High efficiency penetration of antibody-immobilized nanoneedle thorough plasma membrane for in situ detection of cytoskeletal proteins in living cells
© The Author(s) 2016
Received: 12 July 2016
Accepted: 15 October 2016
Published: 3 November 2016
The field of structural dynamics of cytoskeletons in living cells is gathering wide interest, since better understanding of cytoskeleton intracellular organization will provide us with not only insights into basic cell biology but may also enable development of new strategies in regenerative medicine and cancer therapy, fields in which cytoskeleton-dependent dynamics play a pivotal role. The nanoneedle technology is a powerful tool allowing for intracellular investigations, as it can be directly inserted into live cells by penetrating through the plasma membrane causing minimal damage to cells, under the precise manipulation using atomic force microscope. Modifications of the nanoneedles using antibodies have allowed for accurate mechanical detection of various cytoskeletal components, including actin, microtubules and intermediate filaments. However, successful penetration of the nanoneedle through the plasma membrane has been shown to vary greatly between different cell types and conditions. In an effort to overcome this problem and improve the success rate of nanoneedle insertion into the live cells, we have focused here on the fluidity of the membrane lipid bilayer, which may hinder nanoneedle penetration into the cytosolic environment.
We aimed to reduce apparent fluidity of the membrane by either increasing the approach velocity or reducing experimental temperatures. Although changes in approach velocity did not have much effect, lowering the temperature was found to greatly improve the detection of unbinding forces, suggesting that alteration in the plasma membrane fluidity led to increase in nanoneedle penetration.
Operation at a lower temperature of 4 °C greatly improved the success rate of nanoneedle insertion to live cells at an optimized approach velocity, while it did not affect the binding of antibodies immobilized on the nanoneedle to vimentins for mechanical detection. As these experimental parameters can be applied to various cell types, these results may improve the versatility of the nanoneedle technology to other cell lines and platforms.
One of the major reasons for nanoneedle insertion failure can be attributed to the soft and fluidic nature of cellular membranes, which lead to their flexible deformation and an unsuccessful penetration [10, 12]. As membrane fluidity is determined also by the dynamics of lipid molecules that constitute the membrane, and these are affected by temperature, we set here to investigate the effect of temperature, as well as approach velocity, on the success rate of nanoneedle penetration.
In this paper, we test a possibility to improve nanoneedle insertion efficiency by verifying the effect of nanoneedle approaching velocity and operating temperature, focusing on membrane fluidity. With the optimization of these parameters to a variety of cell types, applications of the nanoneedle technology can be expanded to any kind of intracellular investigations in biological and biomedical research.
Results and discussion
Evaluation of nanoneedle penetration into live cells
Insertion of an AFM cantilever-type nanoneedle into the cytosol of live cells has been originally observed during AFM force spectroscopy measurements, evident from a sharp force relaxation peak in the force-distance curve during cell indentation. Successful insertion of nanoneedles into cells has been further confirmed in other studies investigating molecular functions in the cytosol with the use of FRET or molecular beacon [18–20]. Nanoneedle insertion through the plasma membrane has also been directly visualized with use of confocal laser scanning microscope (CLSM) (Fig. 1b) . In some cases, such as in some cell lines and under certain growth conditions, the higher deformability of the plasma membrane prevents a successful penetration of the nanoneedle into the cell. In these cases, the membrane can be observed surrounding the nanoneedle periphery as it indents into the cell, indicating the lack of actual penetration through the membrane (Fig. 1c). However, that is not always clearly visible. A better way to ascertain successful insertion of the nanoneedle through the plasma membrane is to modify the nanoneedle with antibodies specific for intracellular cytoskeletal proteins and observe the force-distance curves, looking out for significant unbinding events during nanoneedle retraction . Here, this force-detection method was employed for direct and prompt evaluation of penetration events, extending the target protein to vimentin, rather than those previously reported such as actin, microtubule and nestin [13–15].
Effect of approaching velocity on nanoneedle penetration
Average of the fishing forces detected in MCF-7 and HeLa cells at various approach velocities: analysis on the data set of Fig. 2c
Approach velocity (µm/s)
Fishing force (nN)
Effect of temperature on nanoneedle penetration
Average of the fishing forces detected in MCF-7 and HeLa cells at various temperature: analysis on the data set of Fig. 3
Fishing force (nN)
The ratio of successful fishing force detection in HeLa cells at low temperature: analysis on the data set of Fig. 3
Detection of fishing forces over threshold* (n out of 30 trials)
Detection of fishing force over threshold* (%)
Average of fishing force over threshold (nN) ± SD
100.7 ± 69.1
58.8 ± 26.8
Stiffness of the plasma membrane was estimated from force-indentation curves during nanoneedle insertion into the cells (Additional file 1: Figure S4). Young’s modulus was calculated by fitting with the Hertz model for initial deformation, to evaluate stiffness of the plasma membrane. Significant differences were not observed in cell stiffness due to temperature changes (Additional file 1: Figure S4) since fluidity of lipid bilayer may not have large contribution to cell stiffness. However, the average cell stiffness under 4 °C condition was higher than that under 37 °C. This tendency is consistent with the decrease in the fluidity (Fig. 4).
From the results for HeLa cells it was suggested that membrane fluidity under low temperature condition would contribute to deeper insertion of nanoneedle into cell. Further validation with other combinations of target intracellular molecules and cell types will be performed in the near future, in order to investigate whether insertion failure is perhaps due to fluidity variations between different cell types. ZZ-BNC, the anchoring agent for the antibody seems to be rigid enough to keep the spherical structure during the temperature change between 5 and 37 °C, since it consists of 80 % protein, 10 % sugar chain and 10 % lipid that derived from endoplasmic reticulum of yeast .
In summary, it is reasonable to conclude that lowering membrane fluidity at lower temperatures enhances penetration efficiency, although other possibilities should be considered. In an attempt to consider other possible mechanisms for enhancing fishing force detection, we followed with examining the effect of temperature on vimentin-antibody unbinding forces.
Lack of temperature effect on antibody-antigen unbinding forces
In this study we aimed to optimize the conditions required for the successful insertion of nanoneedles into live cells, for the purposes of intracellular protein detection, drug/protein delivery, or intracellular manipulation. Both approach velocity and temperature influenced to nanoneedle penetration, especially lowering the temperature was found to greatly improve the detection of unbinding forces, suggesting that alteration in the plasma membrane fluidity led to increase in nanoneedle penetration. As phase transition caused by lowering temperature is general effect for cell membrane, this approach may lead to enhanced versatility in the use of nanoneedles, allowing application of this technology various cell types.
MCF-7 and HeLa cells culture
Human breast cancer cells (MCF-7) and cervical cancer cells (HeLa) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; D5546, Sigma-Aldrich) supplemented with 10 % fetal bovine serum (FBS; Life technologies), 2 mM GlutaMAX (35050-061, Life technologies), gentamycin–amphotericin B (10 and 0.25 µg/ml, GA; R-015-10, Life technologies). The cells were subcultured at 80–90 % confluency every 3 days by following procedures. They were treated with 0.25 % trypsin, 0.01 % EDTA for 3 min at 37 °C and diluted with the medium containing FBS. After centrifuging at 170×g for 5 min at room temperature, the cells were seeded into cell culture flasks (353108, Becton–Dickinson) or dishes (93040, TPP).
Force analysis of cell fishing and adhesion with AFM
Nanoneedles were fabricated from pyramidal silicon AFM cantilevers (ATEC-Cont, Nanosensors) and etched to a cylindrical shape of 200 nm in diameter and 10–15 µm in length, using a focused ion beam (SMI500, Hitachi High-Tech Science). Spring constants (k = 0.1–0.4 N/m) were determined using the thermal fluctuation method prior to each experiment . The silicon surface was cleaned with oxygen plasma in a plasma asher (200 W, 5 min; JPA300, J-science) and treated with 1 % HF for 1 min. After repeating the plasma (10 min) and 1 % HF treatment once again, the nanoneedle was modified by physical adsorption of 50 µg/ml of ZZ-BNC at room temperature for 1 h; ZZ-BNC is bio-nano capsule based anchor to which Fc domain of antibodies can bind [27–29]. Anti-vimentin antibody (V6630, Sigma-Aldrich) was bound to the ZZ-domain of the ZZ-BNC by incubating with 475 µg/ml antibody in PBS at room temperature for 1 h. The antibody-immobilized nanoneedle was rinsed in prior to the use in cell fishing experiments. Force measurements were carried out using a Nanowizard II BioAFM (JPK Instruments AG) with CellHesion® unit that allows long traveling distance over 100 µm. For the cellular membrane penetration test, nanoneedles were inserted to cells at approach velocity of 1–1000 µm/s with a set point of 40 nN, left to dwell within the cells for 2 s, and then evacuated at 10 µm/s. 10 different sites on each cells were targeted for insertion and 3 cells were tested for each cell type. The nanoneedles used for more than 5 cells were washed with 0.05 % Tween 20 in PBS before the use for next cells. Temperature control of the sample was done by dish heater to manage sample dish temperature at 37 °C and by putting donut-shaped ice block of medium in the dish during a measurement at around 4 °C. All of the measurements at low temperature were finished within 15 min.
For unbinding force measurement of vimentin-antibody interaction in vitro, an MPC polymer was used for surface modification of nanoneedles to allow stable linking by covalent bonds [14, 15]. AFM cantilevers (ATEC-Cont) were cleaned with plasma-asher for 5 min at 200 W and rinsed with ultrapure water and ethanol once for each. After cleaning with plasma asher once again, AFM cantilevers were dipped in a solution of 0.05 wt% MPC polymer in dry ethanol containing 1-butanol (95:5) for 10 min at room temperature, followed by baking at 50 °C for 15 min . Following rinsing with ethanol 3 times and with PBS once, needles were then soaked in 475 µg/ml anti-vimentin antibody solution for 1 h and rinsed with PBS 3-times. The remaining active ester of MPC polymer was killed by treating with 10 mM ethanol amine for 30 min at room temperature and rinsed with PBS twice. Vimentin (His-tagged human, SRP5150, Sigma-Aldrich) immobilization to glass surface was done in the same way as the AFM cantilever using MPC polymer but with vimentin solution of 50 µg/ml. Force measurement by AFM was performed with approach/retraction velocity at 1 µm/s in a force mapping mode in which 10 × 30 times measurement was made within area of 10 µm square on the substrate.
Imaging of the cells and the needles by microscopy
Fabricated nanoneedles were cleaned with plasma-asher for 5 min at 200 W and rinsed with ultrapure water and ethanol once for each. After clean with plasma asher once again, the nanoneedle was incubated with MPTMS and the exposed thiol group was further modified with Alexa Fluor 488 maleimide (A1025, Life technologies). For fluorescent visualization, cells were transformed to express Keima-Red protein by transfection of pPM-mKeima-Red. Fluorescence image of cells were obtained with use of confocal laser scanning microscopy system (CLSM; FV-300/IX71, Olympus).
Fluorescence spectroscopy of cells treated with pyrene with temperature variation
Cells were seeded on to collagen coated glass bottom 6-well plate at density of 7 × 105 per well on the day before measurement. After rinsing with PBS, cells were incubated with 1 ml of 15 µM pyrene in a perfusion buffer A (100 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 5 mM d-glucose, 50 mM mannitol, 5 mM HEPES/Tris, pH 7.4) supplemented with 0.08 % Pluronic F-127 for 20 min at 25 °C in the dark. After rinsing with the perfusion buffer A twice, the cells were recovered by incubating with 2 ml of Opti-MEM (31985-062, Life technologies) containing 10 mM HEPES (pH 7.4) and EDTA for 30 min. Fluorescence spectroscopy was performed with excitation light wave length of 360 nm for the emission light wave length range of 380–550 nm at each temperature. Each well was measured three times and the obtained data was averaged. The decreasing of medium temperature down to ca. 4 °C was done by exchanging the medium with cold one and waiting for 5 min after adding frozen medium as described in Additional file 1: Figure S1.
CN conceived the method and organized the research. KS and YM mainly performed experiments and data analysis. MI and SK developed ZZ-BNC, KF and KI developed MPC polymer for antibody immobilization to nanoneedle. RK, AY, YRS suggested research plan for experiments. RK, AY, YRS, CN wrote the manuscript. All authors read and approved the final manuscript.
This research is granted by the Japan Society for the Promotion of Science (JSPS) through KAKENHI Grant Number 26249127 and the “Funding Program for Next Generation World-Leading Researchers (NEXT Program)”, initiated by the Council for Science and Technology Policy (CSTP).
The authors declare that they have no competing interests.
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