SUMMARY Kinesin-1 is a two-headed motor that takes processive 8-nm hand-over-hand steps and transports intracellular cargos towards the plus end of microtubules. Processive motility requires a gating mechanism to coordinate the mechanochemical cycles of the two heads. Kinesin gating involves the 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 from 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.
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.
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.
Mso1 and the SM protein Sec1 aid in the efficient fusion of vesicles at the division site in fission yeast, which is important for proper contractile-ring constriction and plasma-membrane closure during cytokinesis.
steps. Structural dynamics of dynein's AAA þ ring domain during processive stepping are not well understood. We use a combination of polarized TIRF and sub-pixel particle tracking to measure the position and orientation of fluorescent nanorods rigidly attached to AAA5 and AAA6 of the individual dynein ring domains via biotin-NeutrAvidin linkage. We observe rotational changes of the ring and how they correlate with translocation steps. The dynein ring undergoes frequent, small rotations, typically less than 20 degrees, about twice as often as steps. Stepping and tilting both depend on ATP, although some ATP-independent rotations are observed. Rotations which accompany translocation steps have larger magnitudes than ones that are not correlated with steps. Not all steps are correlated with angle changes, either. A heterodimeric mutant construct, with one biotinylated ring carrying a quantum rod and one nonbiotinylated dead-head ring walks slowly, indicating that the rod does not destroy motility. Our results are inconsistent with a purely powerstroke stepping mechanism, analogous to that of myosin which would predict larger angle changes tightly coupled to stepping, but instead they support a winch-like mechanism that involves bending of the coiled-coil stalk under intermolecular torque between the heads in the double-headed bound state. Supported by NIH Grant P015GM087253.
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