Clathrin seems to be dispensable for some endocytic processes and, in several instances, no cytosolic coat protein complexes could be detected at sites of membrane invagination. Hence, new principles must in these cases be invoked to account for the mechanical force driving membrane shape changes. Here we show that the Gb3 (glycolipid)-binding B-subunit of bacterial Shiga toxin induces narrow tubular membrane invaginations in human and mouse cells and model membranes. In cells, tubule occurrence increases on energy depletion and inhibition of dynamin or actin functions. Our data thus demonstrate that active cellular processes are needed for tubule scission rather than tubule formation. We conclude that the B-subunit induces lipid reorganization that favours negative membrane curvature, which drives the formation of inward membrane tubules. Our findings support a model in which the lateral growth of B-subunit-Gb3 microdomains is limited by the invagination process, which itself is regulated by membrane tension. The physical principles underlying this basic cargo-induced membrane uptake may also be relevant to other internalization processes, creating a rationale for conceptualizing the perplexing diversity of endocytic routes.

The homopentameric B-subunit of bacterial protein Shiga toxin (STxB) binds to the glycolipid Gb3 in plasma membranes, which is the initial step for entering cells by a clathrin-independent mechanism. It has been suggested that protein clustering and lipid reorganization determine toxin uptake into cells. Here, we elucidated the molecular requirements for STxB induced Gb3 clustering and for the proposed lipid reorganization in planar membranes. The influence of binding site III of the B-subunit as well as the Gb3 lipid structure was investigated by means of high resolution methods such as fluorescence and scanning force microscopy. STxB was found to form protein clusters on homogenous 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/cholesterol/Gb3 (65∶30∶5) bilayers. In contrast, membranes composed of DOPC/cholesterol/sphingomyelin/Gb3 (40∶35∶20∶5) phase separate into a liquid ordered and liquid disordered phase. Dependent on the fatty acid composition of Gb3, STxB-Gb3 complexes organize within the liquid ordered phase upon protein binding. Our findings suggest that STxB is capable of forming a new membrane phase that is characterized by lipid compaction. The significance of this finding is discussed in the context of Shiga toxin-induced formation of endocytic membrane invaginations.

Membrane proteins are prime drug targets as they control the transit of information, ions, and solutes across membranes. Here, we present a membrane-on-nanopore platform to analyze nonelectrogenic channels and transporters that are typically not accessible by electrophysiological methods in a multiplexed manner. The silicon chip contains 250 000 femtoliter cavities, closed by a silicon dioxide top layer with defined nanopores. Lipid vesicles containing membrane proteins of interest are spread onto the nanopore-chip surface. Transport events of ligand-gated channels were recorded at single-molecule resolution by high-parallel fluorescence decoding.

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A micelle based nanosensor was synthesized and investigated as a ratiometric pH sensor for use in measurements in living cells by fluorescent microscopy. The nanosensor synthesis was based on selfassembly of an amphiphilic triblock copolymer, which was chemically cross-linked after micelle formation. The copolymer, poly(ethylene glycol)-b-poly(2-aminoethyl methacrylate)-b-poly(styrene) (PEG-b-PAEMA-b-PS), was synthesized by isolated macroinitiator atom transfer radical polymerization that forms micelles spontaneously in water. The PAEMA shell of the micelle was hereafter cross-linked by an amidation reaction using 3,6,9-trioxaundecandioic acid cross-linker. The cross-linked micelle was functionalized with two pH sensitive fluorophores and one reference fluorophore, which resulted in a highly uniform ratiometric pH nanosensor with a diameter of 29 nm. The use of two sensor fluorophores provided a sensor with a very broad measurement range that seems to be influenced by the chemical design of the sensor. Cell experiments show that the sensor is capable of monitoring the pH distributions in HeLa cells.