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Cyclic stretch of the substratum using a shape-memory alloy induces directional migration in Dictyostelium cells
 
Yoshiaki Iwadate1,2 and Shigehiko Yumura1
1Department of Functional Molecular Biology, Graduate School of Medicine, Yamaguchi University, Yamaguchi, Japan
2PRESTO, Japan Science and Technology Agency, Saitama, Japan
BioTechniques, Vol. 47, No. 3, September 2009, pp. 757–767
Full Text (PDF)
Supplementary Material
Supplementary Movie 1. Motion of stretching device using SMA as a power source. (.mov)
To generate the power to stretch the elastic sheet, 4 pieces of coiled SMA were connected in parallel to the left edge of the sheet. The time cycle was 30 s. The movie is shown 5× faster than real time.
Supplementary Movie 2. Dictyostelium cells under the cyclic stretching stimuli. (.mov)
This movie was prepared for demonstration. The densities of cells in this movie were higher than that of the trajectory measurement (see Figure 2 in the main text). The time cycle was 30 s. The movie is shown 10× faster than real time.
Supplementary Movie 3. Migration of Dictyostelium cells under the cyclic stretching stimuli. (.mov)
The images where the substratum shrank in Movie 2 were extracted and accumulated sequentially. The movie is shown 450× faster than real time.
Abstract

Living cells are constantly subjected to various mechanical stimulations. They must sense the mechanical aspects of their environment and respond appropriately for proper cell function. In general, cells adhere to substrata. Thus, the cells must receive and respond to mechanical stimuli mainly from the substrata. For example, migrating cells can create their own polarity and migrate in a certain direction even in the absence of any attractive substance. In order to generate such polarity, cells must sense mechanical stimuli from the substrata and transduce these stimuli into intracellular signals. To investigate the relationship between signals derived from mechanical stimuli and related cell functions, one of the most commonly used techniques is the application of mechanical stimuli via stretching of elastic substrata. Here, we developed a new stretching device using a shape-memory alloy (SMA). An SMA has three advantages as an actuator of stretching devices: (i) the cost of the SMA required for the device is inexpensive, ~$30 USD, (ii) the size of an SMA is very small (0.62 mm in diameter and 22 mm in length), and (iii) an SMA does not generate any vibrating noise, which can negatively affect cells. In response to the cyclic stretching by the new stretching device, Dictyostelium discoideum cells migrated perpendicular to the stretching direction and the migrating speed increased significantly. To our knowledge, this is the first report indicating that migrating cells can create their own polarity by the mechanical stimuli from the substrata.

Introduction

Living cells are constantly subjected to a wide variety of mechanical stimulations such as shear flow and substratum strain. They must sense the mechanical aspects of their environment and respond appropriately for proper cell function. For example, blood shear flow activates various cell functions in vascular endothelial cells, such as gene expression, proliferation, and apotosis (1). In the auditory hair cells of vertebrates, stereovilli deflections open mechano-electrical transduction channels and cause changes in the membrane potential (2). To understand the mechanisms of the mechanosensing system in cells, artificial mechanical stimuli—such as sucking of a cell portion using a micro pipette (3,4,5,6), hydrodynamic shear-flow (7,8,9), and manipulation of microbeads attached to a cell with magnetic (10,11) or optical tweezers (12,13)—have been applied (14), leading to many findings, such as the existence of signaling pathways triggered by mechanical stimuli (15).

In general, cells adhere to the substratum via focal adhesion sites. Thus, it seems that the cells receive mechanical stimuli mainly from substrata in the physiological condition (15,16). To mimic this situation, one of the most appropriate techniques for applying mechanical stimuli artificially is to stretch the elastic substratum to which cells adhere. In response to the cyclic stretching of the elastic substratum, intracellular stress fibers in endothelial cells are rearranged perpendicular to the stretching direction (17,18,19,20).

Fast-moving cells, such as Dictyostelium discoideum cells, can create their own polarity and migrate in a certain direction even in the absence of any chemoattractant. In order to generate polarity without any chemoattractant, it is presumed that cells sense mechanical inputs from the substratum. To investigate the relationship between substratum mechanical inputs and migration direction, a device for cyclically stretching the substratum could be a powerful tool. However, migration of fast-moving cells has not, to our knowledge, been observed under cyclic stretching.

A shape-memory alloy (SMA) is a material that remembers its own geometry. After an SMA has been deformed from its original crystallographic configuration, it regains its original geometry by itself during heating. SMA is an appropriate material for the actuator of the stretching device because of the following three reasons: (i) the cost of the SMA required for the device is low, ~$30 USD; (ii) the size required for an SMA for stretching the elastic substratum is very small (0.62 mm in diameter and 22 mm in length), enabling a compact setup (including electrical circuit) which is also advantageous for use with a microscope stage; and (iii) an SMA does not generate any vibrating noise when it moves.

Here, we developed a new stretching device using an SMA as an actuator. Using this new device, we applied cyclic stretching stimuli to migrating Dictyostelium cells. Interestingly, in response to the cyclic stretching, the cells migrated perpendicular to the stretching direction and the migrating speed of the cells increased significantly. To our knowledge, this is the first report indicating that the migrating cells can create their own polarity by the mechanical stimuli from the substrata.

Materials and methods

The overall setup for the new cell stretching device is shown in Figure 1A. The device is composed of an elastic sheet, coiled SMAs, a cooling fan, and an electrical circuit to produce voltage pulses for cyclic contraction of the SMAs.





Preparation of elastic sheets

To apply stretching stimuli to cells via the attached substratum and observe their response, an elastic sheet with optimal elasticity and clearness was prepared as a substratum. We selected an elastomeric polydimethylsiloxane (PDMS) kit (Sylgard 184, Dow Corning Toray, Tokyo, Japan) of Young's modulus 2.2 ± 0.1 MPa (21) as the material for the elastic sheet. The PDMS kit contains two parts, a liquid silicon rubber base and a curing agent, and uses a platinum complex to participate in hydrosilylation of a vinyl functional siloxane polymer by a hydride functional siloxane polymer. The silicone elastomer made from the kit is clear and colorless. A silicon rubber base and a curing agent were mixed at a 10:1 ratio by weight. A 300-mg aliquot of the mixture was poured into a 22 mm × 40 mm × 1 mm mold made from a slide glass (Cat. no. S-2441, Matsunami, Osaka, Japan) with an acrylic frame (Figure 1B). The mixture in the mold form was allowed to solidify at room temperature (23°C) for 2 days. The solidified sheet was peeled off carefully (to facilitate this, the mold was previously prepared with a thin layer of only the silicon rubber base, before the mixture was added). The thickness of the sheet, except for the periphery, was ~200 µm. The periphery was slightly thicker because of the surface tension of the liquid silicone.

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