Cytoplasmic dynein complex is a large multi-subunit microtubule (MT)-associated molecular motor involved in various cellular functions including organelle positioning, vesicle transport and cell division. However, regulatory mechanism of the cell-cycle dependent distribution of dynein has not fully been understood. Here we report live-cell imaging of cytoplasmic dynein in HeLa cells, by expressing multifunctional green fluorescent protein (mfGFP)-tagged 74-kDa intermediate chain (IC74). IC74-mfGFP was successfully incorporated into functional dynein complex. In interphase, dynein moved bi-directionally along with MTs, which might carry cargos such as transport vesicles. A substantial fraction of dynein moved toward cell periphery together with EB1, a member of MT plus end-tracking proteins (+TIPs), suggesting +TIPs-mediated transport of dynein. In late-interphase and prophase, dynein was localized at the centrosomes and the radial MT array. In prometaphase and metaphase, dynein was localized at spindle MTs where it frequently moved from spindle poles toward chromosomes or cell cortex. +TIPs may be involved in the transport of spindle dyneins. Possible kinetochore and cortical dyneins were also observed. These findings suggest that cytoplasmic dynein is transported to the site of action in preparation for the following cellular events, primarily by the MT-based transport. The MT-based transport may have greater advantage than simple diffusion of soluble dynein in rapid and efficient transport of the limited concentration of the protein.

Mutations in ryanodine receptors (RyRs), intracellular Ca²⁺ channels, are associated with deadly disorders. Despite abundant functional studies, the molecular mechanism of RyR malfunction remains elusive. We studied two single-point mutations at an equivalent site in the skeletal (RyR1 R164C) and cardiac (RyR2 R176Q) isoforms using ryanodine binding, Ca²⁺ imaging, and cryo–electron microscopy (cryo-EM) of the full-length protein. Loss of the positive charge had greater effect on the skeletal isoform, mediated via distortion of a salt bridge network, a molecular latch inducing rotation of a cytoplasmic domain, and partial progression to open-state traits of the large cytoplasmic assembly accompanied by alteration of the Ca²⁺ binding site, which concur with the major “hyperactive” feature of the mutated channel. Our cryo-EM studies demonstrated the allosteric effect of a mutation situated ~85 Å away from the pore and identified an isoform-specific structural effect.

Genetic mutations in ryanodine receptors (RyRs), Ca2+-release channels in the sarcoplasmic reticulum essential for muscle contractions, cause various skeletal muscle and cardiac diseases. Because the main underlying mechanism of the pathogenesis is overactive Ca2+ release by gain-of-function of the RyR channel, inhibition of RyRs is expected to be a promising treatment for these diseases. Here, to identify inhibitors specific to skeletal muscle type 1 RyR (RyR1), we developed a novel high-throughput screening (HTS) platform using time-lapse fluorescence measurement of Ca2+ concentrations in the endoplasmic reticulum (ER) ([Ca2+]ER). Because expression of RyR1 carrying disease-associated mutation reduces [Ca2+]ER in HEK293 cells through Ca2+ leakage from RyR1 channels, specific drugs that inhibit RyR1 will increase [Ca2+]ER by preventing such Ca2+ leakage. RyR1 carrying R2163C mutation and R-CEPIA1er, a genetically-encoded ER Ca2+ indicator, were stably expressed in HEK293 cells and time-lapse fluorescence was measured using a FlexStation II fluorometer. False positives were effectively excluded by using cells expressing wild-type (WT) RyR1. By screening 1,535 compounds in a library of well-characterized drugs, we successfully identified four compounds that significantly increased [Ca2+]ER. They include dantrolene, a known RyR1 inhibitor, and three structurally-different compounds; oxolinic acid, 9-aminoacridine and alexidine. All the hit compounds, except for oxolinic acid, inhibited [3H]ryanodine binding of WT and mutant RyR1. Interestingly, they showed different dose-dependencies and isoform specificities. The highly quantitative nature and good correlation with the channel activity validated this HTS platform by [Ca2+]ER measurement to explore drugs for RyR-related diseases.

Ryanodine receptors (RyRs) are Ca²⁺release channels in the sarcoplasmic reticulum of skeletal and cardiac muscles and are essential for muscle contraction. Mutations in genes encoding RyRs cause various muscle and arrhythmogenic heart diseases. Although RyR channels are activated by Ca²⁺, the actual mechanism of Ca²⁺binding remains largely unknown. Here, we report the molecular basis of Ca²⁺binding to RyRs for channel activation and discuss its implications in disease states. RyR1 and RyR2 carrying mutations in putative Ca²⁺and caffeine-binding sites were functionally analysed. The results were interpreted with respect to recent near-atomic resolution RyR1 structures in various ligand states. We demonstrate that a tryptophan residue in the caffeine-binding site controls the structure of the Ca²⁺-binding site to regulate the Ca²⁺sensitivity. Our results reveal the initial step of RyR channel activation by Ca²⁺and explain the molecular mechanism of Ca²⁺sensitization by caffeine and disease-causing mutations.