Intein-mediated cytoplasmic reconstitution of a split toxin enables selective cell ablation in mixed populations and tumor xenografts.

The application of proteinaceous toxins for cell ablation is limited by their high on- and off-target toxicity, severe side effects, and a narrow therapeutic window. The selectivity of targeting can be improved by intein-based toxin reconstitution from two dysfunctional fragments provided their cytoplasmic delivery via independent, selective pathways. While the reconstitution of proteins from genetically encoded elements has been explored, exploiting cell-surface receptors for boosting selectivity has not been attained. We designed a robust splitting algorithm and achieved reliable cytoplasmic reconstitution of functional diphtheria toxin from engineered intein-flanked fragments upon receptor-mediated delivery of one of them to the cells expressing the counterpart. Retargeting the delivery machinery toward different receptors overexpressed in cancer cells enables selective ablation of specific subpopulations in mixed cell cultures. In a mouse model, the transmembrane delivery of a split-toxin construct potently inhibits the growth of xenograft tumors expressing the split counterpart. Receptor-mediated delivery of engineered split proteins provides a platform for precise therapeutic and experimental ablation of tumors or desired cell populations while also greatly expanding the applicability of the intein-based protein transsplicing.

Roles of Mso1 and the SM protein Sec1 in efficient vesicle fusion during fission yeast cytokinesis.

Membrane trafficking during cytokinesis is essential for the delivery of membrane lipids and cargoes to the division site. However, the molecular mechanisms are still incompletely understood. In this study, we demonstrate the importance of uncharacterized fission yeast proteins Mso1 and Sec1 in membrane trafficking during cytokinesis. Fission yeast Mso1 shares homology with budding yeast Mso1 and human Mint1, proteins that interact with Sec1/Munc18 family proteins during vesicle fusion. Sec1/Munc18 proteins and their interactors are important regulators of SNARE complex formation during vesicle fusion. The roles of these proteins in vesicle trafficking during cytokinesis have been barely studied. Here, we show that fission yeast Mso1 is also a Sec1-binding protein and Mso1 and Sec1 localize to the division site interdependently during cytokinesis. The loss of Sec1 localization in mso1Δ cells results in a decrease in vesicle fusion and cytokinesis defects such as slow ring constriction, defective ring disassembly, and delayed plasma membrane closure. We also find that Mso1 and Sec1 may have functions independent of the exocyst tethering complex on the plasma membrane at the division site. Together, Mso1 and Sec1 play essential roles in regulating vesicle fusion and cargo delivery at the division site during cytokinesis.

Oligomerization Affects the Ability of Human Cyclase-Associated Proteins 1 and 2 to Promote Actin Severing by Cofilins.

Actin-depolymerizing factor (ADF)/cofilins accelerate actin turnover by severing aged actin filaments and promoting the dissociation of actin subunits. In the cell, ADF/cofilins are assisted by other proteins, among which cyclase-associated proteins 1 and 2 (CAP1,2) are particularly important. The N-terminal half of CAP has been shown to promote actin filament dynamics by enhancing ADF-/cofilin-mediated actin severing, while the central and C-terminal domains are involved in recharging the depolymerized ADP-G-actin/cofilin complexes with ATP and profilin. We analyzed the ability of the N-terminal fragments of human CAP1 and CAP2 to assist human isoforms of "muscle" (CFL2) and "non-muscle" (CFL1) cofilins in accelerating actin dynamics. By conducting bulk actin depolymerization assays and monitoring single-filament severing by total internal reflection fluorescence (TIRF) microscopy, we found that the N-terminal domains of both isoforms enhanced cofilin-mediated severing and depolymerization at similar rates. According to our analytical sedimentation and native mass spectrometry data, the N-terminal recombinant fragments of both human CAP isoforms form tetramers. Replacement of the original oligomerization domain of CAPs with artificial coiled-coil sequences of known oligomerization patterns showed that the activity of the proteins is directly proportional to the stoichiometry of their oligomerization; i.e., tetramers and trimers are more potent than dimers, which are more effective than monomers. Along with higher binding affinities of the higher-order oligomers to actin, this observation suggests that the mechanism of actin severing and depolymerization involves simultaneous or consequent and coordinated binding of more than one N-CAP domain to F-actin/cofilin complexes.

Kinesin's front head is gated by the backward orientation of its neck linker.

Kinesin-1 is a two-headed motor that takes processive 8-nm hand-over-hand steps and transports intracellular cargos toward the plus-end of microtubules. Processive motility requires a gating mechanism to coordinate the mechanochemical cycles of the two heads. Kinesin gating involves neck linker (NL), a short peptide that interconnects the heads, but it remains unclear whether gating is facilitated by the NL orientation or tension. Using optical trapping, we measured the force-dependent microtubule release rate of kinesin monomers under different nucleotide conditions and pulling geometries. We find that pulling NL in the backward direction inhibits nucleotide binding and subsequent release from the microtubule. This inhibition is independent of the magnitude of tension (2-8 pN) exerted on NL. Our results provide evidence that the front head of a kinesin dimer is gated by the backward orientation of its NL until the rear head releases from the microtubule.