Substrate-initiated, self-inactivating, cell-penetrating poly(disulfide)s (siCPDs) are introduced as general transporters for the covalent delivery of unmodified substrates of free choice. With ring-opening disulfide-exchange polymerization, we show that guanidinium-rich siCPDs grow on fluorescent substrates within minutes under the mildest conditions. The most active siCPD transporters reach the cytosol of HeLa cells within 5 min and depolymerize in less than 1 min to release the native substrate. Depolymerized right after use, the best siCPDs are nontoxic under conditions where cell-penetrating peptides (CPPs) are cytotoxic. Intracellular localization (cytosol, nucleoli, endosomes) is independent of the substrate and can be varied on demand, through choice of polymer composition. Insensitivity to endocytosis inhibitors and classical structural variations (hydrophobicity, aromaticity, branching, boronic acids) suggest that the best siCPDs act differently. Supported by experimental evidence, a unique combination of the counterion-mediated translocation of CPPs with the underexplored, thiol-mediated covalent translocation is considered to account for this decisive difference.

Lessons from surface-initiated polymerization are applied to grow cell-penetrating poly(disulfide)s directly on substrates of free choice. Reductive depolymerization after cellular uptake should then release the native substrates and minimize toxicity. In the presence of thiolated substrates, propagators containing a strained disulfide from asparagusic or, preferably, lipoic acid and a guanidinium cation polymerize into poly(disulfide)s in less than 5 min at room temperature at pH 7. Substrate-initiated polymerization of cationic poly(disulfide)s and their depolymerization with dithiothreitol causes the appearance and disappearance of transport activity in fluorogenic vesicles. The same process is further characterized by gel-permeation chromatography and fluorescence resonance energy transfer.Cell-penetrating peptides (CPPs) are short, polycationic peptides or protein domains that are used by viruses to enter cells. 1,2 Their unique ability to transport linked substrates across lipid bilayer membranes has attracted great interest in biomedical applications. Substrates of varying sizes and properties, e.g., small fluorophores to proteins and quantum dots, have been successfully transported into cells using CPPs. The mechanism of cellular uptake is under debate, currently favored are endocytosxis (i.e., macropinocytosis) or passive diffusion across the membrane, depending on conditions. Multiple, moderately hydrophobic cations seem to be all that is needed. Guanidinium cations, as in arginine, are most common, alternatives include ammonium or phosphonium cations. 1 The originally peptidic oligomer backbone has been extensively varied, covering oligocarbamates, β-peptides and several variations of synthetic polymers. 1 Currently, cell-penetrating poly(disulfide)s are emerging as the cell-penetrating molecules of the future because their cytosolic degradation liberates the substrate and eliminates toxicity, one of the key disadvantages associated with CPPs. [3][4][5] However, cell-penetrating poly-(disulfide)s have so far been used mainly in noncovalent polyplexes for gene transfection, and covalent attachment of substrates would be difficult with their preparation methods. We have found recently that poly(disulfide)s can be grown directly on solid substrates by surface-initiated ring-opening disulfide-exchange polymerization. 6 Therefore, we wondered whether the same methodology could be used to Figure 1). Probes or drugs that contain thiol group but cannot penetrate cells without assistance are the ideal substrates, which could serve as an initiator to be appended with a membrane-active poly(disulfide). Thiolated siRNA, for instance, is commercially available. The generality of this approach promises a conceptually innovative solution for a central current challenge, i.e., the noninvasive, nontoxic delivery of unmodified substrates in welldefined, covalent systems rather than complex, noncovalent formulations. In this initial report on the topic, we describe the design, synthesis and evaluation of propag...

During osmotic changes of their environment, cells actively regulate their volume and plasma membrane tension that can passively change through osmosis. How tension and volume are coupled during osmotic adaptation remains unknown, as their quantitative characterization is lacking. Here, we performed dynamic membrane tension and cell volume measurements during osmotic shocks. During the first few seconds following the shock, cell volume varied to equilibrate osmotic pressures inside and outside the cell, and membrane tension dynamically followed these changes. A theoretical model based on the passive, reversible unfolding of the membrane as it detaches from the actin cortex during volume increase quantitatively describes our data. After the initial response, tension and volume recovered from hypoosmotic shocks but not from hyperosmotic shocks. Using a fluorescent membrane tension probe (fluorescent lipid tension reporter [Flipper-TR]), we investigated the coupling between tension and volume during these asymmetric recoveries. Caveolae depletion and pharmacological inhibition of ion transporters and channels, mTORCs, and the cytoskeleton all affected tension and volume responses. Treatments targeting mTORC2 and specific downstream effectors caused identical changes to both tension and volume responses, their coupling remaining the same. This supports that the coupling of tension and volume responses to osmotic shocks is primarily regulated by mTORC2.

Plasma membrane tension strongly affects cell surface processes, such as migration, endocytosis and signalling. However, it is not known whether membrane tension of organelles regulates their functions, notably intracellular traffic. The ESCRT-III complex is the major membrane remodelling complex that drives Intra-Lumenal Vesicle (ILV) formation on endosomal membranes. Here, we made use of a new fluorescent membrane tension probe to show that ESCRT-III subunits are recruited onto endosomal membranes when membrane tension is reduced. We find that tension-dependent recruitment is associated with ESCRT-III polymerization and membrane deformation in vitro, and correlates with increased ILVs formation in ESCRT-III decorated endosomes in vivo. Finally, we find that endosomal membrane tension decreases when ILV formation is triggered by EGF under physiological conditions. These results indicate that membrane tension is a major regulator of ILV formation and of endosome trafficking, leading us to conclude that membrane tension can control organelle functions.