Togo Shimozawa

Planar polarity of multiciliated ependymal cells involves the anterior migration of basal bodies regulated by non-muscle myosin II

Motile cilia generate constant fluid flow over epithelial tissue, and thereby influence diverse physiological processes. Such functions of ciliated cells depend on the planar polarity of the cilia and on their basal bodies being oriented in the downstream direction of fluid flow. Recently, another type of basal body planar polarity, characterized by the anterior localization of the basal bodies in individual cells, was reported in the multiciliated ependymal cells that line the surface of brain ventricles. However, little is known about the cellular and molecular mechanisms by which this polarity is established. Here, we report in mice that basal bodies move in the apical cell membrane during differentiation to accumulate in the anterior region of ependymal cells. The planar cell polarity signaling pathway influences basal body orientation, but not their anterior migration, in the neonatal brain. Moreover, we show by pharmacological and genetic studies that non-muscle myosin II is a key regulator of this distribution of basal bodies. This study demonstrates that the orientation and distribution of basal bodies occur by distinct mechanisms.

Real-time measurement of the length of a single sarcomere in rat ventricular myocytes: a novel analysis with quantum dots

As the dynamic properties of cardiac sarcomeres are markedly changed in response to a length change of even ∼0.1 μm, it is imperative to quantitatively measure sarcomere length (SL). Here we show a novel system using quantum dots (QDs) that enables a real-time measurement of the length of a single sarcomere in cardiomyocytes. First, QDs were conjugated with anti-α-actinin antibody and applied to the sarcomeric Z disks in isolated skinned cardiomyocytes of the rat. At partial activation, spontaneous sarcomeric oscillations (SPOC) occurred, and QDs provided a quantitative measurement of the length of a single sarcomere over the broad range (i.e., from ∼1.7 to ∼2.3 μm). It was found that the SPOC amplitude was inversely related to SL, but the period showed no correlation with SL. We then treated intact cardiomyocytes with the mixture of the antibody-QDs and FuGENE HD, and visualized the movement of the Z lines/T tubules. At a low frequency of 1 Hz, the cycle of the motion of a single sarcomere consisted of fast shortening followed by slow relengthening. However, an increase in stimulation frequency to 3-5 Hz caused a phase shift of shortening and relengthening due to acceleration of relengthening, and the waveform became similar to that observed during SPOC. Finally, the anti-α-actinin antibody-QDs were transfected from the surface of the beating heart in vivo. The striated patterns with ∼1.96-μm intervals were observed after perfusion under fluorescence microscopy, and an electron microscopic observation confirmed the presence of QDs in and around the T tubules and Z disks, but primarily in the T tubules, within the first layer of cardiomyocytes of the left ventricular wall. Therefore, QDs are a useful tool to quantitatively analyze the movement of single sarcomeres in cardiomyocytes, under various experimental settings.

Actin oligomers at the initial stage of polymerization induced by increasing temperature at low ionic strength: Study with small-angle X-ray scattering

Using small-angle X-ray scattering (SAXS), we have studied the initial stage (nucleation and oligomerization) of actin polymerization induced by raising temperature in a stepwise manner from 1°C to 30°C at low ionic strength (4.0 mg ml−1 actin in G-buffer). The SAXS experiments were started from the mono-disperse G-actin state, which was confirmed by comparing the scattering pattern in q- and real space with X-ray crystallographic data. We observed that the forward scattering intensity I(q → 0), used as an indicator for the extent of poly-merization, began to increase at ∼14°C for Mg-actin and ∼20°C for Ca-actin, and this critical temperature did not depend on the nucleotide species, i.e., ATP or ADP. At the temperatures higher than ∼20°C for Mg-actin and ∼25°C for Ca-actin, the coherent reflection peak, which is attributed to the helical structure of F-actin, appeared. The pair-distance distribution functions, p(r), corresponding to the frequency of vector lengths (r) within the molecule, were obtained by the indirect Fourier transformation (IFT) of the scattering curves, I(q). Next, the size distributions of oligomers at each temperature were analyzed by fitting the experimentally obtained p(r) with the theoretical p(r) for the helical and linear oligomers (2–13mers) calculated based on the X-ray crystallographic data. We found that p(r) at the initial stage of polymerization was well accounted for by the superposition of monomer, linear/helical dimers, and helical trimer, being independent of the type of divalent cations and nucleotides. These results suggest that the polymerization of actin in G-buffer induced by an increase in temperature proceeds via the elongation of the helical trimer, which supports, in a structurally resolved manner, a widely believed hypothesis that the polymerization nucleus is a helical trimer.

Sarcomere Imaging by Quantum Dots for the Study of Cardiac Muscle Physiology

We here review the use of quantum dots (QDs) for the imaging of sarcomeric movements in cardiac muscle. QDs are fluorescence substances (CdSe) that absorb photons and reemit photons at a different wavelength (depending on the size of the particle); they are efficient in generating long-lasting, narrow symmetric emission profiles, and hence useful in various types of imaging studies. Recently, we developed a novel system in which the length of a particular, single sarcomere in cardiomyocytes can be measured at ~30u2009nm precision. Moreover, our system enables accurate measurement of sarcomere length in the isolated heart. We propose that QDs are the ideal tool for the study of sarcomere dynamics during excitation-contraction coupling in healthy and diseased cardiac muscle.

Protein-protein interactions in solution and their interplay with protein specific functions

We summarize recent developments of our small angle scattering studies on protein–protein interactions in solution. We have been focusing especially on representative proteins that function at exceptionally high concentration in human body, whose collective nature, rather than a specialized function of an isolated single molecule, plays an important role for their specific biological functions; for instance, human serum albumin (HSA), actin, and human hemoglobin (Hb). We use a static structure factor analysis, which offers information about spatial distributions of the proteins, as well as a Fourier inversion technique for the form factor, giving a real-space picture of the protein assemblies.

Optimization of Fluorescent Labeling for In Vivo Nanoimaging of Sarcomeres in the Mouse Heart

The present study was conducted to systematically investigate the optimal viral titer as well as the volume of the adenovirus vector (ADV) that expresses α-actinin-AcGFP in the Z-disks of myocytes in the left ventricle (LV) of mice. An injection of 10u2009μL ADV at viral titers of 2 to 4 × 1011 viral particles per mL (VP/mL) into the LV epicardial surface consistently expressed α-actinin-AcGFP in myocytes in vivo, with the fraction of AcGFP-expressing myocytes at ~10%. Our analysis revealed that SL was ~1.90-2.15u2009μm upon heart arrest via deep anesthesia. Likewise, we developed a novel fluorescence labeling method of the T-tubular system by treating the LV surface with CellMask Orange (CellMask). We found that the T-tubular distance was ~2.10-2.25u2009μm, similar to SL, in the healthy heart in vivo. Therefore, the present high-precision visualization method for the Z-disks or the T-tubules is beneficial to unveiling the mechanisms of myocyte contraction in health and disease in vivo.

Planar cell polarity of multiciliated ependymal cells regulated by non-muscle myosin II

Subcerebral projection neurons (SCPNs) in layer 5 (L5) are the major output neurons of the neocortex. They receive their major interlayer excitatory inputs from layer 2/3 (L2/3) neurons. The connection between these two groups of neurons is important to understand the functional mechanism of neocortex and has been extensively studied. However, it has been technically difficult and time-consuming to assess their connectivity in large scale; it usually requires recording electrophysiological data from a large number of neurons in the two layers. In addition, identification of SCPNs requires either assessment of their electrophysiological properties or retrograde tracing from subcerebral areas. Here, we propose a less technical and less time-consuming method utilizing ChR2-assisted circuit mapping and transgenic mice with readily identifiable SCPNs. GFP was expressed in the transgenic mice under the control of the promoter of a SCPN marker. We confirmed that GFP was exclusively expressed in SCPNs in L5 with two methods: retrograde tracing from pons and simultaneously staining GFP with a SCPN marker. ChR2 was exclusively expressed in L2/3 excitatory neurons by in utero electroporation. Then we mapped the excitatory inputs to L5 SCPNs from L2/3 in acute brain slices by scanning the laser in L2/3 while recording EPSCs from GFP (+) neurons. The inputs from an area of 350 um x 750 um of L2/3 to a L5 SCPN could be mapped in less than 5 min. The spatial resolution of the map was improved by minimizing simultaneous spike induction in multiple L2/3 neurons. This was achieved by minimizing the laser power, reducing the density of ChR2 (+) neurons and reducing ChR2 from axons. This simple experimental system enables high-resolution mapping of functional inputs to L5 SCPNs with minimal cost.