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Physiology and Biochemistry |
Institut de recherche en biologie végétale, Département de sciences biologiques, Université de Montréal, 4101 rue Sherbrooke est, Montréal, Québec H1X 2B2, Canada
Received for publication March 10, 2006. Accepted for publication July 3, 2006.
ABSTRACT
From a mechanical point of view, plant and hyphal cells are more complex than their animal counterparts because the variety of structural components determining cellular architecture is broader. In addition to cytoskeletal elements and the plasma membrane, the cell wall and turgor pressure equip plant and hyphal cells with structures analogous to an exoskeleton and a hydroskeleton, respectively. To quantify the physical properties of plant and hyphal cells, researchers have developed a plethora of experimental methods. This review provides an overview of experimental approaches that have been used to measure turgor pressure and to determine the mechanical properties of the plant cell wall at the subcellular level. It is completed by a glimpse into the arsenal of techniques that has been used to characterize the physical properties of cytoskeletal elements. These have mostly been used on animal cells, but we hope they will find their way into plant cell research. Finally, assays and tests to measure the generation of forces by cells and subcellular structures are discussed.
Key Words: cell biomechanics • cell wall • cytomechanics • cytoskeleton • hypha • plant cell • tip growth • turgor
The mechanical behavior of organisms and organs is determined by the physical properties of their building blocks, the cells and their components. Cells act as mechanical structures because their morphogenesis results from local compliance with applied forces (Harold, 2005
). They are able to resist external physical forces such as gravity and to exert forces by way of growth processes. On the other hand, cells are also influenced by external physical forces. These play a fundamental role in the regulation of cell functions, including gene induction, protein synthesis, cell growth, and death. We have long known that tissues differentiate, grow, and remodel in response to changes in mechanical forces. The formation of tension wood resulting from stress induced by gravity is only one of numerous examples. Tension wood is characterized by biochemical alterations in the cell walls and morphological changes of those cells that are strategically located to resist the stress generated by the weight of a plant organ. To appreciate the mechanical aspect of cellular architecture and to understand how cells are able to perceive and to react to applied mechanical forces, we need to investigate and quantify their physical properties. The elucidation of these structure–function relationships will contribute to our understanding of the mechanisms that govern the adaptation of an organism to its environment.
The principal concept in quantitative mechanophysical studies is the application of calibrated loads or deformations on an object and the subsequent quantification of the object's response. On a macroscopic scale, the physical properties of wood are tested for example by using a three-point bending test in which a wooden beam is placed on two supports located at both ends and subsequently bent by a deforming force applied to the center. The response of the beam to the flexural load provides information on its physical properties such as elasticity and failure resistance. Another type of stress is the application of a tensile stress on a strip of material such as a rubber band. The change in length resulting from the application of such a stress expresses the elasticity of the material. An assessment of physical properties at the cellular and subcellular level uses the same mechanical principles, which presents a technical challenge because experimental devices in the appropriate dimension have to be developed.
Depending on the structure under investigation and the precise question to be answered, different approaches have been realized to deform cells or cellular structures and to measure their reactions. Deformations can either be local or global, and they can address either an individual cellular component or the cell as a physical entity. This review summarizes some of the experimental techniques and methodological approaches that have been developed to measure the physical properties of cells and cellular components. The focus will be on approaches used for plant and hyphal cells, but because of its technical advancement occasional glimpses into the progress achieved with mammalian cells are also provided.
All eukaryotic cells share at least two structural building elements that compose cellular architecture and determine cellular morphology: the cytoskeleton and the plasma membrane. The cytoskeletal proteins function as rod-shaped building blocks that are able to resist both compression and tensile stresses. Furthermore, these polymers are dynamic and hence able to actively exert forces through their assembly and disassembly as well as by way of motor proteins. The plasma membrane surrounds the cell and from a mechanical point of view can be modeled as an envelope characterized by its pliability and elasticity.
In plants, fungi, and water molds, two additional features contribute to the mechanics of cellular structure—the cell wall and turgor pressure. The cell wall surrounds the cell and, depending on its biochemical composition, forms a more or less rigid case. The presence of this case allows the establishment of considerable pressure in the protoplast, mainly by swelling of the vacuole. From an architectural point, plant and fungal cells are therefore much more complex than animal cells, and all of the subcellular, structural building blocks and their interactions have to be considered to quantitatively assess the mechanical parameters governing cellular functioning. In the following, experimental approaches to assess the physical properties and the mechanical behavior of the three most important elements determining structure in plant and hyphal cells are discussed: the cytoskeleton, the cell wall, and turgor pressure. The final section reviews devices that are used to measure dynamic cellular activities and the generation of forces at the cellular and subcellular levels.
BUCKLING AND BENDING: THE CYTOSKELETON IS AN ELASTIC SCAFFOLD
The elements of the cytoskeleton have multiple mechanical functions. Among others, they form the rails along which intracellular transport takes place, and they govern cell division and provide the force to contract or to move the leading edge of many animal cells forward (Mitchison and Cramer, 1996
). Cytoskeletal elements are also involved in the mechanotransduction of mechanical stresses (Wang et al., 1993
). In animal cells the cytoskeleton has an important architectural function because it determines the shape of the cell. This structural function is particularly obvious in cells that are able to undergo repeated deformations such as erythrocytes on their way through minute capillaries. In plant cells, maintenance of overall cellular shape does not seem to be an important cytoskeletal function given that plant protoplasts generally round up after having been isolated from the surrounding cell wall. Detailed studies on this topic are lacking, however. Some of the better known mechanical functions of the plant cell cytoskeleton include the interaction with statoliths during graviperception and the formation of the mitotic spindle during cell division, functions that are associated with providing internal structure.
The question of how the cytoskeletal elements interact to provide a cell with structure that is able to resist deforming forces has mostly been examined using mammalian cells because of the absence of a surrounding cell wall. This permits the deformation of cytoskeletal elements from the outside through the membrane. Another reason for the relative abundance of biomechanical data on mammalian cells is the fact that they allow quantitative studies on the change in the physical properties of cells and biomolecules that occur with the progression of certain human diseases such as malaria, sickle cell anemia, and cancer. This research therefore is strongly supported in the medical field (Wang and Thampatty, 2006
). For an excellent review of the fundamental approaches to cytoskeletal mechanics in terms of flexible polymers and networks, refer to Boal (2002)
.
One of the methods used to probe the mechanical properties of a cell and in particular of the cytoskeleton is micropipette aspiration—measuring the suction pressure necessary to partially or wholly suck a single cell into a micropipette (Fig. 1A). The diameter of the pipette may range from less than 1 µm to 10 µm, and video microscopy is used to record the shape change of the cell (Hochmuth, 2000
). Besides measuring the underlying cytoskeleton, this technique also measures the membrane elasticity. This approach to completely deform the cell, and thus the entire cytoskeletal scaffold, is particularly relevant in those mammalian cells, such as erythrocytes, for which the ability to repeatedly undergo shape changes is pivotal for cellular functioning. Other techniques to globally change the shape of cells in order to gain insight into cellular mechanics are based on manipulations of cells using microneedles (Dennerll et al., 1989
) or optical tweezers (Hénon et al., 1999
). The latter technique uses a laser to trap, control, and manipulate minute objects or particles; hence its other name, optical trap. The principle of this technique is the light pressure (or gradient force) that moves a particle with higher refractive index than that of the surrounding medium to the focal point of the laser beam and exerts a net force to keep it there (Fig. 1B). Optical tweezers also have been used to probe the elasticity of single DNA molecules (Bustamante et al., 2003
), protein–protein interactive forces (Litvinov et al., 2002
), and the mechanical properties of cytoskeletal elements in vitro (Kuo et al., 1995
; Kurachi et al., 1995
; Tsuda et al., 1996
). A list of other applications can be found in Lang and Block (2003)
.
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). The variety of experimental approaches illustrates the versatility of the optical tweezer method. It has the advantage of not necessitating any physical contact with the sample, but the drawbacks are possible photodamage of the biological samples and the relatively low maximum force (a few hundred pN) achievable.
Because of this low achievable force, the optical trap is unlikely to be able to cope with the forces necessary to deform walled plant cells in their entirety. Nevertheless, it most certainly has potential for the investigation of the plant cell cytoskeleton by displacing objects within the cytoplasm. In the early days of the optical trap, it was employed to investigate the viscoelastic properties of the plant cell cytoplasm (Ashkin and Dziedzic, 1989
) as well as that of Hechtian strands (Buer et al., 2000
). The effect of cytoskeletal structure and the movement of organelles such as statoliths has been investigated in Chara rhizoids. The laser-guided displacement of statoliths led to conclusions about the forces acting on individual organelles by axially oriented actin filaments (Leitz et al., 1995
). Many more applications in plant cells can be envisioned given that the (primary) cell wall does not interfere with the functionality of the laser.
Other experimental approaches to measure the physical properties of the cytoskeleton in vivo are applied from the outside of the cell but act more locally and thus provide information with subcellular resolution. Among them is atomic force microscopy (AFM), a technique that can act as both a powerful imaging tool and a force sensor with piconewton force resolution. AFM is based on a very sharp, tiny tip mounted at the end of a flexible cantilever (Fig. 1C). The force interacting between the tip and the sample surface deflects the cantilever, and a laser beam is then reflected off the back of the cantilever and recorded by a photodiode. The sensitivity of the application is high enough to be able to measure interactions between individual biomolecules (Willemsen et al., 2000). As far as intact cells are concerned, the spatial distribution of the stiffness of cellular surfaces can be mapped using the Hertz contact theory, which provides analytical solutions to the stress distribution at the interface between and within two elastic bodies. In mammalian cells, cytoskeletal fibers underlying the cell membrane can thus be "seen" and their rigidity quantified through the relatively pliable plasma membrane (Simon et al., 2004
).
This approach to measure cytoskeletal mechanical properties from the outside is of rather limited use for plant cells because of the surrounding cell wall, which is likely to partially or completely mask the stiffening effect of the cytoskeletal elements. It might be possible in situations where the surrounding wall is very thin or absent such as in isolated plant cell protoplasts. A technique similar to AFM, micro-indentation, has been used with thin-walled pollen tubes (Geitmann et al., 2004
; Geitmann and Parre, 2004
; Bolduc et al., 2006
). A glass stylus is connected to a horizontal beam with a known bending constant, which gauges an object's resistance to deformation (Fig. 1D). Being in the order of a few micrometers, the deformation amplitude is bigger than that common for the AFM. Importantly, due to the length of the micro-indenter stylus, the horizontal beam is completely out of focus and allows simultaneous microscopic observation of the deformed cellular region (Fig. 2). It is thus possible to monitor the deformation proper as well as cellular dynamic processes in the inverted microscope on which the indenter is mounted. This is of particular interest in rapidly growing cells such as pollen tubes or fungal hyphae, because the position of the deforming stylus relative to the growing apex of the cell changes constantly.
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). In pollen tubes, the cellular stiffness measured by micro-indentation coincides well with the non-uniform distribution of actin filaments in these cells. However, this parameter has yet to be discriminated from the effect of the non-uniform distribution of the mechanical properties of the surrounding cell wall (Geitmann and Parre, 2004
). This example illustrates that in plant cells, quantification of the cytoskeletal mechanics by way of loads applied from the outside requires complex analysis of the data, even in thin-walled cells.
Approaches that act within the cell might therefore be more promising for the quantification of cytoskeletal mechanics in plant cells. In addition to optical tweezer applications, several other methods are of potential interest for plant cells. In magnetic bead rheometry, ferromagnetic beads a few micrometers in diameter are taken up by living cells through phagocytosis and tracked optically while exposed to a magnetic field (Fig. 1E). The movements provide information on the viscoelastic moduli of the cytoplasm (Bausch et al., 1999
). Because plant cells generally are not capable of phagocytosis, insertion of such a bead could probably be achieved with particle gun bombardment or microinjection.
A similar principle is realized in magnetic rotational microrheology, which is based on the analysis of the rotational dynamics of chains of magnetic endosomes in response to a rotation of the external magnetic field (Fig. 1F) (Wilhelm, 2003
). Magnetic nanoparticles are placed inside living cells via endocytosis, subsequently concentrating in the endocytotic compartments. Endosome movements provide information on the viscosity governing the long-time flow of the medium surrounding the endosome chains and on the relaxation time, reflecting the proportion of solidlike vs. liquidlike behavior—properties that reflect the configuration of the cytoskeleton.
Instead of the forces applied by a magnetic field, the natural Brownian motion of particles embedded in the cytoskeletal network is monitored in laser tracking microrheology. Using a focused, low-power laser beam, a probe particle is tracked by monitoring its forward-scattered light with a quadrant photodiode detector, which allows the calculation of the viscoelastic shear moduli of the cytoplasm (Yamada et al., 2000
).
Almost all of the methods investigating cytoskeletal mechanics that are described in this article have been applied only on animal cells so far. In particular, those that apply deforming forces on the cytoskeleton by displacing organelles or particles embedded in the cytoplasm are suitable for use in walled plant and hyphal cells because interference from the cell wall is avoided. These methods are therefore suitable strategies to be employed in future plant cell research.
UNDER PRESSURE: HYDRAULIC FORCES BASED ON TURGOR
The concept of a stable structure being generated by the interaction between the internal pressure and a surrounding case is realized in certain animal organs, but in the plant and fungal kingdoms it is a fundamental architectural principle of all organisms. By putting cell walls under tension stress, the turgor functions analogous to a hydroskeleton, thus contributing significantly to the mechanical properties of plant organs, especially those that are herbaceous. The precise control of the pressure serves to allow for structural stability and to generate growth and movement. The technical principle behind most growth processes in plant cells is an irreversible increase in cell volume driven by water uptake. This water influx is driven by a difference in the water potential between the protoplast and the apoplast, that in turn is caused by stress relaxation in the cell wall temporarily reducing the turgor (Schopfer, 2006
). On the other hand, many plant movements are based on the ability of certain strategically located cells to rapidly increase and decrease their volume through water uptake or loss in a reversible fashion, regulated by the osmotic pressure of the protoplast. The opening and closing of stomatal guard cells and the leaf and branch movements caused by bulliform cells and pulvini are only few examples in which this mechanical principle allows movements at the individual cellular or organellar level.
Turgor and water movement therefore play important roles in various physiological functions and present an important challenge as far as quantification. Turgor has been measured with various indirect methods that are based on the quantification of the difference between the osmotic pressure of the protoplast and that of the surrounding medium, because in fully turgid cells osmotic pressure is approximately equal to the turgor pressure in the cell. Psychrometric techniques are based on the fact that the vapor pressure of water is lowered as its water potential is reduced. Psychrometers measure the water vapor pressure of a solution or plant sample on the principle that evaporation of water from a surface cools the surface (Boyer, 1995
). Many variations of the techniques exist, such as isopiestic psychrometry, in which the vapor pressure of a tissue is compared to that of a droplet of a defined solution (Nonami et al., 1987
).
Other approaches to indirectly assess turgor pressure include incipient plasmolysis, in which the osmotic potential can be quantified by balancing a tissue with a defined external solution so that it is neither turgid nor flaccid (Fig. 1G). With the incipient cytorrhysis or cell collapse technique, the same principle is used; tissues or cells are exposed to a range of external solutions with different concentrations of osmotica to determine at what osmotic value the cells collapse (Fig. 1G). This technique was used to determine the turgor pressure in the penetration organs of fungal hyphae, the appressoria, which can reach up to 8 MPa (Howard and Valent, 1996
). To put this number in perspective, an inflated car tire typically has a pressure of 0.2 MPa and household water pressure is c. 0.3 MPa. Various osmometric methods (e.g., freezing point osmometry, melting point osmometry, vapor pressure deficit osmometry) use the principle that changes in the solute content alter the physical characteristics of a solution (e.g., freezing and melting points) (Kaminskyj et al., 1992
) (Fig. 1G).
The pressure bomb is a direct method to quantify the turgor pressure in the vascular system of shoots or parts thereof. An external pressure is applied until water extrudes from the organ, indicating the pressure necessary to balance the xylem pressure (Turner, 1988
).
While providing accurate numbers, none of the aforementioned methods allowed the direct measurement of turgor pressure in real time at the level of a single cell. Direct measurements only became possible with the development of the pressure probe. The first pressure probe was a water- and air-filled glass microcapillary devised to make direct measurements of turgor in the giant-celled alga Nitella (Green, 1968
). With the application of Boyle's Law, cellular pressure was estimated based upon the compression of an air bubble. The technique was then improved by the group of Zimmermann and Steudle who replaced the air bubble with an electronic pressure sensor attached to an oil-filled capillary (Fig. 1H). Their model also included a piston that allowed turgor in the cell to be varied artificially, thus allowing for the assessment of parameters such as hydraulic conductivity, cell wall volumetric elastic modulus, and the half time for water exchange. These parameters were first determined for a number of giant-celled alga such as Nitella, Chara, and Acetabularia, in which values for turgor typically ranged from 0.1 to 0.6 MPa (Zimmermann and Steudle, 1978
; Tomos, 1988
). Further improvements of the device finally allowed the measurement of much smaller cells so that higher plant cells such as stomatal guard cells could be studied (Hüsken et al., 1978
; Franks, 2003
). Values in well-watered higher plants were found to range from below 0.1 MPa to over 1 MPa. Plant cell walls therefore have to provide considerable mechanical resistance against these pressures.
While having provided important information, the pressure probe method necessitates the insertion of a micropipette into the cell; hence it is invasive and prone to artefacts. A non-invasive method of assessing turgor for a single cell was developed by Lintilhac and Wei, although its applicability is limited to surface cells with rather thin cell walls. They calibrated the cell's ability to bear a spherical load and related the load-induced deformation of its surface with the pressure inside the cell—the same principle used in the medical device that measures intraocular pressure (Fig. 1I). Pressure is quantified by calibrating the size of the contact area between the deforming glass sphere and the underlying cell and can thus be assessed optically (Fig. 3). This tonometer approach has been successfully used for plant epidermal cells; if the cells are turgid, this non-invasive method produces results similar to those obtained with the pressure probe (Lintilhac et al., 2000
; Wei et al., 2001
).
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The cell wall represents the most prominent structural feature that distinguishes plant and hyphal cells from animal cells. By surrounding themselves with a more or less rigid structure analogous to an exoskeleton, plant cells generate tissues that are structurally very different from their animal counterparts. From a mechanical point of view, in plant tissues the cell wall is the dominant load-bearing feature that determines structure. Depending on its biochemical composition, the cell wall can be a rather rigid shell or a pliable and expandable coat. Two mechanical strategies can be identified. (1) The ability of the cell wall to resist tensile stress allows for the establishment of a turgor-based hydroskeleton and thus the generation of hydraulic erectile forces that are able to withstand applied stresses. This principle is predominantly realized in cells with primary cell walls. (2) Secondary cell walls typical of sclerenchymatic tissue are equipped to not only resist tensile, but also compressive stresses. In this case, the living protoplast and thus the turgor is dispensable for the cell's mechanical functioning and fully differentiated cells are often devoid of any cell component other than the cell wall.
Mechanical tests of cell walls have been carried out on various levels of organization: tissue, cell and molecule. Most studies used whole plant tissues, which are easy to handle, but the underlying cellular structure precludes direct interpretation in terms of cell wall material properties (Cosgrove, 1989
, 1993
; Kutschera, 1996
). On the other end of the scale, individual components of the plant cell wall have been purified or synthesized to measure their respective biochemical and biomechanical properties in vitro. The mechanical properties of those components that in isolated form are of commercial interest, such as cellulose and pectin, have been especially well characterized via tensile or compression tests or rheometric assays (Jarvis, 1984
; Endress, 1996
; Chanliaud and Gidley, 1999
; Edge et al., 2000
). Depending on the character of the material and the physical property to be assessed, the type of the test differs. In tensile and compression tests, typically applied for the crystalline component cellulose, blocks or strips of the purified compound are clamped into an extensiometer and subjected to a deforming stress that stretches or compresses the object. The reaction of the specimen block is quantified and allows conclusions on its physical properties such as the Young's modulus of elasticity.
Viscoelastic materials such as pectin gels are commonly tested with rheometers, that measure the viscosity of the substance, by plunging an object into the substance or by subjecting the material to an oscillating shear stress. This last method is also known as small deformation oscillatory rheology. A layer of the test substance is placed between two plates, one of which applies sinusoidal strains to the material. The movement transferred to the opposite plate is then registered and the behavior characterized (Endress, 1996
; Whitney et al., 1999
). Key parameters obtained by this method include the in-phase (elastic or storage) component of the shear modulus and the dynamic viscosity.
To physically test cell walls in their natural state, large deformation tensile testing is generally performed by measuring the deformation of isolated cell walls or entire tissue blocks in response to forces applied externally, such as by extensiometers or instron-type stress–strain analyzers (Cosgrove, 1989
, 1993
; Kutschera, 1996
; Schopfer, 2006
). These are based on the same principle as the tensile tests for cell wall components in vitro. Although such measurements can give answers concerning mechanical properties, the extensions measured cannot always directly be related to the growth of living cells in a quantitative way because the applied stress in most cases is unidirectional, whereas the in vivo stress in the cell wall due to turgor pressure is multidirectional.
While approaches to studying cell wall components in vitro or entire cell walls in the form of tissue blocks have provided important information, quantitative physical data obtained for individual living cells are scarce. There are several reasons for this lack. Most plant cells are present in the form of aggregates forming a tissue. Isolation of a single cell from such a tissue almost inevitably disturbs the cell wall structure, even though some authors have succeeded in minimizing this effect by mechanically peeling away individual cells instead of chemically macerating them (Keckes et al., 2003
). Secondly, while it is relatively easy to clamp a piece of tissue for a creep or bending experiment, the same manipulation at a cellular level is not trivial, simply because of the size of the specimen. The development of micro- and nanomanipulation methods has therefore been an important step to obtain information at this scale. Some cells are big enough, however, to allow for manual excision of strips of cell wall that can be clamped into an extensiometer. An example is Chara, for which a detailed multi-azimuthal description of the anisotropic elastic modulus of the walls has been produced (Wei et al., 2006
).
Another strategy to apply stress on cell walls of individual cells is their compression with a micromanipulation probe. This technique has allowed the estimation of the Young's modulus of their cell walls (Wang et al., 2004
). The authors placed living tomato cells grown in suspension culture into a device that compressed the entire cell between two plates (Fig. 1J). Using a theoretical model for the force-deformation data, they were able to analyze the physical properties of the cell wall. A similar principle was used with a displacement transducer to obtain the volumetric elastic modulus of the cell walls of the huge Chara internode cells (Steudle et al., 1982
).
Another method that has been used to measure the elastic modulus of the cell wall in living cells is the turgor pressure probe. As turgor changes, the cell volume will change by an amount determined by the volumetric elastic modulus. The elastic modulus is therefore measured by determining the relative change in volume (measured by microscopy) in response to a change in turgor applied by the pressure probe (Tomos and Leigh, 1999
).
A different concept lies behind nano-indentation and modulation force microscopy (an application of the AFM) for the quantitative assessment of the physical properties of plant and fungal cell walls. As mentioned earlier, the principle of such a technique is the application of a cycle of unloading and loading in the form of a minute, local deformation and the monitoring of the elasticity and plasticity response of the deformed material (Figs. 1C, 1K). Only very few applications have been published on living plant and hyphal cells so far. Among these is an AFM study of the elastic properties of the cell wall of fungal hyphae (Zhao et al., 2005
). AFM has mostly been applied to dead plant cells such as wood fibers. Given that wood fibers do not contain a living protoplast at maturity, the authors were able to cut open the cells horizontally and scan the section by applying local deforming forces parallel to the cell surface. This method allowed them to assess the elastic properties of the individual cell wall layers in the fiber cell wall (Wimmer and Lucas, 1997
; Wimmer et al., 1997
; Clair et al., 2003
) (Fig. 4). The mechanical properties of wood fiber cell walls were also assessed using tensile forces in combination with x-ray analysis of the microfibril angle (Keckes et al., 2003
).
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, b
). While this device delivers deformations with higher amplitudes and on a larger surface than does an AFM tip, they are still small enough for spatial resolution of these cylindrical cells (Fig. 1D). The experiments revealed that the non-uniform distribution of various cell wall components and their configurations are in part responsible for the characteristic gradient of physical properties along the longitudinal axis of pollen tubes. GROWTH AND INVASION: GENERATION OF CELLULAR FORCES
Although the study of cellular architecture means understanding a relatively static structure, cells are by no means motionless. They are able to move, change their shapes, form extensions, and divide. In all of these activities, cellular forces are involved in generating the necessary changes in cellular structure. In mammalian cells, contractile movements such as cell crawling have been measured using ruffling tests, that are based on the ruffling of a substratum caused by the traction forces exerted by attached cells (Oliver et al., 1999
). The forces generated by the cells can be calibrated by measuring the distortions caused by microneedles with known bending constants. These experiments have demonstrated the central role of the cytoskeleton in generating the forces necessary for cellular movements in animal cells (Condeelis, 1993
; Oliver et al., 1994
).
Plant cells rarely contract or move in an amoeboid manner, but they do share with animal cells an ability to grow, change shape, and form cellular protrusions. In animal cells, the formation of cellular protrusions is powered by the cytoskeleton, either by way of motor proteins (myosin, kinesin) (Ishijima et al., 1991
) or by the polymerization of the cytoskeletal elements proper (Theriot, 2000
). It was therefore important to quantify the propulsion and tensile forces that can actually be generated by the assembly (or disassembly) of individual cytoskeletal elements (Cooper, 1991
). Such studies have been performed in vitro on microtubules that were assembled from purified monomers and made to interact with flexible or rigid barriers (Fig. 5A). The subsequent buckling of the rod-shaped microtubule polymers was then analyzed to determine the force on individual microtubule ends (Dogterom and Yurke, 1997
). These studies have helped us to understand the mechanical function of pushing and pulling forces exerted by microtubules in the positioning of chromosomes and in generating force at the cell cortex (Dogterom et al., 2005
).
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). Whether or not the cytoskeletal elements contribute more directly by providing propulsive forces is very poorly understood even though speculations for possible mechanisms were put forward (Money, 1997
). These speculations were based on the finding that almost turgor-less cells were able to grow. In most situations, however, growing cells are turgid, and it is difficult to envisage that cytoskeletal polymerization forces play a significant mechanical role in this process. Instead, the mechanics of plant cell growth is presumed to be based mainly on an increase in the extensibility of the cell wall, which temporarily reduces turgor, thus causing water uptake that leads to an increase in cell volume (Cosgrove, 1993
; Kutschera, 2000
; Schopfer, 2006
). Monitoring of osmotic and turgor pressures and the quantification of local cell wall extensibility are therefore important approaches for the study of cellular dynamics and morphogenesis in plants.
In many cases, not only do the propulsive forces driving cellular expansion have to act against cellular limitations such as the plasma membrane and/or the plant cell wall, but the cells also have to grow against mechanical resistance generated by the surrounding substrate. In no cell type is this more obvious than in tip-growing cells that typically invade relatively solid substrates: root hairs in the soil, pollen tubes in the transmitting tissue, and hyphae in a nutritional substrate. While the cells often use chemical means such as secreting enzymes or inducing cell death (in the case of the transmitting tissue) to soften the growth substrate, unless these complete liquefy the substrate, the cells will likely meet substantial mechanical resistance that has to be overcome. Nowhere is this performance more impressive than in the hyphal appressorium. This specialized structure differentiates at the end of the fungal germ tube and penetrates mechanical obstacles such as epidermal plant tissue covered by thick cuticles or mammalian skin tissue (Ravishankar et al., 2001
; MacDonald et al., 2002
).
Attempts to measure the penetration force exerted by fungal appressoria have been made using various techniques (Bastmeyer et al., 2002
). In very early studies Miyoshi (1895)
allowed the penetration peg of the appressorium to perforate gold membranes (chosen because gold is inert and thus not prone to enzymatic digestion) and compared this with the forces necessary for a hyphal-shaped glass needle to breach the membrane. More recently, Bechinger et al. (1999)
used an optical waveguide for the same purpose (Fig. 5B). The principle of this technique relies on total internal reflection that is realized in structures such as glass fibers and allows light propagation. The intensity of reflected light depends on the precise thickness of the waveguide, and deformations caused by the penetration peg pushing against it can thus be detected and quantified. Provided that the elastic constants of the waveguide are known, the force exerted by the penetration peg can be calculated. Unfortunately, this technique necessitates tight adherence of an appressorium to the waveguide and will thus be of limited use for other types of tip-growing cells.
A different method was therefore used to quantify the invasive forces produced by hyphae that do not form penetration pegs. The group of Money (2001)
placed a miniature strain gauge in front of a growing hyphal apex and recorded the force exerted by the advancing cell. When the hypha contacted the beam, the electrical output from the strain gauge immediately changed in proportion to the applied force. The pressure exerted by the cell was then derived from dividing the measured force by the area in contact with the beam (Fig. 5C).
An indirect method to measure penetration force is the agar penetration assay that has been applied to both fungal hyphae (Bastmeyer et al., 2002
) and pollen tubes (Gossot and Geitmann, unpublished manuscript). The capacity of tip-growing cells to grow within or to invade media stiffened with various concentrations of agarose allows conclusions on the role of various factors such as cell wall rigidity, turgor, or cytoskeleton for the exertion of an invasive force (Figs. 5D, 6). The use of a penetrometer, that measures the mechanical resistance of the agarose, allows quantification of the penetration force.
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The quantification of the physical properties of cells and subcellular structures represents a challenge for practical experimentation because of the small size of the specimens. A significant variety of approaches has been realized, but more sophisticated methods are certain to be developed, especially considering that in the recent years micro- and nanotechniques have become increasingly available. I hope that researchers in the field of plant cell biomechanics will take advantage of the developments made in the animal field and also pursue new experimental strategies. The presence of the plant cell wall certainly poses conceptual challenges for the application of techniques such as atomic force microscopy directed at the cytoskeletal elements. However, progress in the analysis of complex data using advanced modeling strategies and technical approaches such as optical tweezers and microrheology that circumvent the interference by the cell wall represent promising avenues for future research. These approaches will lead to a better understanding of the mechanical aspects of cell physiology such as cellular growth, mechanotransduction, and perception of mechanical and physical signals.
FOOTNOTES
2 anja.geitmann{at}umontreal.ca 
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