A Uniaxial Loading Device for Studying Mechanoresponses of Single Plant Cell

doi:10.4236/eng.2013.510B085

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Engineering, 2013, 5, 416-419

http://dx.doi.org/10.4236/eng.2013.510B085 Published Online October 2013 (http://www.scirp.org/journal/eng)

A Uniaxial Loading Device for Studying Mechanor esponses

of Single Plant Cell*

Liqing Zhu1,2, Xue Fu3,4, Jie Yan5, Junyu Liu5

1School of Chemistry and Chemical Engineering, Chongqing University of Science and Technology, Chongqing, China;

2College of Bioengineering Chongqing University, Chongqing, China

3School of Chemistry and Chemical Engineering, Southwest Petroleum University, Chengdu, China

4Key Laboratory of Biorheological Science and Technology (Chongqing University), Ministry of Education, Chongqing, China

5College of Bioengineering Chongqing University, Chongqing, China

Email: c qz [email protected], [email protected], [email protected], [email protected]

Received 2013

ABSTRACT

A system which consists of a loading chamber unit, displacement sensor, data collector and processor, and a feedback

control, was established for applying mechanical forces to single plant cells. The method works by compressing an agar

cell-suspension block between parallel surfaces through using a force-feedback control circuit coupled to a microchip,

delivering the pre-defined. The actual controlled stimulus is achieved whilst measuring the force being imposed on the

cell, and its deformation. The Arabidopsis protoplasts were utilized to test the system. It provides an experimental ap-

proach to investigate the mechanoresponses of plant cells in vitro conditions.

Keywords: Mechanical Loading; Plant Cell; Chamber; Feedback Control

1. Introduction

Plants are unable to escape from an unfavorable envi-

ronment, they must respond to both endogenous and ex-

ogenous stimulus starting with a transduction of mecha-

nical perturbation into a biological signal and ending into

a global response as modification of growth, which is

known as thigmomorphogenesis [1]. Besides, plants use

regulated cell expansion to adapt their form to environ-

mental conditions. In contrast with most non-living ma-

terial systems, the cell structure is a dyn amic system that

adapts to its local mechanochemical environment, the

mechanical behavior of a living plant cell can’t be cha-

racterized only in terms of mechanical properties [2].

Observation of the patterns of cell expansion and wall

orientation is challenging because it occurs on several

scales, which range from the cellular to the tissue level,

and ultimately to a consideration of the whole plant. In

particular, application of ex ternal mechanical stimuli can

induce biochemical reactions, including the synthesis of

new biomolecules and the enhanced interaction among

biomolecules that can generate mechanical forces [3].

Despite the experimental sophistication and computa-

tional approaches in cell and molecular biology, the me-

chanisms by which mechanical stimuli could influence

cell signaling processes are poorly understood [4,5]. In

the past few decades, engineering fundamentals have been

used to design devices for probing the mechanical prop-

erties of living cells [6,7]. Here our focus is on the de-

velopment of mechanical stimulating system, which pro-

vided organismal survival conditions and measurable forces

delivery. In the present work, the authors report the use

of compressive force generated by a controllable loading

system as a tool to investigate cell in situ with the actual

stress and alteration of living cells’ geometry [8]. Our de -

sign of this system will allow us to conduct real-time stu-

dies for cellular responses to compress forces on samples.

Specifically, we want to satisfy the following criteria:

1) The system has to be small enough to be allowed

conventional and fluorescence microscopic observation

at high res olution .

2) The system should be a sustainable plant cell cul-

ture platform which can support the regeneration of a

small portion of cells in a long term culture.

3) The precision of linearity of displac ement sensor for

the system has to be accurate enough to be achieved to

conduct accurate compress stress analysis with an upper

limit of 300 mN.

2. Equipment and Method Development

1) System Design

The basic loading technique was that described by

*Supported by the Science and Technology Project of Chongqing Mu-

nicipal Education Commission, China (Project No. KJ121403).

L. Q. ZHU ET AL.

417

Lintilhac et al. [9]. An in-vitro experimental systems are

capable of generating controlled and well-defined me-

chanical forces were developed, which as a typical of

micro loading and measuring system, the basic compo-

nents of this apparatus are: loading unit, displacement or

deformation sensor, data collector and processor, and

feedback control. The conceptual simple schematic of

structure of the system is shown in Figure 1.

2) Device Fabrication

The chamber of the apparatus was constructed consist-

ing of one 2.0 mm thick deck of cast acrylic (30.0 mm ×

90.0 mm).

The deck has one threaded holes, in which a connect-

ing rod was set for connected to the stepper motor. To

enable accurate, sustainable application of forces, a step-

per motor (57BYG250A, stepper angular distance: 0.9˚/

1.8˚, operating voltage: 4.5 - 7.5 V) that mounted to the

top of pedestal was utilized to automate the movement of

applying connecting rod, which served as the source to

the deformation of test specimens. The stepper motor has

a threaded rotor which engages a threaded lead screw,

and the lead screw in turn is attached to a guide rod

which could slide freely along the axis of the guide way

(to maintain alignment of the slide relative to the medium

planar plate). The guide way enable positioning planar

motion of the subsequently connected applying rod, ap-

plying bafﬂe (20.0 mm × 15.0 mm) that can be gradually

and accurately energized to move toward and away from

the vertical slide of the sample in order to provide a di-

rect forces (ranged from 50 to 300 mN) to the test spe ci-

mens disposed in the chamber. A schematic diagram of

the apparatus is shown in Figure 2.

3) Feedback control technique

In general, for recording the force-displacement data

during the application of stress, sensor to simultaneously

monitor load and displacement is required, respectively.

Automatic strain measurement described in the work was

adopted with half-bridge circuit, which is composed of

some strain sensitive electrical resistance strain gauges.

The strain gauges (sensitivity factor: 2.12% ± 1%) them-

selves were placed on the surface of a piece of leaf spring,

whose one end was anchored on the guide rod and an-

other bolted to a certain position of the applying rod,

amplifying and transforming the displacement signal to

strain gauges. It is easy to find the relationship between

the e maximal actuator strain εmax and the force F, which

applied on the free end of leaf spring:

max 2

6Fl

Ebh

(1)

Where E is its elastic modulus, l, b and h are its length,

width and height of the rectangular se ction of leaf spring ,

respectively. When the external forces are applied, the

maximal strain will be produced close to the root of leaf

spring. Equation (1) can be used to obtain the consequen-

tial strain agar.

The altered resistance signals were collected and re-

gulated by the strain indicator (BZ2201, Beidai River

Land Science and Technology Ltd.), and then transferred

to the control and monitoring subsystem, thereby the dis-

placement could be recognized directly from the test spe-

cimens into a data acquisition system. Specifically, the

single channel dynamic strain indicator is a versatile,

Samples

Micro-probe

Stepper

motor

Strain

gauges

Strain

inductor

Stepper motor

drive circuit

Micro controller unit

Display Input

Figure 1. Block diagram of simple structure of the system.

Figure 2. Schematic diagram of the apparatus.

Vertical Base Plate

Guide Rod

Chamber

Microscope

Sample

Applying Baffle

Leaf Spring

Strain Gauge

Horizontal Transparent Base Plate

Stepper MotorLead Screw

L. Q. ZHU ET AL.

418

high-precision laboratory-type instrument developed for

use with strains gauges and strain gauge-based transduc-

ers to correcting for the spread in gauge resistance and

gauge factor. For overcoming the circumstances and to

compensate the thermal effect, temperature compensa-

tion circuit was designed mainly by means of two exter-

nal temperature compensating gauges.

The micro-controller unit (MCU) based closed-loop

control subsystem was built to measure and manipulate

this device. The data acquisition and control unit consists

of a multifunction single chip (SPCE061A, Sunplus Tech-

nology Company Limited) [10], which provides seven

channel 10-bit high-speed analog-to-digital converters

(ADC), and two 10-bit digital-to-analog con ve rt e r (D AC)

output channels, combined with higher processing speed

(up to 49.152 MHz) . The voltage r egulator SPY0029 was

utilized as power source to provide 3.3 V d.c. based on

the operating voltage of single chip that ranged from 2.6

to 3.6 V. The input signal comes from a microchip pro-

gram based on the DAC, it could predeﬁne the desired

force level. In addition, the I/O channel provides output

electronically control as well as impulse wave signal. A

four phase stepper motor component is driven to deliver

and maintain a desired precise force with the uniaxial

movement. This feedback control circuit provides a real-

time visual indication on the digital display of the strain

indicator output, the signal of the strain gauge and the

contr ol si gnal woul d be compa red whil e the for ce is sensed.

3. System Test

The loading system must be calibrated by verifying the

accuracy and precision of linearity of the displacement

sensor. To verify this device, the protoplasts isolated from

the wild-type Arabidopsis thaliana (ecotype Columbia

Col-0) rosette leaves were retained as experimental ma-

terial, Protoplasts were then immobilized by gently swirl-

ing them into low-melting-point agar in MS medium sup-

plemented with 0.4 mol∙l−1 mannitol, 0.1% MES, sucrose,

2,4-dichlorophenoxyacetic acid, and self-conditioned me-

dium harvested from suspension cultured Arabidopsis

cells [11]. Then the loaded test specimen which embed-

ded with living cells was removed and chipped into slices

of approximately 2.0 - 5.0 mm thick, and it was placed

onto the chamber of the mechanical loading apparatus

described above.

Hamant has reported that microtubule orientation in

the shoot apical meristem was found to follow the orien-

tation of stress patterns in the organ [12], this conclusion

supports the prior viewpoint that th e lo c al microinduction

of expansin expression and resulting cell wall softening

is sufﬁcient to induce morphogenetic processes, leading

to the initiation of leaf structures from the shoot apical

meristem [13]. Continuous uniaxial compressive force (at

approximately 50 - 300 mN) was imposed on the oppo-

site sides of agar block, time ranged from 60 seconds to

1200 seconds, this allowed a microscope to be brought

close to the cell under test. Our device could play a role

in the relative research field. The applying baffle dis-

placement and the cell deformation could be observed,

and a force-deformation curve generated. As shown in

Figure 3, the deformation of embedded protoplasts under

constant mechanical strain is visible in the substrate, the

minor axis will be the potential division plane. A 20%

stain of the agar matrix could induce a significant defor-

mation of the single cell without cell wall (Figure 4;

control versus loaded). It should be noted that the proto-

plast all show a preferential orientation (Orientation =

angle between stress vector and minor axis) with their

long axis perpendicular to the principle stress vector, and

a 15-mins loading of such intensity did not influence the

viability of the subjected protoplasts in the subsequent

24h’s culture.

4. Results and Discussion

An instrument, which has potential to provide mechani-

cal property information at the individual cell Level has

been devised that is capable of delivering static or cyclic

compression or tension of variable duration to cells cul-

tured in vitro. A lack of published data on properties of

Arabidopsis protoplasts prevents a comparison of the

Figure 3. Control experiment to test the influence of t he me -

chanical stimuli to the protoplasts elongation. Contrast con-

trol group, the loaded group show that the aspect (Aspect =

minor axis/major axis) of embedded protoplasts altered sig-

nificantly. Bar = 50 μm.

Figure 4. 20 mins constant mechanical strain (80%) induced

protoplast deformation-Aspects, Orientation and Viability.

Compare the orientation of loaded group relative to control

group, it is reduc ed significantly. Ot herwise, the viability of

both groups rarely changed.

L. Q. ZHU ET AL.

419

other parameters. However, the values of this method

should be a function of the state of the cell will allow us

to model mechanical stress on cell and cytoskeleton in

studyin g plant cell mechanorespons e s.

5. Acknowledgements

The authors thank Chongqing University of Science and

Technology for financial support for this work.

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