentin fibrils and squiggles within the same cytoplasmic regions. This suggests that there are different factors regulating the dynamic properties of different types of IFs within the same cytoplasmic regions.

The ␣4 laminin subunit is a component of endothelial cell basement membranes. An antibody (2A3) against the ␣4 laminin G domain stains focal contact-like structures in transformed and primary microvascular endothelial cells (TrHBMECs and HMVECs, respectively), provided the latter cells are activated with growth factors. The 2A3 antibody staining colocalizes with that generated by ␣v and ␤3 integrin antibodies and, consistent with this localization, TrHBMECs and HMVECs adhere to the ␣4 laminin subunit G domain in an ␣v␤3-integrin-dependent manner. The ␣v␤3 integrin/2A3 antibody positively stained focal contacts are recognized by vinculin antibodies as well as by antibodies against plectin. Unusually, vimentin intermediate filaments, in addition to microfilament bundles, interact with many of the ␣v␤3 integrin-positive focal contacts. We have investigated the function of ␣4-laminin and ␣v␤3-integrin, which are at the core of these focal contacts, in cultured endothelial cells. Antibodies against these proteins inhibit branching morphogenesis of TrHBMECs and HMVECs in vitro, as well as their ability to repopulate in vitro wounds. Thus, we have characterized an endothelial cell matrix adhesion, which shows complex cytoskeletal interactions and whose assembly is regulated by growth factors. Our data indicate that this adhesion structure may play a role in angiogenesis.

Much effort has been expended on analyzing how microfilament and microtubule cytoskeletons dictate the interaction of cells with matrix at adhesive sites called focal adhesions (FAs). However, vimentin intermediate filaments (IFs) also associate with the cell surface at FAs in endothelial cells. Here, we show that IF recruitment to FAs in endothelial cells requires β3 integrin, plectin and the microtubule cytoskeleton, and is dependent on microtubule motors. In CHO cells, which lack β3 integrin but contain vimentin, IFs appear to be collapsed around the nucleus, whereas in CHO cells expressing β3 integrin (CHOwtβ3), vimentin IFs extend to FAs at the cell periphery. This recruitment is regulated by tyrosine residues in the β3 integrin cytoplasmic tail. Moreover, CHOwtβ3 cells exhibit significantly greater adhesive strength than CHO or CHO cells expressing mutated β3 integrin proteins. These differences require an intact vimentin network. Therefore, vimentin IF recruitment to the cell surface is tightly regulated and modulates the strength of adhesion of cells to their substrate.

Phosphorylation of keratin intermediate filaments (IF)is known to affect their assembly state and organization; however, little is known about the mechanisms regulating keratin phosphorylation. In this study, we demonstrate that shear stress, but not stretch, causes disassembly of keratin IF in lung alveolar epithelial cells (AEC) and that this disassembly is regulated by protein kinase C ␦-mediated phosphorylation of keratin 8 (K8) Ser-73. Specifically, in AEC subjected to shear stress, keratin IF are disassembled, as reflected by their increased solubility. In contrast, AEC subjected to stretch showed no changes in the state of assembly of IF. Pretreatment with the protein kinase C (PKC) inhibitor, bisindolymaleimide, prevents the increase in solubility of either K8 or its assembly partner K18 in shearstressed AEC. Phosphoserine-specific antibodies demonstrate that K8 Ser-73 is phosphorylated in a time-dependent manner in shear-stressed AEC. Furthermore, we showed that shear stress activates PKC ␦ and that the PKC ␦ peptide antagonist, ␦ V1-1, significantly attenuates the shear stress-induced increase in keratin phosphorylation and solubility. These data suggested that shear stress mediates the phosphorylation of serine residues in K8, leading to the disassembly of IF in alveolar epithelial cells. Importantly, these data provided clues regarding a molecular link between mechanically induced signal transduction and alterations in cytoskeletal IF.