Subscriber access provided by Technical University of Munich University Library
Article
Motility of microtubules on the inner surface of water-in-oil emulsion droplets Mikako Tsuji, Arif Md. Rashedul Kabir, Masaki Ito, Daisuke Inoue, Kenta Kokado, Kazuki Sada, and Akira Kakugo Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b01550 • Publication Date (Web): 03 Oct 2017 Downloaded from http://pubs.acs.org on October 5, 2017
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Langmuir is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 28
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Langmuir
Motility of microtubules on the inner surface of water-in-oil emulsion droplets Mikako Tsujia, Arif Md. Rashedul Kabirb, Masaki Itoa, Daisuke Inoueb, Kenta Kokado a,b,Kazuki Sadaa,b,* and Akira Kakugo a,b,*
a
Graduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo
060-0810, Japan, b
Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan
*
Corresponding author
E-mail: kakugo@ sci.hokudai.ac.jp;
[email protected] Phone and Fax: +81-11-706-3474
1 ACS Paragon Plus Environment
Langmuir
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Abstract The water-in-oil emulsion systems have recently been attracting much attention in various fields. However, functionalization of the water-in-oil emulsion systems, which is required for expanding their applications in industry and research, has been challenging. We now demonstrate functionalization of a water-in-oil emulsion system by anchoring a target protein molecule. A microtubule associated motor protein kinesin-1 was successfully anchored to the inner surface of water-in-oil emulsion droplets by employing the specific interaction of the nickel-nitrilotriacetic acid (NTA)-histidine tag. The microtubules exhibited a gliding motion on the kinesin functionalized inner surface of the emulsion droplets, which confirmed success of the functionalization of the water-in-oil emulsion system. This result would be beneficial in exploring the roles of biomolecular motor systems in the cellular events that take place at the cell membrane and might also contribute to expanding the nanotechnological applications of biomolecular motors and water-in-oil emulsion systems in the future.
2 ACS Paragon Plus Environment
Page 2 of 28
Page 3 of 28
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Langmuir
Introduction The water-in-oil (w/o) emulsion systems have been gaining much interest in various fields, particularly for biological systems since they offer a very small confined space similar to living cells and allow the study of mechanisms of various cellular events1-4. In particular, spatial constraints of the aqueous solution as a droplet in oil has provided many cellular mimics for investigating the cellular morphology, governed by the self-organization of cytoskeletal components and the cellular events that take place on the surface of a cell membrane. For example, Baumann et al. investigated the biomolecular motor mediated organization of microtubules encapsulated in small aqueous droplets in oil5. The degree of confinement was found to be directly correlated to the motor mediated reorganization of the microtubules into a cortical array or asters. By using confined microtubules in water droplets in the w/o emulsion, Sanchez et al. reported the autonomous rearrangement of microdroplets6. In another example, the surface of the water droplet in a w/o emulsion was employed to reproduce the cortices of actin in cells which revealed symmetry breaking of the actin cortices induced by myosin motors7. On another front, reconstructed biomolecular motor systems, microtubule-kinesin and actin-myosin, permit demonstration of the in vitro motility assay where biomolecular motors (kinesin or myosin) are adsorbed on a substrate surface and the associated cytoskeletal filaments (microtubules or actins) exhibit a gliding motion on the biomolecular motor carpet in the presence of adenosine triphosphate (ATP)8,9. In recent years, the in vitro motility assay 3 ACS Paragon Plus Environment
Langmuir
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
has provided valuable insights into the biophysical and chemo-mechanical properties of motor protein systems as well as their self-organization which are of great physiological interest10-13. The in vitro motility assay has been a standard, reproducible and perfect technique for testing new methods incorporating abiotic and biological materials14,15. However, in many examples of the in vitro motility assay of the microtubule-kinesin system, the gliding motion of microtubules is generally demonstrated on a planar surface or micro-patterned flat surfaces with various architectures with the view to control movement of the microtubules16-18. However, the gliding motion of microtubules on an encapsulated deformable substrate has never been reported, for which functionalization of the deformable substrate by biomolecular motors is the first step. Demonstration of such a gliding assay of microtubules would be important for understanding the involvement of cortical motor proteins in the cellular events occurring at the cell membrane19-21. In this study, we report immobilization of a biomolecular motor kinesin on the inner surface of water droplets in a w/o emulsion system and demonstrate the gliding motion of microtubules on the kinesin-coated inner surface of the droplets. Our strategy for immobilization of the kinesins on the interface of the w/o emulsion is based on the high binding affinity between the histidine tag (His-tag) and NTA in the presence of Ni2+ 22. We used a surfactant with the NTA-moiety as the hydrophilic part at the oil-water interface as an anchor for kinesins containing the His-tag. Attachment of the microtubules to the inner 4 ACS Paragon Plus Environment
Page 4 of 28
Page 5 of 28
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Langmuir
surface of the water droplets in oil was realized through interaction with the anchored kinesins. The microtubules, adsorbed on the inner surface of the droplets, demonstrated a gliding motion on the kinesins in the presence of adenosine triphosphate (ATP). The motility of the microtubules on the inner surface of the w/o emulsion droplets confirms that the kinesins are successfully immobilized on the inner surface of the w/o emulsion droplets and the kinesins retained their enzymatic activity; as a result the microtubules being able to exhibit the motility. This study, which demonstrates fixation of kinesins on an encapsulated deformable substrate, would be useful for investigating the role of cortical motor proteins in cellular events occurring at the cell membrane.
5 ACS Paragon Plus Environment
Langmuir
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Experimental Tubulin and kinesin purification Tubulin was purified from porcine brain using a highly concentrated PIPES buffer (1 M PIPES, 20 mM EGTA, and 10 mM MgCl2; pH adjusted to 6.8 using KOH). The PIPES buffer and 80 mM BRB80 buffer were prepared using PIPES from Sigma, and the pH was adjusted using KOH23. Green fluorescent protein-fused kinesin-1 consisting of the first 560 amino acid residue of human kinesin-1 (GFP-kinesin) and histidine tag (His-tag) was prepared by partially modifying the previously reported expression and purification method24. For the lysis of E. coli used to express the GFP-kinesin, benzonase was used to degrade the nucleic acids. After binding the proteins to Ni–NTA resin that has an affinity to the His-tag of GFP-kinesin, the resin was washed with a series of buffers differing in their pH from 8.0 to 6.0 in a step-wise fashion. The target protein was eluted from the resin by washing it with a buffer containing 250 mM imidazole-Cl. The buffer of the effluent was exchanged with microtubule binding buffer (25 mM PIPES, 250 mM NaCl, 1 mM EGTA, 2 mM MgCl2, 0.1 mM ATP, 1mM DTT; pH 6.8 by KOH) using a PD-10 column (GE Healthcare). After the buffer exchange, the GFP-kinesin solution was supplemented with 0.5 mM GTP, 4 mM AMPPNP, 0.5 U mL−1 apyrase and 10 µM paclitaxel. This GFP-kinesin solution (2 mL) was mixed with the microtubules obtained by polymerization of the tubulins (56 µM, 1 mL) and subsequent ultra-centrifuging at 200,000g for 30 min at 37 °C. The GFP-kinesins were bound to the microtubules by incubating at 25 °C for 15 min. The GFP-kinesin bound microtubules were 6 ACS Paragon Plus Environment
Page 6 of 28
Page 7 of 28
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Langmuir
centrifuged at 300,000g for 30 min (30 °C) after placing it on an equal volume of cushion buffer, which is a microtubule buffer supplemented with 30% (w/v) sucrose. The pellet of the microtubules was suspended for releasing the kinesins in the buffer. The microtubules were again pelleted by ultra-centrifuging and the supernatant yielded the purified GFP-kinesin which was stored at -80 °C after snap freezing in liquid nitrogen.
Preparation of fluorescence dye labeled tubulins and microtubules The rhodamine-labeled tubulin was prepared using the tetramethylrhodamine succinimidyl ester (TAMRA-SE; Invitrogen) according to the standard technique25. Rhodamine-tubulin was obtained by chemical cross-linking and the labeling ratio was 1:1. This ratio was determined by measuring the concentration of the protein and tetramethylrhodamine. The concentration of the protein was measured according to the Bradford method using bovine serum albumin (BSA)
as
the
standard.
The
concentration
of
tetramethylrhodamine
was
spectrophotometrically determined by measuring the absorbance at 555 nm using a NanoDrop 2000c spectrophotometer (Thermo Scientific). A mixture of the TAMRA-SE labeled tubulin and non-labelled tubulin (at a molar ratio of 1:9) at a total concentration of 70 µM was incubated at 37 oC for 30 min to prepare the microtubules. After polymerization, the microtubules were diluted to the concentration of 4 µM using taxol buffer (1 mM taxol in BRB80). 7 ACS Paragon Plus Environment
Langmuir
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 8 of 28
Preparation of emulsion (oil phase) 1,2-Dioleoyl-sn-glycero-3-phosphocholine
(DOPC;
purchased
1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic
from
TCI)
and
acid)succinyl]
(ammonium salt) (DGS-NTA purchased from Avanti®) solutions were prepared at concentrations of 5 mg/mL and 10 mg/mL respectively by dissolving in chloroform (Kanto Chemical Co.). The DOPC and DGS-NTA solutions were mixed so that the final concentrations of DOPC and DGS-NTA became 1.8 and 0.2 mM, respectively. The chloroform was allowed to evaporate under vacuum evaporation for 1 hour at room temperature. A 100 µL aliquot of mineral oil (Sigma) was added to the dried lipid and the mixture (oil phase) was sonicated for 1 hour at in ice cold water bath and kept in an ice box until it was used for the experiment. Next, 20 µL of 4 µM microtubules in taxol buffer was applied to 200 µL of cusion buffer (10 μM taxol, 60 % (v/v) glycerol, 80 mM PIPES, 1 mM MgCl2, 1 mM EGTA) in a 1.5 mL eppendorf tube. The mixture was centrifuged at 310,000g (rotor P65A, Hitachi Centrifuges) at 37 oC for 1 hour. After centrifugation, the supernatant was removed and the microtubule pellet was dissolved in 20 µL of droplet buffer (44 mM PIPES, 10 mM MgCl2, 3 mM EGTA, 5 mM ATP, 40 mM 2-mercaptoethanol, 50 mM KCl, 1 μM Taxol, 20 mM glucose, 1 mg/mL glucose oxidase, 0.5 mg/mL catalase, 0.5 mg/mL BSA, 0.25 mg/mL casein).
8 ACS Paragon Plus Environment
Page 9 of 28
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Langmuir
Preparation of water phase The water phase was prepared by mixing the GFP-kinesin containing his-tag (in BRB80 buffer), microtubules (in droplet buffer), and nickel sulfate solution (Wako; prepared in water). In the water phase, the concentrations of the GFP-kinesin, microtubules, Ni2+ and ATP were 0.2, 2, 1 µM and 5 mM, respectively. The oil phase and water phase were then mixed at a volume ratio of 9:1 and vigorously vortexed for 1 min at room temperature.
Motility of microtubules on a slide glass A flowcell was prepared by placing a cover glass (18×18 mm2; Matsunami Inc.) on a slide glass (26×76 mm2) equipped with a pair of spacers to form a chamber with the approximate dimensions 4×18×0.1 mm3 (W×L×H). A single layer of Parafilm was used to fix the spacer-separated glasses by heating. The flowcell was filled with 0.2 mg mL-1 anti-GFP antibody (Invitrogen) and incubated for 15 min, followed by a wash with 10 µL of wash buffer (80 mM PIPES, 1 mM EGTA, 1 mM MgCl2, 0.5 mg mL-1 casein; pH adjusted to 6.8 with KOH). After incubating for 5 min to block the remaining glass surface, 10 µL of kinesin buffer (200 nM kinesin, ∼80 mM PIPES, 40 mM NaCl, 1 mM EGTA, 1 mM MgCl2, 0.5 mg mL-1 casein, 1 mM DTT, 10 µM paclitaxel; pH 6.8) was introduced, and incubated for 10 min to allow the kinesins to bind to the antibody. The flowcell was washed with 20 µL of M_BRB buffer (∼80 mM PIPES, 1 mM EGTA, 1 mM MgCl2, 0.5 mg mL-1 casein, 1 mM DTT, 10 µM 9 ACS Paragon Plus Environment
Langmuir
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
paclitaxel, 1% DMSO, 4.5 mg mL-1 D-glucose, 50 UmL-1 glucose oxidase, 50 UmL-1 catalase; pH 6.8). Next, 10 µL of the microtubule solution (200 nM) was introduced and incubated for 10 min, followed by washing with 20 µL of M_BRB buffer. Finally, the motility assay was initiated by applying 10 µL of ATP-buffer (∼80m MPIPES, 1 mM EGTA, 1 mM MgCl2, 0.5 mg mL-1 casein, 1 mM DTT, 10 µM paclitaxel, 1% DMSO, 4.5 mgmL-1 D-glucose, 50 UmL-1 glucose oxidase, 50 UmL-1 catalase, 5 mM ATP; pH 6.8).
Microscopic observation and image analysis Samples were visualized by two-color TIRF microscopy (Nikon) equipped with an Apo TIRF 60 × 1.49 N.A. oil objective and a confocal laser scanning microscope (CLSM). Images were captured using a cooled CMOS camera (Neo sCMOS; Andor) connected to a PC. For the observation under a CLSM, 5 µL of a w/o emulsion was cast on a cover glass and observed. For the observation by the TIRF, a flowcell with the dimension of (18×3×0.08) mm3 was first prepared by sandwiching a cover glass and a slide glass. A 10 µL emulsion was then applied to the flowcell, and the flowcell was placed in an inert chamber to remove any oxygen and prevent oxidative damage of the samples by keeping them in the inert environment.26 After passing nitrogen gas for 30 min through the chamber, emulsion droplets were observed by total internal reflection fluorescence (TIRF). Imaging of the microtubules and GFP-kinesins using the TIRF was performed with a 900-ms exposure time. The images and movies, 10 ACS Paragon Plus Environment
Page 10 of 28
Page 11 of 28
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Langmuir
captured by the TIRF microscopy, were analyzed using image analysis software (ImageJ). Tracking of the gliding microtubules and measurement of their velocity were performed using an ImageJ plugin ‘MTrackJ’. For measuring the velocity from the movies, the gliding microtubules were continuously tracked for one minute. The velocity of the microtubules was steady over the period of investigation.
Determination of diffusion coefficient of lipids The diffusion coefficient of the lipids was determined from fluorescence recovery after the photobleaching (FRAP) experiments. The experiments were performed at the molar composition of DOPC:DGS-NTA:DOPE-Atto555=90:10:0.05. The captured images were analyzed to estimate the diffusion coefficient of the lipids according to a previous report27.
11 ACS Paragon Plus Environment
Langmuir
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 12 of 28
Results and discussion The strategy for immobilization of the kinesins and motility of the microtubules on the kinesin-coated inner surface of the water droplets is schematically shown in the Figure 1. As the first step, we confirmed that the kinesins are anchored to the inner surface of the emulsion droplets
through
a
Ni-NTA-His
tag
1,2-dioleoyl-sn-glycero-3-phosphocholine
interaction. (DOPC)
The and
oil
phase
containing
1,2-dioleoyl-sn-glycero-3-
[(N-(5-amino-1-carboxypentyl) iminodiacetic acid)succinyl] (DGS-NTA) as the anchoring lipid with the NTA moiety as the hydrophilic part were mixed with an aqueous buffer of the kinesin-containing 6X-His tag (GFP-kinesin) at the ratio of 9:1 (v/v). This was followed by vigorous stirring using a vortex mixer for 1 min. The prepared w/o emulsion was then observed using a confocal laser scanning microscope (CLSM). Although the size of the water droplets was widely distributed (62.4±37.2 µm (mean±standard deviation); see also Fig. S1), we considered the droplets with diameters of ~50-120 µm in order to eliminate bending of microtubules inside the droplets. In the presence of Ni2+ ions in the aqueous phase, the kinesins should be anchored to the oil-water interface through coordination to the Ni2+ with the His-tag moiety of the GFP-kinesins and the NTA moiety of DGS-NTA. To confirm binding of the GFP-kinesins to the interface of the w/o emulsion, the droplets were observed using the CLSM (Figure 2). From the fluorescence of the GFP, localization of the GFP-kinesins on the inner surface of the water droplets was confirmed. In the absence of the Ni2+, no localization of the GFP-kinesins took place at the interface, and the kinesins were 12 ACS Paragon Plus Environment
Page 13 of 28
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Langmuir
found to be distributed with an aggregated form in the inner aqueous phase. This result clearly indicated that the anchoring of the GFP-kinesins containing the His-tag can be simply controlled by employing or eliminating the Ni2+ from the aqueous phase. We then attempted to incorporate the microtubules, with an average length of ~24.3±7.4 µm (average±standard deviation, n=50), into the aqueous droplets containing ATP according to the procedure already described. Representative CLSM images, as shown in Figure 3, revealed that the microtubules as well as the GFP-kinesins were localized on the inner surface of the w/o emulsion droplets in the presence of Ni2+. On the other hand, aster-like aggregated structures5 were observed when no Ni2+ was present in the droplets (Figure 3, see also Figures S2, S3, and S4). Thus localization of the microtubules to the interface of the w/o emulsion droplets can be controlled by controlling the availability of Ni2+ inside the droplets. Finally, by monitoring the motility of the microtubules using a total internal reflection fluorescence (TIRF) microscope in the presence of ATP, the activity of the GFP-kinesins anchored to the droplet surface and the encapsulated microtubules was investigated. We prepared a flowcell, as described in the experimental section, and added the w/o emulsion droplets containing the anchored kinesins and microtubules to the flowcell, which were then monitored by the TIRF microscope (Figures 4a, 4b). In any field of view, most of the microtubules (>90 %) were found to move on the surface of the aqueous droplets on which the GFP-kinesins were anchored. We compared the trajectories and velocity of the 13 ACS Paragon Plus Environment
Langmuir
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
microtubules (Figures 4c-4f) moving on the inner surface of an emulsion droplet to that of microtubules gliding on the surface of a solid substrate (glass) as a reference (see supporting movie 1 and 2). The mean velocity of the microtubules on the inner surface of the emulsion droplets was 0.34±0.17 µm/s (average±standard deviation; number of microtubules considered, n=100), which is within the range of velocity of the kinesin driven microtubules reported in literature28,29, but 1.2 fold slower than that on a solid substrate observed in our study (0.43±0.05 µm/s, n=100). The difference in velocity of the microtubules on the two substrates was found to be statistically significant (p
