Name: development of microfluidic devices to study the elongation capability of tip-growing plant cells in extremely small spaces
Uploaded: 2018-05-22T12:30:00
Description: Watch this Scientific Journal Video about Development of Microfluidic Devices to Study the Elongation Capability of Tip-growing Plant Cells in Extremely Small Spaces at JoVE.com

We thank H. Tsutsui and D. Kurihara for providing us with transgenic plants, including the T. fournieriRPS5Ap::H2B-tdTomato line and the A. thaliana UBQ10pro::H2B-mClover line, respectively. This work was supported by the Institute of Transformative Bio-Molecules of Nagoya University and the Japan Advanced Plant Science Network. Financial support for this work was provided by grants from the Japan Science and Technology Agency (ERATO project grant no. JPMJER1004 for T.H.), a Grant-in-Aid for Scientific Research on Innovative Areas (Nos. JP16H06465 and JP16H06464 for T.H.), and Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for challenging Exploratory Research (grant no. 26600061 for N.Y. and grant nos. 25650075 and 15K14542 for Y.S.).

1. Fabrication of the PDMS Microdevice to Examine Growing Pollen Tubes and Moss Protonemata
NOTE: We used a maskless photolithography instrument to prepare PDMS molds on silicon wafers. The details regarding the operation of the system are omitted in this manuscript. A standard photolithography technique9 using a photomask may also be used to create the PDMS molds described in this manuscript.
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	<li>Pour 11&#160;g of pre-polymer PDMS mixture (elastomer base:curing...

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Several critical steps in the protocol need to be followed precisely to obtain the results presented above. First, the PDMS layer and glass bottom dish surfaces must both be treated with plasma for a sufficient amount of time before bonding. Otherwise, the PDMS layer may locally detach from the glass surface while tip-growing cells are crossing the microgaps. Another crucial step in the root hair and moss protonemata protocol is the sterilization of the microdevice. Normally, root hairs and moss protonemata cells need to...

After pollen grains germinate on a stigma, each grain produces a single pollen tube that carries sperm cells to the egg cell and the central cell in the ovule for double fertilization. Pollen tubes elongate through the style and eventually reach the ovule by sensing multiple guidance cues along their way1. During the elongation, pollen tubes encounter a series of physical barriers; the transmitting track is filled with cells, and pollen tubes must enter the minute micropylar opening of the ovule to reach their target (Figure 1A)2. Therefore, pollen tubes must have the ability to penetrate physical obstacles, while tolerating the compressive stress from their surroundings. Root hairs are another type of tip-growing plant cell that must withstand physical obstacles in the environment, in the form of packed soil particles (Figure 1B).
Various mechanical properties of the pollen tube have been studied, including turgor pressure and stiffness of the cell&#39;s apical region, which can be measured using the incipient plasmolysis method3,4 and cellular force microscopy (CFM)5,6, respectively. However, these methods alone do not reveal whether pollen tubes are capable of elongating through physical barriers along their growth paths. An alternative technique that allows pollen tube elongation to be monitored in vivo is two photon microscopy7. However, with this method, it is difficult to observe the morphological changes in individual pollen tubes deep inside the ovule tissue. Additionally, root hair growth in soil can be visualized using X-ray computed tomography (CT) and magnetic resonance imaging (MRI)8, albeit with low resolution. Here, we present a method that can be used to acquire high-resolution images of a cell&#39;s deformation process on a conventional microscope.
The overall goal of the method described here is to visualize the elongation capability of tip-growing plant cells, including pollen tubes, root hairs, and moss protonemata, in extremely small spaces. As the poly-dimethylsiloxane (PDMS) microdevices presented in this manuscript are optically transparent and air permeable, we can culture living cells inside the device and observe their growth behaviors under a microscope. It is also possible to create micro ~ nanometer scale spaces by the soft lithography technique9 with the use of molds. These features allow us to study the elongation capability of tip-growing plant cells in a physically confined environment.
In this work, we constructed 1 &#181;m wide gaps (4 &#181;m in height) in microfluidic devices and examined the ability of pollen tubes to penetrate these artificial obstacles that are much smaller than the diameter of the cylindrical pollen tube (approximately 8 &#181;m). This experimental platform enables us to visualize the pollen tube&#39;s response to microgaps and capture time-lapse images of the response, which track the cell&#39;s deformation process. We also developed the microdevices that can be used to investigate the penetration capability of root hairs and moss protonemata. Several microdevices have been reported to date that enable the visualization of plant root10,11,12,13 and moss protonemata14 growth at high resolution. In our device, a series of root hair growth channels are perpendicularly connected to a root growth chamber, and individual root hairs (approximately 7 &#181;m in diameter) are guided to fluidic channels with a 1 &#181;m wide gap. We also cultured moss protonemata (approximately 20 &#181;m in diameter) in a microdevice containing microgaps to examine their responses to these physical barriers. The proposed microfluidic-based approach allows us to explore the capability of various tip-growing plant cells to elongate through extremely small spaces, which cannot be examined by any other currently available method.

<table border="0" cellpadding="0" cellspacing="0" width="927">
	<tbody>
		<tr height="18">
			<td height="18" style="height:19px;width:216px;">PDMS</td>
			<td style="width:165px;">Dow Corning Co.</td>
			<td style="width:119px;">Sylgard184</td>
			<td style="width:427px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Murashige &#38; Skoog Medium</td>
			<td style="width:165px;">Wako Pure Chemical</td>
			<td style="width:119px;">392-00591</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MES</td>
			<td style="width:165px;">Dojindo</td>
			<td style="width:119px;">345-01625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sucrose</td>
			<td style="width:165px;">Wako Pure Chemical</td>
			<td style="width:119px;">196-00015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50 mm glass-bottom dish</td>
			<td style="width:165px;">Matsunami Glass</td>
			<td style="width:119px;">D210402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">35 mm glass-bottom dish</td>
			<td style="width:165px;">Iwaki&#160;</td>
			<td style="width:119px;">3971-035</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Surgical blade</td>
			<td style="width:165px;">Feather</td>
			<td style="width:119px;">No.11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">biopsy punches</td>
			<td style="width:165px;">Harris</td>
			<td style="width:119px;"></td>
			<td>Uni-Core</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gel loading tips</td>
			<td style="width:165px;">Bio-Bik</td>
			<td style="width:119px;">124-R-204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Inverted Microscope</td>
			<td style="width:165px;">Olympus</td>
			<td style="width:119px;">IX83</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:22px;width:216px;">CSU-W1</td>
			<td style="width:165px;">Yokogawa Electric</td>
			<td style="width:119px;"></td>
			<td>No Catalog number is avairable for this customized microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MetaMorph imaging software</td>
			<td style="width:165px;">Molecular Devices</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

development of microfluidic devices to study the elongation capability of tip-growing plant cells in extremely small spaces

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior in such restrictive conditions. To address this issue, we have developed growth chambers for tip-growing plant cells that contain a series of narrow, micro-fabricated gaps (~1 &#181;m) in a poly-dimethylsiloxane (PDMS) substrate. This transparent material allows the user to monitor tip elongation processes in individual cells during microgap penetration by time-lapse imaging. Using this experimental platform, we observed morphological changes in pollen tubes as they penetrated the microgap. We captured the dynamic changes in the shape of a fluorescently labeled vegetative nucleus and sperm cells in a pollen tube during this process. Furthermore, we demonstrated the capability of root hairs and moss protonemata to penetrate the 1 &#181;m gap. This in vitro platform can be used to study how individual cells respond to physically constrained spaces and may provide insights into tip-growth mechanisms.

As illustrated in Figure 1, tip-growing plant cells encounter a series of physical barriers along their growth paths in vivo. The microfluidic in vitro cell culture platforms presented in this study enabled the examination the of tip-growing process in three types of plant cells (pollen tubes, root hairs, and moss protonemata) through 1 &#181;m artificial gaps (Figure 3, Figure 4, <...

Watch this Scientific Journal Video about Development of Microfluidic Devices to Study the Elongation Capability of Tip-growing Plant Cells in Extremely Small Spaces at JoVE.com

Development of Microfluidic Devices to Study the Elongation Capability of Tip-growing Plant Cells in Extremely Small Spaces

We describe a method to investigate the capability of tip-growing plant cells, including pollen tubes, root hairs, and moss protonemata, to elongate through extremely narrow gaps (~1 &#181;m) in a microfluidic device.

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior ...

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