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Trimethylamine N-oxide (TMAO), one of osmolytes, has been shown to enhance protein stability. It has been reported that TMAO protects the interaction between microtubules and kinesin from thermal degradation. We examined here how much TMAO protects the actomyosin motility from thermal denaturation. To assay the effects of TMAO on the thermal stability of myosin, we measured the velocity of actin filament sliding along myosin which was heat-treated in the presence of TMAO. Myosin incubated for 20 min at 40 ℃ in the absence of TMAO didn’t cause the actin filament sliding, but myosin which was heat-treated in the presence of 600 mM TMAO caused the actin filament sliding. These results indicate that TMAO protects the actomyosin motility from thermal denaturation.

2019年度衝撃波シンポジウム (2020年3月4日-6日. 神戸大学 海事科学研究科（深江キャンパス)）神戸市, 兵庫県, Symposium on Shock Waves in Japan, FY2019 (March 3-4, 2020. Kobe University), Kobe, Hyogo Japan, 2019年度衝撃波シンポジウムは、新型コロナウイルスの感染拡大防止のため開催中止, 資料番号: SA6000148028, レポート番号: 1B3-5[若]

The hatching rate and the distribution of actin filaments were observed in mouse blastocysts whose ability of actin polymerization had been suppressed by cytochalasin B (CB), in order to examine the role of actin filaments in blastocyst hatching. The hatching rate of blastocysts developed from morulae in a medium containing 0.4μg/ml CB was 9.7%, which was significantly lower than the 78.1% of control blastocysts developed from morulae in a medium without CB. The fluorescence showing the presence of actin filaments was observed in the cytoplasm of trophectoderm and inner-cell-mass cells in blastocysts, and was especially strong in the peripheral cytoplasm where two trophectoderm cells adhering to each other. Also actin fluorescence was much brighter in protruded trophectoderm cells at the region of small hole or slit in the zona pellucida of hatching blastocysts. Such the distribution of actin filaments was similar in CB-treated and control blastocysts in each hatching period. On the other hand, the rate of CB-treated blastocysts in which most trophectoderm cells possess the fluorescence of actin filaments did not differ from that of control blastocysts in the post-hatching period, whereas such the rates of CB-treated blastocysts in the pre-hatching and hatching periods (34.1 and 46.5%) were significantly lower than the 95.0 and 82.9% of control blastocysts, respectively. The CB-treated blastocysts in the pre-hatching and hatching periods had many trophectoderm cells devoid of the fluorescence of actin filaments. From these findings, it was suggested that the polymerization of actin is essential for blastocyst hatching, and that actin filament-mediated movements of trophectoderm cells contribute to the hatching by facilitating the protrusion of trophectoderm cells from a small hole in the zona pellucida and also by enlarging the protrusion for slit formation in the zona pellucida., サイトカラシンB（CB）で処置してアクチンの重合を抑制したマウス胚盤胞について、ハッチング能とアクチンフィラメントの分布を調べ、胚盤胞ハッチングに果たすアクチンフィラメントの役割を検討した。0.4μg/mlのCBを含む培養液で桑実胚から発生した胚盤胞のハッチング率は9.7％であり、対照のCBを含まない培養液で発生した胚盤胞の78.1％に比べ、有意に低かった。アクチンフィラメントの存在を示す特異蛍光は、栄養膜細胞と内細胞塊細胞の細胞質にみられ、栄養膜細胞同士が接する部位の細胞膜直下の細胞質で特に強かった。また、ハッチング中の胚盤胞において、この蛍光は透明帯の小孔から突出した栄養膜細胞および透明帯の裂け目付近の栄養膜細胞で特に強かった。このようなアクチンフィラメントの分布は、CB処置した胚盤胞と対照の胚盤胞の間で相違なかった。また、ほとんどの栄養膜細胞がアクチンフィラメントの存在を示す特異蛍光を有している胚盤胞の割合は、ハッチング後では、CB処置したものと対照のものとの間で相違なく、それぞれ75.0％と92.0％であった。一方、ハッチング前とハッチング中の期間において、ほとんどの栄養膜細胞がアクチンフィラメントの存在を示す特異蛍光を有しているCB処置胚盤胞の割合は、それぞれ34.1および46.5％であり、対照の胚盤胞の95.0および82.9％に比べて有意に低かった。以上のように、CBで処置してアクチンの重合を抑制したマウス胚盤胞では、ハッチング前とハッチング中にアクチンフィラメントを欠く栄養膜細胞が多数出現したために、このような胚盤胞ではハッチングの開始と完了に不可欠な透明帯での小孔形成と裂け目形成に必要な栄養膜細胞の運動が阻害され、結果としてハッチング能が低下したものと思われた。従って、アクチンフィラメントを介した栄養膜細胞の運動は、胚盤胞のハッチング過程の進行に重要な役割を果たしていることが考えられた。

In animal cultured cells, it has been reported that gravity change affects cell function and cell proliferation. But, the response mechanism of gravity change has not been known yet. A point of mechanism is what is sensor for response of gravity change. It is hypothesized that gravity change is sensed by cytoskeleton and transduced into biochemical signal via integrin, whose cytoplasmic domain is bind to one of cytoskeleton, actin filament. Integrin is major receptor for extracellular matrix molecules and is responsible not only for cell-extracellular matrix adhesion, but also for transducer of mechanical stimulation into biochemical signal as mechanoreceptor. To verify the hypothesis, a correlation between cell spreading activity and hypergravity-dependent increase of DNA synthesis in animal cultured cells was examined using one of extracellular matrix molecule, fibronectin. Baby hamster kidney (BHK) cells in the experiments, and cultured BHK cells on fibronectin-coated dishes were used. In this system, hypergravity condition (75 G, 20 h) increased 10 to 50 percent of DNA synthesis correlated with fibronectin-dependent increase of cell spreading activity using 0 to 100 microgram/ml fibronectin. Since integrin and actin filament play an important role in cell spreading, this result suggested that integrin and actin filament are involved in hypergravity response. To examine mechanism of hypergravity response in more detail, hypergravity response was observed in shorter period (2 h). Hypergravity condition (75 G, 2 h) also increased about 50 percent of DNA synthesis in BHK cells. Cytochalasin B, which inhibits actin polymerization, abolished the increase of DNA synthesis, indicating that actin filament plays a role in hypergravity response. To examine whether signal transduction via integrin is involved in hypergravity response, amount of phosphorylated tyrosin residue, which is responsible for signal transduction via integrin, was observed in BHK cells. This result showed that hypergravity increased the level of phosphorylated tyrosine residue and phosphorylation of src and paxillin. In addition, hypergravity promoted the formation of stress fiber in BHK cells. These results demonstrate that hypergravity response is mediated by a cell-adhesive molecule, integrin and cytoskeleton in BHK cells., 資料番号: AA0045438008

Changes in the cytoskeletal network alter the mechanical properties of cells that are essential for functions such as locomotion and cytokinesis. The transmembrane receptors and intracellular signals have been extensively studied on cytoskeletal changes in response to extracellular stimuli. After the procedure of percutaneous transluminal coronary angioplasty (PTCA) and highfrequency rotational atherectomy (PTCR) for stenosis of blood vessels, restenosis may be observed. The restenosis were generated under the proliferation and migration of vascular smooth muscle (VSM) cells. In addition, until recently it has been expected that the physiological regulation of VSM cells was dependent on L-type Ca^ (Ca_1) channel activity and structure of the cytoskeleton. To clarify the interactions between the structure of the cytoskeleton and activity of Ca_1 channel in VSM cell, we used electrophysiological and morphological experiments concomitantly. We investigated the effect of disruption of the actin filaments and the microtubules on Ca_1 channel activity (Ca^ current) of cultured VSM cells (A7r5 cell line) using whole cell voltage clamp. The results of immunostaining using anti-$ \alpha $-actin and anti-$ \alpha $-tubulin antibodies showed that colchicines disrupted both actin filaments and microtubules, while cytochalasin D and nocodazole disrupted only actin filaments, and microtubules respectively. Ca^ current was greatly reduced in cells that were exposed to colchicines or cytochalasin D but not to nocodazole. When the actin filaments were stabilized by phalloidine, the inhibition of Ca^ current was not observed. Actin filaments disruption of VSM cells inhibits Ca_1 channel activity, whereas the microtubules disruption gave no effect on the activity. These results suggested that the maintenance of physiological function in VSM cells involve the functional-structural relationship between the Ltype Ca^ channel and cytoskeleton. The development of restenosis is dependent on the pathophysiological proliferation and then migration of the VSM cells could be prevented by control of L-type Ca^ channel activity and stabilization of the cytoskeleton in VSM cells., 論文

Changes in the cytoskeletal network alter the mechanical properties of cells that are essential for functions such as locomotion and cytokinesis. The transmembrane receptors and intracellular signals have been extensively studied on cytoskeletal changes in response to extracellular stimuli. After the procedure of percutaneous transluminal coronary angioplasty (PTCA) and highfrequency rotational atherectomy (PTCR) for stenosis of blood vessels, restenosis may be observed. The restenosis were generated under the proliferation and migration of vascular smooth muscle (VSM) cells. In addition, until recently it has been expected that the physiological regulation of VSM cells was dependent on L-type Ca^ (Ca_1) channel activity and structure of the cytoskeleton. To clarify the interactions between the structure of the cytoskeleton and activity of Ca_1 channel in VSM cell, we used electrophysiological and morphological experiments concomitantly. We investigated the effect of disruption of the actin filaments and the microtubules on Ca_1 channel activity (Ca^ current) of cultured VSM cells (A7r5 cell line) using whole cell voltage clamp. The results of immunostaining using anti-$ \alpha $-actin and anti-$ \alpha $-tubulin antibodies showed that colchicines disrupted both actin filaments and microtubules, while cytochalasin D and nocodazole disrupted only actin filaments, and microtubules respectively. Ca^ current was greatly reduced in cells that were exposed to colchicines or cytochalasin D but not to nocodazole. When the actin filaments were stabilized by phalloidine, the inhibition of Ca^ current was not observed. Actin filaments disruption of VSM cells inhibits Ca_1 channel activity, whereas the microtubules disruption gave no effect on the activity. These results suggested that the maintenance of physiological function in VSM cells involve the functional-structural relationship between the Ltype Ca^ channel and cytoskeleton. The development of restenosis is dependent on the pathophysiological proliferation and then migration of the VSM cells could be prevented by control of L-type Ca^ channel activity and stabilization of the cytoskeleton in VSM cells.

Article, 信州医学雑誌 39(6): 717-729(1991)