Estimation of crossbridge-state during cardiomyocyte beating using second harmonic generation.

Estimation of dynamic change of crossbridge formation in living cardiomyocytes is expected to provide crucial information for elucidating cardiomyopathy mechanisms, efficacy of an intervention, and others. Here, we established an assay system to dynamically measure second harmonic generation (SHG) anisotropy derived from myosin filaments depended on their crossbridge status in pulsating cardiomyocytes. Experiments utilizing an inheritable mutation that induces excessive myosin-actin interactions revealed that the correlation between sarcomere length and SHG anisotropy represents crossbridge formation ratio during pulsation. Furthermore, the present method found that ultraviolet irradiation induced an increased population of attached crossbridges that lost the force-generating ability upon myocardial differentiation. Taking an advantage of infrared two-photon excitation in SHG microscopy, myocardial dysfunction could be intravitally evaluated in a Drosophila disease model. Thus, we successfully demonstrated the applicability and effectiveness of the present method to evaluate the actomyosin activity of a drug or genetic defect on cardiomyocytes. Because genomic inspection alone may not catch the risk of cardiomyopathy in some cases, our study demonstrated herein would be of help in the risk assessment of future heart failure.

Recent Advances in Raman Spectral Imaging in Cell Diagnosis and Gene Expression Prediction.

Normal and tumor regions within cancer tissue can be distinguished using various methods, such as histological analysis, tumor marker testing, X-ray imaging, or magnetic resonance imaging. Recently, new discrimination methods utilizing the Raman spectra of tissues have been developed and put into practical use. Because Raman spectral microscopy is a non-destructive and non-labeling method, it is potentially compatible for use in the operating room. In this review, we focus on the basics of Raman spectroscopy and Raman imaging in live cells and cell type discrimination, as these form the bases for current Raman scattering-based cancer diagnosis. We also review recent attempts to estimate the gene expression profile from the Raman spectrum of living cells using simple machine learning. Considering recent advances in machine learning techniques, we speculate that cancer type discrimination using Raman spectroscopy will be possible in the near future.

Valosin-containing protein regulates the stability of fused in sarcoma granules in cells by changing ATP concentrations.

Fused in sarcoma (FUS), a DNA/RNA-binding protein, undergoes liquid-liquid phase separation to form granules in cells. Aberrant FUS granulation is associated with neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal lobar degeneration. We found that FUS granules contain a multifunctional AAA ATPase, valosin-containing protein (VCP), which is known as a key regulator of protein degradation. FUS granule stability depends on ATP concentrations in cells. VCP ATPase changes the FUS granule stability time-dependently by consuming ATP to reduce its concentrations in the granules: VCPs in de novo FUS granules stabilize the granules, while long-lasting VCP colocalization destabilizes the granules. The proteolysis-promoting function of VCP may subsequently dissolve the unstabilized granules. We propose that VCP colocalized to the FUS granules acts as a timer to limit the residence time of the granules in cells.

Mitochondria-associated myosin 19 processively transports mitochondria on actin tracks in living cells.

Mitochondria are fundamentally important in cell function, and their malfunction can cause the development of cancer, cardiovascular disease, and neuronal disorders. Myosin 19 (Myo19) shows discrete localization with mitochondria and is thought to play an important role in mitochondrial dynamics and function; however, the function of Myo19 in mitochondrial dynamics at the cellular and molecular levels is poorly understood. Critical missing information is whether Myo19 is a processive motor that is suitable for transportation of mitochondria. Here, we show for the first time that single Myo19 molecules processively move on actin filaments and can transport mitochondria in cells. We demonstrate that Myo19 dimers having a leucine zipper processively moved on cellular actin tracks in demembraned cells with a velocity of 50 to 60 nm/s and a run length of ∼0.4 µm, similar to the movement of isolated mitochondria from Myo19 dimer-transfected cells on actin tracks, suggesting that the Myo19 dimer can transport mitochondria. Furthermore, we show single molecules of Myo19 dimers processively moved on single actin filaments with a large step size of ∼34 nm. Importantly, WT Myo19 single molecules without the leucine zipper processively move in filopodia in living cells similar to Myo19 dimers, whereas deletion of the tail domain abolished such active movement. These results suggest that Myo19 can processively move on actin filaments when two Myo19 monomers form a dimer, presumably as a result of tail-tail association. In conclusion, Myo19 molecules can directly transport mitochondria on actin tracks within living cells.

Quantitation of Cell Membrane Permeability of Cyclic Peptides by Single-Cell Cytoplasm Mass Spectrometry.

Cyclic peptides (CPs) have attracted attention as next-generation drugs because they possess both cell-permeable potential as small molecules and specific affinity similar to antibodies. As intracellular molecules are important targets of CPs, quantitation of the intracellular retention and transmembrane permeability of CPs is necessary for drug development. However, permeated CPs within cells cannot be directly assessed by conventional permeability assays using methods such as artificial membranes and cell monolayers. Here, we propose a new approach using single-cell cytoplasm mass spectrometry (SCC-MS). After cells were incubated with CPs, the cytoplasm was directly collected from a single cell using a microneedle followed by nanoelectrospray ionization mass spectrometry detection of the CPs. The height of the CP peak was plotted against time and fitted with a simple function, y = a(1 - e-bx), to calculate the apparent permeability coefficient (Papp) for both the influx and efflux directions. MCF-7 cells were selected as model cancer cells and cultured with cyclosporin A (CsA) and its demethylated analogs (dmCsA-1, -2, and -3) as model CPs. Papp values (10-6 cm/s) obtained from cells incubated with 50 µM CPs ranged from 0.017 to 0.121 for influx and 0.20 to 1.48 for efflux. The higher efflux ratio was possibly caused by efflux transporters such as P-glycoprotein, a well-known receptor of CsA. The equilibrated intracellular concentration of CPs was estimated to be as low as 4.1-6.8 µM, which showed good consistency with the high efflux ratio. SCC-MS is promising as a reliable permeability assay for next-generation CP-based pharmaceuticals.

Multiplexed 129Xe HyperCEST MRI Detection of Genetically Reconstituted Bacterial Protein Nanoparticles in Human Cancer Cells.

Gas vesicle nanoparticles (GVs) are gas-containing protein assemblies expressed in bacteria and archaea. Recently, GVs have gained considerable attention for biotechnological applications as genetically encodable contrast agents for MRI and ultrasonography. However, at present, the practical use of GVs is hampered by a lack of robust methodology for their induction into mammalian cells. Here, we demonstrate the genetic reconstitution of protein nanoparticles with characteristic bicone structures similar to natural GVs in a human breast cancer cell line KPL-4 and genetic control of their size and shape through expression of reduced sets of humanized gas vesicle genes cloned into Tol2 transposon vectors, referencing the natural gas vesicle gene clusters of the cyanobacteria planktothrix rubescens/agardhii. We then report the utility of these nanoparticles as multiplexed, sensitive, and genetically encoded contrast agents for hyperpolarized xenon chemical exchange saturation transfer (HyperCEST) MRI.

An improved fluorescent protein-based expression reporter system that utilizes bioluminescence resonance energy transfer and peptide-assisted complementation.

Fluorescent protein-based reporter systems are used to track gene expression in cells. Here, we propose a modified bioluminescence resonance energy transfer (BRET) reporter as a maturation-less reporter that utilizes a peptide-assisted complementation strategy. Using effective dimerized peptides obtained from library-versus-library screening with more than 4000 candidates, rapid activation of the reporter was achieved.

Advanced CUBIC tissue clearing for whole-organ cell profiling.

Tissue-clearing techniques are powerful tools for biological research and pathological diagnosis. Here, we describe advanced clear, unobstructed brain imaging cocktails and computational analysis (CUBIC) procedures that can be applied to biomedical research. This protocol enables preparation of high-transparency organs that retain fluorescent protein signals within 7-21 d by immersion in CUBIC reagents. A transparent mouse organ can then be imaged by a high-speed imaging system (>0.5 TB/h/color). In addition, to improve the understanding and simplify handling of the data, the positions of all detected cells in an organ (3-12 GB) can be extracted from a large image dataset (2.5-14 TB) within 3-12 h. As an example of how the protocol can be used, we counted the number of cells in an adult whole mouse brain and other distinct anatomical regions and determined the number of cells transduced with mCherry following whole-brain infection with adeno-associated virus (AAV)-PHP.eB. The improved throughput offered by this protocol allows analysis of numerous samples (e.g., >100 mouse brains per study), providing a platform for next-generation biomedical research.

Single-Cell Screening of Tamoxifen Abundance and Effect Using Mass Spectrometry and Raman-Spectroscopy.

Monitoring drug uptake, its metabolism, and response on the single-cell level is invaluable for sustaining drug discovery efforts. In this study, we show the possibility of accessing the information about the aforementioned processes at the single-cell level by monitoring the anticancer drug tamoxifen using live single-cell mass spectrometry (LSC-MS) and Raman spectroscopy. First, we explored whether Raman spectroscopy could be used as a label-free and nondestructive screening technique to identify and predict the drug response at the single-cell level. Then, a subset of the screened cells was isolated and analyzed by LSC-MS to measure tamoxifen and its metabolite, 4-Hydroxytamoxifen (4-OHT) in a highly selective, sensitive, and semiquantitative manner. Our results show the Raman spectral signature changed in response to tamoxifen treatment which allowed us to identify and predict the drug response. Tamoxifen and 4-OHT abundances quantified by LSC-MS suggested some heterogeneity among single-cells. A similar phenomenon was observed in the ratio of metabolized to unmetabolized tamoxifen across single-cells. Moreover, a correlation was found between tamoxifen and its metabolite, suggesting that the drug was up taken and metabolized by the cell. Finally, we found some potential correlations between Raman spectral intensities and tamoxifen abundance, or its metabolism, suggesting a possible relationship between the two signals. This study demonstrates for the first time the potential of using Raman spectroscopy and LSC-MS to investigate pharmacokinetics at the single-cell level.

Kinesin-binding-triggered conformation switching of microtubules contributes to polarized transport.

Kinesin-1, the founding member of the kinesin superfamily of proteins, is known to use only a subset of microtubules for transport in living cells. This biased use of microtubules is proposed as the guidance cue for polarized transport in neurons, but the underlying mechanisms are still poorly understood. Here, we report that kinesin-1 binding changes the microtubule lattice and promotes further kinesin-1 binding. This high-affinity state requires the binding of kinesin-1 in the nucleotide-free state. Microtubules return to the initial low-affinity state by washing out the binding kinesin-1 or by the binding of non-hydrolyzable ATP analogue AMPPNP to kinesin-1. X-ray fiber diffraction, fluorescence speckle microscopy, and second-harmonic generation microscopy, as well as cryo-EM, collectively demonstrated that the binding of nucleotide-free kinesin-1 to GDP microtubules changes the conformation of the GDP microtubule to a conformation resembling the GTP microtubule.

Myosin X is recruited to nascent focal adhesions at the leading edge and induces multi-cycle filopodial elongation.

Filopodia protrude from the leading edge of cells and play important roles in cell motility. Here we report the mechanism of myosin X (encoded by Myo10)-induced multi-cycle filopodia extension. We found that actin, Arp2/3, vinculin and integrin-ß first accumulated at the cell's leading edge. Myosin X was then gathered at these sites, gradually clustered by lateral movement, and subsequently initiated filopodia formation. During filopodia extension, we found the translocation of Arp2/3 and integrin-ß along filopodia. Arp2/3 and integrin-ß then became localized at the tip of filopodia, from where myosin X initiated the second extension of filopodia with a change in extension direction, thus producing long filopodia. Elimination of integrin-ß, Arp2/3 and vinculin by siRNA significantly attenuated the myosin-X-induced long filopodia formation. We propose the following mechanism. Myosin X accumulates at nascent focal adhesions at the cell's leading edge, where myosin X promotes actin convergence to create the base of filopodia. Then myosin X moves to the filopodia tip and attracts integrin-ß and Arp2/3 for further actin nucleation. The tip-located myosin X then initiates the second cycle of filopodia elongation to produce the long filopodia.

In vivo analysis of protein crowding within the nuclear pore complex in interphase and mitosis.

The central channel of the nuclear pore complex (NPC) is occupied by non-structured polypeptides with a high content of Phe-Gly (FG) motifs. This protein-rich environment functions as an entropic barrier that prevents the passage of molecules, as well as the binding sites for karyopherins, to regulate macromolecular traffic between the nucleoplasm and the cytoplasm. In this study, we expressed individual Nups fused with a crowding-sensitive probe (GimRET) to determine the spatial distribution of protein-rich domains within the central channel in vivo, and characterize the properties of the entropic barrier. Analyses of the probe signal revealed that the central channel contains two protein-rich domains at both the nucleoplasmic and cytoplasmic peripheries, and a less-crowded central cavity. Karyopherins and other soluble proteins are not the constituents of the protein-rich domains. The time-lapse observation of the post-mitotic reassembly process also revealed how individual protein-rich domains are constructed by a sequential assembly of nucleoporins.

Human myosin VIIa is a very slow processive motor protein on various cellular actin structures.

Human myosin VIIa (MYO7A) is an actin-linked motor protein associated with human Usher syndrome (USH) type 1B, which causes human congenital hearing and visual loss. Although it has been thought that the role of human myosin VIIa is critical for USH1 protein tethering with actin and transportation along actin bundles in inner-ear hair cells, myosin VIIa's motor function remains unclear. Here, we studied the motor function of the tail-truncated human myosin VIIa dimer (HM7AΔTail/LZ) at the single-molecule level. We found that the HM7AΔTail/LZ moves processively on single actin filaments with a step size of 35 nm. Dwell-time distribution analysis indicated an average waiting time of 3.4 s, yielding ∼0.3 s-1 for the mechanical turnover rate; hence, the velocity of HM7AΔTail/LZ was extremely slow, at 11 nm·s-1 We also examined HM7AΔTail/LZ movement on various actin structures in demembranated cells. HM7AΔTail/LZ showed unidirectional movement on actin structures at cell edges, such as lamellipodia and filopodia. However, HM7AΔTail/LZ frequently missed steps on actin tracks and exhibited bidirectional movement at stress fibers, which was not observed with tail-truncated myosin Va. These results suggest that the movement of the human myosin VIIa motor protein is more efficient on lamellipodial and filopodial actin tracks than on stress fibers, which are composed of actin filaments with different polarity, and that the actin structures influence the characteristics of cargo transportation by human myosin VIIa. In conclusion, myosin VIIa movement appears to be suitable for translocating USH1 proteins on stereocilia actin bundles in inner-ear hair cells.

Activated full-length myosin-X moves processively on filopodia with large steps toward diverse two-dimensional directions.

Myosin-X, (Myo 10), is an unconventional myosin that transports the specific cargos to filopodial tips, and is associated with the mechanism underlying filopodia formation and extension. To clarify the innate motor characteristic, we studied the single molecule movement of a full-length myosin-X construct with leucine zipper at the C-terminal end of the tail (M10FullLZ) and the tail-truncated myosin-X without artificial dimerization motif (BAP-M101-979HMM). M10FullLZ localizes at the tip of filopodia like myosin-X full-length (M10Full). M10FullLZ moves on actin filaments in the presence of PI(3,4,5)P3, an activator of myosin-X. Single molecule motility analysis revealed that the step sizes of both M10FullLZ and BAP-M101-979HMM are widely distributed on single actin filaments that is consistent with electron microscopy observation. M10FullLZ moves on filopodial actin bundles of cells with a mean step size (~36 nm), similar to the step size on single actin filaments (~38 nm). Cartesian plot analysis revealed that M10FullLZ meandered on filopodial actin bundles to both x- and y- directions. These results suggest that the lever-arm of full-length myosin-X is flexible enough to processively steps on different actin filaments within the actin bundles of filopodia. This characteristic of myosin-X may facilitate actin filament convergence for filopodia production.

Predictive diagnosis of the risk of breast cancer recurrence after surgery by single-particle quantum dot imaging.

In breast cancer, the prognosis of human epidermal growth factor receptor 2 (HER2)-positive patients (20-25%) has been dramatically improved by the clinical application of the anti-HER2 antibody drugs trastuzumab and pertuzumab. However, the clinical outcomes of HER2-negative cases with a poor prognosis have not improved, and novel therapeutic antibody drugs or diagnostic molecular markers of prognosis are urgently needed. Here, we targeted protease-activated receptor 1 (PAR1) as a new biomarker for HER2-negative patients. The developed anti-PAR1 antibody inhibited PAR1 activation by matrix metalloprotease 1 and thereby prevented cancer-cell migration and invasion. To estimate PAR1 expression levels in HER2-negative patient tissues using the antibody, user-friendly immunohistochemistry with fluorescence nanoparticles or quantum dots (QDs) was developed. Previously, immunohistochemistry with QDs was affected by tissue autofluorescence, making quantitative measurement extremely difficult. We significantly improved the quantitative sensitivity of immunohistochemistry with QDs by using an autofluorescence-subtracted image and single-QD imaging. The immunohistochemistry showed that PAR1 expression was strongly correlated with relapse-free survival time in HER2-negative breast cancer patients. Therefore, the developed anti-PAR1 antibody is a strong candidate for use as an anticancer drug and a prognostic biomarker for HER2-negative patients.

SH3 domain of c-Src governs its dynamics at focal adhesions and the cell membrane.

We studied the role of the Src SH3 domain in its dynamics at the cell membrane using site-directed mutagenesis and live cell imaging. Physiologically, cell proliferation and migration require the expression of Src family kinases. Hyperactivation of Src molecules has been detected in various cancer cells. Although the activation mechanism of Src has been intensively studied, the dynamics of Src at the cell membrane are still unclear. Although Src molecules also exist at various cellular locations, we found that activated Src molecules are mainly localized at peripheral cell adhesion sites. Src phosphorylation status and subdomain conformations are thought to regulate Src activation and translocation. In this study, we analyzed the single-molecule dynamics of wild-type Src and SH2- and SH3-mutated Src at the cell membrane. Introducing mutations in the SH3 domain resulted in reduced Src motility at the cell membrane, both inside and outside of focal adhesions. Disruption of the actin cytoskeleton resulted in less diffusive Src movement at the cell membrane. We demonstrate that, inside focal adhesions, the SH3 domain enhanced dissociation of Src from the adhesion site and disruption of the SH3 domain altered the distribution of Src at the cell membrane. Inside focal adhesions, kinase activity of Src was essential for the Src mobility reduction by SH3 domain mutation, suggesting that rapid mobility of Src at focal adhesions mediated by the SH3 domain is catalytic-activity-dependent. These findings show that the SH3 domain of Src governs the dynamics of Src at the cell membrane and may be involved in rapid signal transduction in cells.

Visualizing cell state transition using Raman spectroscopy.

System level understanding of the cell requires detailed description of the cell state, which is often characterized by the expression levels of proteins. However, understanding the cell state requires comprehensive information of the cell, which is usually obtained from a large number of cells and their disruption. In this study, we used Raman spectroscopy, which can report changes in the cell state without introducing any label, as a non-invasive method with single cell capability. Significant differences in Raman spectra were observed at the levels of both the cytosol and nucleus in different cell-lines from mouse, indicating that Raman spectra reflect differences in the cell state. Difference in cell state was observed before and after the induction of differentiation in neuroblastoma and adipocytes, showing that Raman spectra can detect subtle changes in the cell state. Cell state transitions during embryonic stem cell (ESC) differentiation were visualized when Raman spectroscopy was coupled with principal component analysis (PCA), which showed gradual transition in the cell states during differentiation. Detailed analysis showed that the diversity between cells are large in undifferentiated ESC and in mesenchymal stem cells compared with terminally differentiated cells, implying that the cell state in stem cells stochastically fluctuates during the self-renewal process. The present study strongly indicates that Raman spectral morphology, in combination with PCA, can be used to establish cells' fingerprints, which can be useful for distinguishing and identifying different cellular states.

Four-dimensional spatial nanometry of single particles in living cells using polarized quantum rods.

Single particle tracking is widely used to study protein movement with high spatiotemporal resolution both in vitro and in cells. Quantum dots, which are semiconductor nanoparticles, have recently been employed in single particle tracking because of their intense and stable fluorescence. Although single particles inside cells have been tracked in three spatial dimensions (X, Y, Z), measurement of the angular orientation of a molecule being tracked would significantly enhance our understanding of the molecule's function. In this study, we synthesized highly polarized, rod-shaped quantum dots (Qrods) and developed a coating method that optimizes the Qrods for biological imaging. We describe a Qrod-based single particle tracking technique that blends optical nanometry with nanomaterial science to simultaneously measure the three-dimensional and angular movements of molecules. Using Qrods, we spatially tracked a membrane receptor in living cells in four dimensions with precision close to the single-digit range in nanometers and degrees.

Rab6a releases LIS1 from a dynein idling complex and activates dynein for retrograde movement.

Cytoplasmic dynein drives the movement of a wide range of cargoes towards the minus ends of microtubules. We previously demonstrated that LIS1 forms an idling complex with dynein, which is transported to the plus ends of microtubules by kinesin motors. Here we report that the small GTPase Rab6a is essential for activation of idling dynein. Immunoprecipitation and microtubule pull-down assays reveal that the GTP bound mutant, Rab6a(Q72L), dissociates LIS1 from a LIS1-dynein complex, activating dynein movement in in vitro microtubule gliding assays. We monitor transient interaction between Rab6a(Q72L) and dynein in vivo using dual-colour fluorescence cross-correlation spectroscopy in dorsal root ganglion (DRG) neurons. Finally, we demonstrate that Rab6a(Q72L) mediates LIS1 release from a LIS1-dynein complex followed by dynein activation through an in vitro single-molecule assay using triple-colour quantum dots. Our findings reveal a surprising function for GTP bound Rab6a as an activator of idling dynein.

Multilayered, core/shell nanoprobes based on magnetic ferric oxide particles and quantum dots for multimodality imaging of breast cancer tumors.

Multilayered, core/shell nanoprobes (MQQ-probe) based on magnetic nanoparticles (MNPs) and quantum dots (QDs) have been successfully developed for multimodality tumor imaging. This MQQ-probe contains Fe(3)O(4) MNPs, visible-fluorescent QDs (600 nm emission) and near infrared-fluorescent QDs (780 nm emission) in multiple silica layers. The fabrication of the MQQ-probe involves the synthesis of a primer Fe(3)O(4) MNPs/SiO(2) core by a reverse microemulsion method. The MQQ-probe can be used both as a fluorescent probe and a contrast reagent of magnetic resonance imaging. For breast cancer tumor imaging, anti-HER2 (human epidermal growth factor receptor 2) antibody was conjugated to the surface of the MQQ-probe. The specific binding of the antibody conjugated MQQ-probe to the surface of human breast cancer cells (KPL-4) was confirmed by fluorescence microscopy and fluorescence-activated cell sorting analysis in vitro. Due to the high tissue permeability of near-infrared (NIR) light, NIR fluorescence imaging of the tumor mice (KPL-4 cells transplanted) was conducted by using the anti-HER2 antibody conjugated MQQ-probe. In vivo multimodality images of breast tumors were successfully taken by NIR fluorescence and T(2)-weighted magnetic resonance. Antibody conjugated MQQ-probes have great potential to use for multimodality imaging of cancer tumors in vitro and in vivo.