Cell-penetrating peptides like the cationic human immunodeficiency virus-1 trans-acting activator of transcription (TAT) peptide have the capability to traverse cell membranes and to deliver large molecular cargoes into the cellular interior. We used optical sectioning and state-of-the-art single-molecule microscopy to examine the passive membrane permeation of fluorescently labeled TAT peptides across the membranes of giant unilamellar vesicles (GUVs). In GUVs formed by phosphatidylcholine and cholesterol only, no translocation of TAT up to a concentration of 2 microM into the GUVs could be observed. At the same peptide concentration, but with 40 mol % of anionic phosphatidylserine in the membrane, rapid translocation of TAT peptides across the bilayers was detected. Efficient translocation of TAT peptides was observed across GUVs containing 20 mol % of phosphatidylethanolamine, which is known to induce a negative curvature into membranes. We discovered that TAT peptides are not only capable of penetrating membranes directly in a passive manner, but they were also able to form physical pores with sizes in the nanometer range, which could be passed by small dye tracer molecules. Lipid topology and anionic charge of the lipid bilayer are decisive parameters for pore formation.

The force generated by the actomyosin cytoskeleton controls focal adhesion dynamics during cell migration. This process is thought to involve the mechanical unfolding of talin to expose cryptic vinculin-binding sites. However, the ability of the actomyosin cytoskeleton to directly control the formation of a talin–vinculin complex and the resulting activity of the complex are not known. Here we develop a microscopy assay with pure proteins in which the self-assembly of actomyosin cables controls the association of vinculin to a talin-micropatterned surface in a reversible manner. Quantifications indicate that talin refolding is limited by vinculin dissociation and modulated by the actomyosin network stability. Finally, we show that the activation of vinculin by stretched talin induces a positive feedback that reinforces the actin–talin–vinculin association. This in vitro reconstitution reveals the mechanism by which a key molecular switch senses and controls the connection between adhesion complexes and the actomyosin cytoskeleton.

No abstract

Cell-penetrating peptides like the cationic HIV1 TAT peptide are able to translocate across cell membranes and to carry molecular cargoes into the cellular interior. For most of these peptides, the biophysical mechanism of the membrane translocation is still quite unknown. We analyzed HIV1 TAT peptide binding and mobility within biological model membranes. To this end, we generated neutral and anionic giant unilamellar vesicles (GUVs) containing DPPC, DOPC, and cholesterol and containing DPPC, DOPC, cholesterol, and DPPS (DOPS), respectively. First, we characterized the mobility of fluorescently labeled lipids (TR-DHPE) within liquid-ordered and liquid-disordered lipid phases by single-molecule tracking, yielding a D LO of 0.6 ( 0.05 μm 2 /s and a D LD of 2.5 ( 0.05 μm 2 /s, respectively, as a reference. Fluorescently labeled TAT peptides accumulated on neutral GUVs but bound very efficiently to anionic GUVs. Single-molecule tracking revealed that HIV1 TAT peptides move on neutral and anionic GUV surfaces with a D N,TAT of 5.3 ( 0.2 μm 2 /s and a D A,TAT of 3.3 ( 0.2 μm 2 /s, respectively. TAT peptide diffusion was faster than fluorescent lipid diffusion, and also independent of the phase state of the membrane. We concluded that TAT peptides are not incorporated into but rather floating on lipid bilayers, but they immerged deeper into the headgroup domain of anionic lipids. The diffusion constants were not dependent on the TAT concentration ranging from 150 pM to 2 μM, indicating that the peptides were not aggregated on the membrane and not forming any "carpet".In the past decade, specific peptides and proteins have been shown to penetrate cell membranes while retaining their normal biological activity by a process called protein transduction (1, 2). The peptide sequences, which could be identified as being responsible for the translocation capability, were designated as protein transduction domains (PTDs) 1 (3), cell-penetrating peptides (CPPs) (4), or Trojan horse peptides (5). Descriptions of the internalization process range from energy-independent cell penetration of membranes to endocytic uptake (1, 6-9). Certainly, the process varies for different types of PTDs. In general, however, the biophysical mechanism of the plasma membrane translocation of the PTDs is not well understood. A highly charged cationic domain is common to all PTDs and essential for translocation. However, it is well-known that charged molecules cannot cross the lipid bilayer by passive diffusion due to the high Born charging energy encountered in a medium with a low dielectric constant. Cells usually utilize special transport systems such as ion carriers, channels, and ATP-coupled pumps to regulate the flow of ions and charged molecules across their membranes. From a physicalchemical point of view, it is therefore very surprising that short peptides containing a high percentage of cationic amino acids can nevertheless cross the plasma membrane of living cells by pathways that mostly appear to function without energy consumpti...

Cell-matrix adhesion plays a major role during cell migration. Proteins from adhesion structures connect the extracellular matrix to the actin cytoskeleton, allowing the growing actin network to push the plasma membrane and the contractile cables (stress fibers) to pull the cell body. Force transmission to the extracellular matrix depends on several parameters including the regulation of actin dynamics in adhesion structures, the contractility of stress fibers, and the mechanosensitive response of adhesion structures. Here we highlight recent findings on the molecular mechanisms by which actin assembly is regulated in adhesion structures and the molecular basis of the mechanosensitivity of focal adhesions.

Many lantibiotics use the membrane bound cell wall precursor Lipid II as a specific target for killing Gram-positive bacteria. Binding of Lipid II usually impedes cell wall biosynthesis, however, some elongated lantibiotics such as nisin, use Lipid II also as a docking molecule for pore formation in bacterial membranes. Although the unique nisin pore formation can be analyzed in Lipid II-doped vesicles, mechanistic details remain elusive. We used optical sectioning microscopy to directly visualize the interaction of fluorescently labeled nisin with membranes of giant unilamellar vesicles containing Lipid II and its various bactoprenol precursors. We quantitatively analyzed the binding and permeation capacity of nisin when applied at nanomolar concentrations. Specific interactions with Lipid I, Lipid II and bactoprenol-diphosphate (C55-PP), but not bactoprenol-phosphate (C55-P), resulted in the formation of large molecular aggregates. For Lipid II, we demonstrated the presence of both nisin and Lipid II in these aggregates. Membrane permeation induced by nisin was observed in the presence of Lipid I and Lipid II, but not in the presence of C55-PP. Notably, the size of the C55-PP-nisin aggregates was significantly smaller than that of the aggregates formed with Lipid I and Lipid II. We conclude that the membrane permeation capacity of nisin is determined by the size of the bactoprenol-containing aggregates in the membrane. Notably, transmitted light images indicated that the formation of large aggregates led to a pinch-off of small vesicles, a mechanism, which probably limits the growth of aggregates and induces membrane leakage.

In many mechanosensitive biological processes, actin-binding proteins (ABPs) sense the force generated by the actomyosin cytoskeleton and respond by recruiting effector proteins. We developed an in vitro assay, with pure proteins, to observe the force-dependent binding of a protein to a cryptic binding site buried in the stretchable domain of an ABP. Here we describe the protocol to study the actomyosin-dependent binding of vinculin to the ABP talin. In this assay, talin is immobilized in 5-μm-diameter disc-shaped islands, which are regularly spaced by 35 μm and micropatterned on a glass coverslip. In response to the force generated by an actomyosin network, talin extension reveals cryptic vinculin-binding sites (VBSs). To follow this reaction, fluorescent proteins are visualized by total internal refection fluorescence (TIRF) microscopy. EGFP-vinculin fluorescence in talin-coated discs reveals the binding of vinculin to stretched talin. Actomyosin structures are visualized by the fluorescence of Alexa Fluor 594-labeled actin. This protocol describes the purification of the proteins, the preparation of the chamber in which talin is coated on a micropatterned surface, and the biochemical conditions to study several kinetic parameters of the actomyosin-dependent binding of vinculin to talin. A stable actomyosin network is used to measure the steady-state dissociation of vinculin from talin under constant force. In the presence of α-actinin-1, actomyosin cables undergo cycles of force application and release, allowing the measurement of vinculin dissociation associated with talin re-folding. Expression and purification of the proteins requires at least 3 weeks. The assay can be completed within 1 d.

Focal adhesions (FAs) mechanically couple the extracellular matrix (ECM) to the dynamic actin cytoskeleton, via transmembrane integrins and actin-binding proteins. The molecular mechanisms by which protein machineries control force transmission along this molecular axis, i.e. modulating integrin activation and controlling actin polymerization, remain largely unknown. Talin is a major actin-binding protein that controls both the inside-out activation of integrins and actin-filament anchoring and thus plays a major role in the establishment of the actin-ECM mechanical coupling. Talin contains three actinbinding domains (ABDs). The N-terminal head domain contains both the F3 integrin-activating domain and ABD1, while the C-terminal rod contains the actin-anchoring ABD2 and ABD3. Integrin binding is regulated by an intramolecular interaction between the N-terminal head and a Cterminal five-helix-bundle (R9). Whether talin ABDs regulate actin polymerization in a constitutive or regulated manner has not been fully explored. Here, we combine kinetics assays using fluorescence spectroscopy and single actin filament observation in TIRF microscopy, to examine relevant functions of the three ABDs of talin. We find that the N-terminal ABD1 blocks actin filament barbed end elongation while ABD2 and ABD3 do not show any activity. By mutating residues in ABD1, we find that this activity is mediated by a positively charged surface that is partially masked by its intramolecular interaction with R9. Our results also demonstrate that, once this intramolecular interaction is released, integrinbound talin head retains the ability to inhibit actin assembly.Cell adhesion to the extracellular matrix plays a critical role in many physiological functions such as cell migration, invasion or epithelial basement membrane attachment. Among the multiple adhesion structures, focal adhesions (FAs) play a major role (1,2). These multiprotein complexes couple various extracellular matrices to the actin cytoskeleton via the transmembrane heterodimeric αβ integrins and actin-binding proteins (ABPs) (3,4). The control of actin polymerization by ABPs is thought to play an important role to initiate the formation of the actomyosin stress fibers and control their tension by modulating their elongation. We showed previously that vinculin blocks actin filament barbed end elongation (5), while others reported that VASP promotes the elongation of actin filament barbed ends in a processive-like manner (6,7). Several formins may also play a role in the formation and elongation of Talin head inhibits actin assembly 2 stress fibers (8). However, despite these isolated characterizations, the respective roles of the multiple ABPs and their coordination in this process are poorly understood. The actin-binding protein talin plays a major role in FAs (9) ( Figure S1). First it acts very early to activate integrins. In this process the N-terminal PTB (phosphotyrosine binding) domain, located in the head domain of talin, also known as the F3 subdomain of the ...