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Directional Bleb Formation in Spherical Cells Under Temperature Gradient Biophysical Journal Volume 109 July 2015 355–364 355 Article Directional Bleb Formation in Spherical Cells under Temperature Gradient Kotaro Oyama,1,* Tomomi Arai,1 Akira Isaka,1 Taku Sekiguchi,1 Hideki Itoh,1,2 Yusuke Seto,1 Makito Miyazaki,1 Takeshi Itabashi,1 Takashi Ohki,1 Madoka Suzuki,3,4,* and Shin’ichi Ishiwata1,3,4,* 1Department of Physics, School of Advanced Science and Engineering, Waseda University, Tokyo, Japan; 2Institute of Medical Biology, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore; 3WASEDA Bioscience Research Institute in Singapore (WABIOS), Singapore, Singapore; and 4Organization for University Research Initiatives, Waseda University, Tokyo, Japan ABSTRACT Living cells sense absolute temperature and temporal changes in temperature using biological thermosensors such as ion channels. Here, we reveal, to our knowledge, a novel mechanism of sensing spatial temperature gradients within single cells. Spherical mitotic cells form directional membrane extensions (polar blebs) under sharp temperature gradients (R~0.065C mmÀ1; 1.3C temperature difference within a cell), which are created by local heating with a focused 1455-nm laser beam under an optical microscope. On the other hand, multiple nondirectional blebs are formed under gradual temper- ature gradients or uniform heating. During heating, the distribution of actomyosin complexes becomes inhomogeneous due to a break in the symmetry of its contractile force, highlighting the role of the actomyosin complex as a sensor of local temperature gradients. INTRODUCTION Temperature-sensitive Ca2þ signalingisusedbysingle directional morphological response coupled with asym- cells to respond to temporal temperature changes. Tran- metric protein dynamics. sient receptor potential channels are representative biolog- ical thermosensors in cells (1), but other Ca2þ channels MATERIALS AND METHODS such as ryanodine receptor (2), IP3 receptor (3,4), and Orai1-STIM1 complex (5) also sense temporal tempera- Cell culture ture changes. This thermosensing function of ion channels HeLa cells were cultured in flasks (AGC Techno Glass, Shizuoka, Japan) in is used to remotely control neural activity with radiofre- Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine quency magnetic field local heating (6,7). Local heating serum (FBS), 100 units mLÀ1 penicillin, and 100 mgmLÀ1 streptomycin itself can also induce local gene expression (8), membrane at 37 C with 5% CO2. DMEM, FBS, penicillin, and streptomycin were pur- depolarization (9,10), and muscle contraction (11–13). chased from Thermo Fisher Scientific (Waltham, MA). 1–2 days before the These local heating techniques have a strong potential experiments, cells were seeded on a glass dish (AGC Techno Glass). for the spatial control of cell functions. However, except for thermotaxis (14–17), biological responses to local Experimental solutions temperature gradients remain unclear. In vitro studies Cells on the glass dish were rinsed twice with 1 mL Leibovitz’s L-15 have revealed that temperature gradients affect cytoskel- medium (Thermo Fisher Scientific) containing 10% FBS and incubated etal polymerization (18,19), molecular motor activities in 2 mL L-15 medium containing 10% FBS under the microscope for (20–22), and the distribution of biomolecules including 15 min at room temperature (25 5 1C), at 35 5 0.5C, or at a physiolog- DNAandRNA(23,24), but few studies have observed ical temperature (37 5 0.5 C) adjusted by a thermostatically controlled these biomolecular dynamics in living cells under temper- incubator on the sample stage (INUG2-ONICS; Tokai Hit, Shizuoka, Japan). ature gradients (25,26). To elucidate the system for sensing temperature gradients, we applied a local temper- ature gradient to single living cells and observed the Drug treatments Cytoskeleton (actin filament and microtubule) and motor protein (myosin II) inhibitors were mixed into L-15 medium containing 10% FBS 15 min before observation. Actin polymerization was inhibited by 500 nM (final Submitted February 3, 2015, and accepted for publication June 9, 2015. concentration) latrunculin B (Calbiochem, Merck KGaA, Darmstadt, Ger- *Correspondence: [email protected] or suzu_mado@aoni. many) and 10 mM cytochalasin D (Wako Pure Chemical Industries, Osaka, waseda.jp or [email protected] Japan). Actin polymerization was promoted by 50 nM jasplakinolide (Cal- m Kotaro Oyama, Tomomi Arai, and Akira Isaka contributed equally to this biochem). Microtubules were depolymerized with 10 M nocodazole m work. (Merck KGaA) or stabilized with 20 M paclitaxel (taxol) (Sigma-Aldrich, St. Louis, MO). Myosin II ATPase was inhibited with 100 mM blebbistatin Kotaro Oyama’s and Tomomi Arai’s present address is Department of Cell (Toronto Research Chemicals, Ontario, Canada). Rho-kinase (ROCK) and Physiology, The Jikei University School of Medicine, 3-25-8 Nishi-shinba- the myosin light chain kinase inhibitors were 10 mM Y-27632 (Wako shi, Minato-ku, Tokyo 105-8461, Japan. Ó 2015 by the Biophysical Society 0006-3495/15/07/0355/10 $2.00 http://dx.doi.org/10.1016/j.bpj.2015.06.016 356 Oyama et al. Pure Chemical Industries) and 20 or 50 mM ML-7 (Sigma-Aldrich), respec- The temperature dependence of the fluorescence intensity was measured tively. The stock solutions of latrunculin B, cytochalasin D, jasplakinolide, by a fluorescence spectrophotometer (F-4500, Hitachi High-Technologies, nocodazole, taxol, blebbistatin, Y-27632, and ML-7 were 500 mM, 10 mM, Tokyo, Japan) at various temperatures. The temperature in the cuvette was 50 mM, 10 mM, 20 mM, 100 mM, 10 mM, and 20 mM, respectively. controlled by a precision thermostatic circulator (AB-1600, ATTO, Tokyo, Y-27632 was dissolved in sterile water, and the others were dissolved in Japan) and measured by a digital thermometer (ASF-250T, AS ONE, dimethyl sulfoxide (DMSO) (Sigma-Aldrich). The stock solutions were Osaka, Japan). The excitation and emission wavelengths were respectively stored at À20C. Spherical interphase cells were prepared by rinsing twice 555 nm and 578–580 nm for TMR-dextran, 540 nm and 565–568 nm for with 1 mL phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 1.5 mM Alexa Fluor 555-dextran, and 365 nm and 612–618 nm for Eu-TTA. The À1 KH2PO4, 8.1 mM Na2HPO4) containing 0.2 g L EDTA and 0.025% relative fluorescence intensity of TMR-dextran normalized at 25.1 C was trypsin (Thermo Fisher Scientific) and incubated for 1 min at 25C, and fitted with the quadratic function y ¼ 1.31 Â 10À4 x2 – 2.47 Â 10À2 x þ the solution was replaced by 2 mL L-15 medium containing 10% FBS 1.53, where x and y are the temperature [C] and normalized fluorescence for observation. intensity, respectively (Fig. S1 A in the Supporting Material). The fluores- cence intensity of Alexa Fluor 555-dextran normalized at 24.9C was fitted with y ¼ 2.36 Â 10À4 x2 – 3.66 Â 10À2 x þ 1.76 (Fig. S1 A), and that of Eu- À À Optical setup TTA normalized at 25.1C was fitted with y ¼ 3.33 Â 10 4 x2 – 5.17 Â 10 2 x þ 2.09 (Fig. S1 A). The spatial temperature gradients were fitted with the The microscope with local heating systems was previously described (11). logarithmic function y ¼a ln (x/x0) þ b, where x, x0, and y were the dis- Confocal fluorescence images were captured with a confocal unit (CSU-X1, tance from the heat source [mm], 1 [mm] as a reference, and temperature Yokogawa Electric, Tokyo, Japan), an electron multiplying charge-coupled [C], respectively (4). The positive numbers a and b were determined device camera (iXon-Ultra, Andor Technologies plc, Belfast, Northern with Microsoft Excel. This fitting is based on a model that steady-state tem- Ireland), and an objective lens (PlanApo N 60Â/1.45 Oil, Olympus, Tokyo, perature distribution T(r) [K] around a cylindrical heat source is described Japan) attached to the inverted microscope (IX-70, Olympus). The excita- in T(x1) ¼ T(x2)–Q/(2plk) Â ln(x1/x2), where x1 [m] and x2 [m] are the dis- tion light sources were 488 nm (Sapphire, Coherent, Santa Clara, CA) tances from a center of heat source, Q [W] is a derived power, l [m] is a and 561 nm (Sapphire, Coherent). The confocal unit was set up with a height of a cylindrical heat source, and k [W mÀ1 KÀ1] is thermal conduc- YOKO-T405/488/561 dichroic mirror (Semrock, Rochester, NY) and tivity of medium. T(x) around a spherical heat source, T(x1) ¼ T(x2) þ YOKO-FF01-520/32 and YOKO-FF01-617/73 emission filters (Semrock). Q/(4pk) Â (1/x1 –1/x2), was not well fitted with the spatial temperature gra- The excitation and emission light passed through an FF409-Di03 dichroic dients measured in this study. mirror (Semrock) in the microscope. Europium (III) thenoyltrifluoroaceto- nate trihydrate (Eu-TTA), fluorescent beads, blebbistatin, calcein, and ethidium homodimer-1 (EthD-1) were excited by a mercury lamp Visualization of actin, myosin, and the cell (Olympus) with excitation filters for Eu-TTA (BP360–370, Olympus) or membrane others (BP470–490, Olympus). Fluo-4 was excited by a SPECTRA Light Engine (485/20 nm, Lumencor, Beaverton, OR). For epifluorescence micro- Actin-red fluorescent protein (RFP) and tubulin-green fluorescent protein scopy of Eu-TTA, an FF409-Di03 dichroic mirror (Semrock) was used. For (GFP) were expressed by a baculovirus expression system with CellLights epifluorescence microscopy of fluorescent beads, blebbistatin, fluo-4, cal- (Thermo Fisher Scientific) 1 day before observation. F-actin was visualized cein, and EthD-1, a DM505 dichroic mirror (Olympus) and a BA515IF by Lifeact-RFP (28) or Lifeact-GFP. Myosin II was visualized by mCherry- excitation filter (Olympus) were included in the microscope, and fluores- human myosin, with light chain 2 fluorescently modified (mCherry-MRLC). cence was observed by epifluorescence microscopy with an electron The plasmids containing Lifeact-RFP, Lifeact-GFP, or mCherry-MRLC þ multiplying charge-coupled device camera (iXon EM 897, Andor Tech- were transfected with Lipofectamine LTX (Thermo Fisher Scientific) nologies plc) and the same objective lens used for confocal microscopy.
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