Departments of Biology/Biotechnology (C.S.B., P.J.W.)
and Physics (G.A.S.), Worcester Polytechnic Institute, 100 Institute Road, Worcester, Massachusetts 01609-2280
Optical
microsurgical techniques were employed to investigate the mechanical
properties of Hechtian strands in tobacco (Nicotiana tabacum) and Ginkgo biloba callus cells. Using
optical tweezers, a 1.5-µm diameter microsphere coated with
concanavalin A was inserted though an ablated hole in the cell wall of
a plasmolyzed cell and attached to a Hechtian strand. By displacing the
adhered microsphere from equilibrium using the optical trapping force,
the tensions of individual strands were determined. Measurements were
made using both normal and cold-hardened cells, and in both cases, tensions were on the order of 10
12 N. Significant
differences were found in the binding strengths of cold-hardened and
normal cultured cells. An increased number density of strands in
cold-hardened G. biloba compared with normal cultured
cells was also observed. Although no Hechtian strands were detected in
any Arabidopsis callus cells, strands were present in leaf epidermal
cells. Finally, the movement of attached microspheres was monitored
along the outside of a strand while cycling the osmotic pressure.
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INTRODUCTION |
Following plasmolysis of plant cells, it is often possible to
detect thread-like strands connecting the cell wall to the plasmalemma, as first described by Pringsheim (1854)
and Nägeli (1855). Hecht (1912), however, received credit for the discovery and the filaments were subsequently called Hechtian strands. Several authors have cited
examples of Hechtian strands, since these early descriptions in various
organisms, including algae, fungi, and higher plants (Bower, 1883;
Burgess, 1971; Goosen-De Roo, 1973; Pont-Lezica et al., 1993; De Boer
et al., 1994; Henry et al., 1996; Niu et al., 1996; Bachewich and
Heath, 1997). To our knowledge, only three reports have described
Hechtian strands in callus cells (Johnson-Flanagan and Singh, 1986;
Chang et al., 1996; Buer, 1998).
Little is known about the function, composition, or physical properties
of Hechtian strands. Their cross-sectional diameter ranges from 30 to
250 nm (Sitte, 1963; Oparka et al., 1994; Bachewich and Heath, 1997)
and the length varies with the degree of plasmolysis and location
within the individual cell. Oparka et al. (1994) suggested the strands
conserve excess plasmalemma during plasmolysis. Using a fractal
analysis with a time-dependent fractal dimension, they showed that
Hechtian strands could account for the loss of surface area of the
plasmalemma as plasmolysis occurs. On the other hand, Pont-Lezica et
al. (1993) suggested two mechanical functions of the strands:
maintenance of cell polarity by limiting slippage of the cytoplasm
against the cell wall, and organization of deplasmolysis events
providing an orderly restoration of cell function.
The strand's outer layer is plasmalemma, as seen by transmission
electron microscopy using phosphotungstic acid (Sitte, 1963; Oparka et al., 1994; Bachewich and Heath, 1997). Some authors (Drake et
al., 1978; Attree and Sheffield, 1985; Chang et al., 1996) have
observed esterase activity within the strands using fluorescein
diacetate (FDA), suggesting that they contain cytoplasm. Oparka et al.
(1994), however, did not detect esterase activity in the strands of
onion epidermal cells. 3,3'-Dihexyloxacarbocyanine iodide
[DiOC6(3)] fluorescence suggested that
the interior of the strands is ER (Quader and Schnepf, 1986; Oparka et
al., 1994). This fluorochrome is a known marker for mitochondria and ER
in animal cells (Terasaki and Reese, 1992). Staehelin (1997) reviewed the functional domains of the ER and described a plasmalemma
"anchoring domain." These sites have been shown to resist
plasmolysis and remain coupled to the cell wall during the formation of
Hechtian strands (Gunning and Steer, 1996; Oparka et al., 1996). Hepler et al. (1990) found no direct adhesion of the ER to the plasmalemma in
lettuce using electron microscopy, although very close associations were present.
Schindler et al. (1989) hypothesized that wall-to-membrane linker
proteins (integrin- and vitronectin-like) were binding sites of the
plasmalemma to the cell wall. Canut et al. (1998) showed that
RGD-containing peptides inhibited Hechtian strand formation in onion
epidermal cells and altered the shape of the plasmalemma in plasmolyzed
Arabidopsis callus cells. Others have suggested that motor proteins
associated with the ER (actin filaments, Knebel et al., 1990, and
Lichtscheidl et al., 1990; myosin, Liebe and Quader, 1994) may
also provide adhesion points and subsequent Hechtian strand formation
(Gunning and Steer, 1996).
Particularly interesting to our study were inferences that Hechtian
strands undergo changes during cold hardening. Scarth (1941) and
Johnson-Flanagan and Singh (1986) noted that the strands in unhardened
cells broke more easily and were less numerous than in hardened cells.
Their results suggested that strands become more elastic following cold acclimation.
A complete understanding of cold acclimation and freezing tolerance is
lacking despite several decades of research. The induction of freezing
tolerance and its loss during warming varies substantially between
species, complicating this knowledge. Cold acclimation is necessary for
cold-hardy plants to survive freezing temperatures. Several changes in
physiological characteristics occur during the acclimation process (for
recent reviews, see Alberdi and Corcuera, 1991; Hughes and Dunn, 1996;
Li and Chen, 1998). For example, cold-responsive genes are activated
(Thomashow, 1998), plasmalemma lipids show increased levels of
unsaturation after growth at low temperatures (Tokuhisa et al., 1998),
and the lipid composition and the ratio of lipid to protein in
membranes change (Hughes and Dunn, 1996). Plants that can cold harden
generally avoid membrane destabilization because changes in the lipids
of the plasmalemma increase the cryostability of the cell (Uemura and
Steponkus, 1998). A working hypothesis is that lipids in cold
acclimated cells provide more elasticity to the plasmalemma, thereby
allowing it to withstand rehydration during thaw cycles (Alberdi and
Corcuera, 1991).
The time scale to achieve maximum hardiness varies between species. In
spinach, a maximum freezing tolerance (approximately 
18°C) was
achieved after 1 to 3 weeks of low temperature exposure, whereas
deacclimation was completed within 1 week (Guy, 1990). Hedera
helix required 6 weeks to reach maximum cold tolerance and
deacclimated in 1 week. Locust began achieving tolerance in August and
reached maximum cold tolerance by mid-November in a natural setting in
Japan, whereas a loss of tolerance took 2 months (Guy, 1990). The most
rapid change has been shown to occur in as little as 1 d after low
temperature exposure in Arabidopsis (Gilmore et al., 1988).
Using laser microsurgery and optical micromanipulation techniques, we
have, for the first time to our knowledge, determined the elastic
properties of Hechtian strands in hardened and unhardened cells. By
adhering a microsphere to a strand and displacing the strand using
"optical tweezers" (Ashkin et al., 1986), one may measure the
tension and spring constant of a strand. Optical tweezers use laser
light to produce non-contact forces in the piconewton range, and are
capable of positioning small particles with submicrometer accuracy (for
reviews, see Svoboda and Block, 1994; Hoffmann, 1996; Sheetz, 1998).
Intracellular forces have previously been indirectly measured using
organelles trapped in a focused laser beam (Ashkin and Dziedzic, 1989;
Grabski et al., 1994; Schindler, 1995; Felgner et al., 1996), but the
force was not calibrated.
Based on earlier reported lipid changes in cold-hardened cells, we
expected to find differences between the elastic properties of the
strands in our investigation: callus cells of a non-hardy species,
tobacco (Nicotiana tabacum), and a cold-hardy species, Ginkgo biloba. Surprisingly, we found no significant
difference in the spring constant, which was in the range of
k = 107 N
m1, of either species after exposures to cold
temperatures for prolonged periods of time. However, we did measure a
marked increase in the binding strength between microspheres coated
with concanavalin A (Con A) and strands following cold acclimation:
14% for N. tabacum and 31% for G. biloba.
Similar to results from Scarth (1941) and Johnson-Flanagan and Singh
(1986), we detected an increased number density of strands in
cold-hardened G. biloba compared with the other species we
studied. In addition to our measurements of tension and binding
strength, we developed a means to observe physiological dynamics of a
strand during deplasmolysis/replasmolysis cycles by monitoring multiple
microspheres attached to individual Hechtian strands.
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MATERIALS AND METHODS |
Plant Cell Culture
Ginkgo biloba callus, initiated from a seedling
hypocotyl, was grown according to the method of Carrier et al. (1990)
and Johnson (1994) in liquid Murashige and Skoog Minimum Organics (MSMO) basal salts (Linsmaier and Skoog, 1965) supplemented with Suc
(3%, w/v) and agitated at 100 rpm on a rotary shaker. Kinetin (1 mg
L1) and naphthylacetic acid (NAA) (2 mg
L1) were added as plant growth regulators, and
the pH was adjusted to 6.0 with 2 M NaOH before autoclaving.
Tobacco (Nicotiana tabacum) callus was maintained on
semi-solid (Gelrite, Adams Scientific, West Warwick, RI, or Phytagel, Sigma Chemical, St. Louis; 0.4%, w/v) Murashige and Skoog (MS) basal
salts medium (Murashige and Skoog, 1962), supplemented with Suc (3%,
w/v) and casein hydrolysate (1 g L1). Kinetin
(0.2 mg L1) and indole-3-acetic acid (IAA) (2 mg L1) were added as growth regulators. The pH
was adjusted to 5.8 before autoclaving.
Arabidopsis ecotype Columbia callus was initiated from seedlings and
maintained in culture according to the methods of Akama et al. (1992).
Hechtian strands were observed in cells of epidermal peels of the
oldest leaves from 10-d-old seedlings.
All plant tissues were grown under continuous cool-white fluorescent
lights (20 µmol m2
s1) at 25°C. Hechtian strands were found most
readily in actively growing cells, so all tissues were subcultured at
2- to 4-week intervals. Cold-hardened cells were grown at 25°C for 1 week following subculture and then transferred to and maintained at
5°C ± 2°C at similar light levels for a minimum of 1 week
before experimentation.
Visual observations of cytoplasmic streaming and
plasmolysis/deplasmolysis were used to determine cell viability. Only
living cells are capable of cytoplasmic streaming and can deplasmolyze from a plasmolyzed state (Lee-Stadelmann and Stadelmann, 1989; Kuroda,
1990).
Callus cell plasmolysis was accomplished using a step gradient varied
from 0.1 
0.2 0.3 0.4 M NaCl with a minimum of 5-min (usually 10 min) intervals between steps. All concentrations of
the step gradient also contained 10 mM Tris (pH 7.2), 10 mM CaCl2, and 10 mM
MnCl2. Plasmolysis/deplasmolysis cycle
experiments were performed using a smaller step: 0.4 0.35 0.3 M NaCl.
Microsphere Insertion
The optical system (Fig. 1),
included a pulsed (30 Hz), seeded Nd:YAG laser (model GCR 170, Spectra-Physics, Mountain View, CA) with second and third harmonic
generation capabilities (for details, see Buer et al., 1998). The
stationary fundamental Gaussian beam (1064 nm, long pulse mode,
200-µs duration) was used for trapping polystyrene microspheres, and
the frequency tripled beam (355 nm, Q-switched, 6-ns duration pulse)
was used to ablate a hole through the cell wall. The laser beams were
collimated with uncoated lenses (BK-7 glass), directed through a series
of beam steering prisms (BK-7 glass), and focused with a ×100
(numerical aperture 1.3) oil immersion microscope objective (Neofluar,
Carl Zeiss, Thornwood, NY) to a nearly diffraction-limited spot (about 1 wavelength in diameter). The objective was also used to image the
working area onto a CCD camera, and provided a depth of field of
approximately 1 µm. A beam stop was used to prevent the simultaneous illumination of the sample at both wavelengths. Optical trapping and
ablation operations were performed following the method of Buer et al.
(1998) using a flow chamber (Walcerz and Diller, 1991) to immobilize
the cells and allow variable osmotic concentrations.
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Figure 1.
Schematic of the optical system. A Nd:YAG laser
supplies a Q-switched UV beam for ablating a hole through the cell wall
and a long-pulsed infrared beam for optical trapping of polystyrene
microspheres. The beam is directed to a ×100 microscope objective, and
the beam forms a focal spot within a chamber containing callus cells.
Backlighting the chamber allows the objective to image the trapping
region onto a CCD camera.
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Preparation of Con A-Coated Microspheres
Microspheres were surfactant-free, carboxylated white polystyrene
latex of diameter (D) = 1.50 ± 0.08 µm (P/N
7-1600, Interfacial Dynamics, Portland, OR). Even though the particles
are charged, the hydrophobic latex binds strongly to hydrophobic
molecules. The negative surface charge is pH dependent (stable at
neutral to high pH). The microspheres were coated with Con A (type V, product no. C7275, Sigma) following the protocol of Kucik et al. (1991). Microspheres (100-µL suspension) were transferred into 1 mL
of Con A (2 mg mL1 in 200 mM Tris, pH 7.2), shaken on ice for 3 h,
centrifuged (10,000g, 3 min), resuspended in bovine serum
albumin (BSA) (5 mg mL1 in 200 mM Tris, pH 7.2), and washed twice by
centrifugation. The microspheres were resuspended in a 10-mL solution
of 0.4 M NaCl, 10 mM Tris,
pH 7.2, 10 mM CaCl2, 10 mM MnCl2
(Ca2+ and Mn2+ are
essential cofactors for Con A activity; Goldstein and Poretz, 1986),
and calcofluor (0.01%, w/v, De Boer et al., 1994), resulting in a
final concentration of approximately 2 × 1010 microspheres mL1.
The microspheres and Con A solution were stored at 4°C. Freshly coated microspheres were prepared for each day's experiments. Unused
Con A was stored desiccated below 0°C. Negative controls (uncoated
and BSA-coated microspheres) verified that Con A was the binding factor
to Hechtian strands.
Calibration of Trapping Force
The effective trapping force exerted on a microsphere may be
determined by calibrating the system against a viscous drag force (Wright et al., 1994). We used a computer-controlled actuator (StepperMike, model 18510, Oriel, Stratford, CT) to accelerate (a 
1 µm s2) the entire
trapping chamber (including the fluid within it) so that a trapped
stationary particle would experience viscous drag from the moving
fluid. To minimize vibrations, a compact translation stage assembly was
used. Microscope chambers (CoverWell PCI-0.5, Grace Bio-Labs, Sunrise,
OR) were used instead of the massive flow chamber. A no. 11/2
coverslip (25 × 25 mm) was used to seal the top of the microscope
chamber and allow light transmission. The critical velocity at which
viscous drag overcomes the trapping force
(vcr) was obtained from the actuator
controller display. The maximum effective trapping force
(Ftrap = Fdrag) was calculated from Stokes'
Law: Fdrag = 6
µRpvcr,
where µ is the viscosity of the surrounding aqueous solution
(approximately 1.20 × 103 Ns
m2 for 0.4 M NaCl,
20°C; Munson et al., 1994), and Rp = 1/2D = 0.75 ± 0.04 µm is the radius of
the microsphere. This experiment was repeated at least 20 times at a
given laser beam energy (Ebeam) to
obtain an average relation with Ftrap,
resulting in a precision of ±15% (Fig.
2). The value of
Ebeam was varied from the trapping threshold of 50 µJ to 350 µJ (or until damage occurred). The
calibration differs with trapping depth due to optical aberrations. To
account for the decreased trapping strength with increasing depth, we made measurements at depths of 20, 40, 60, and 80 µm below the coverslip. At depths <20 µm boundary effects perturb the calibration (Mehta et al., 1998), and at depths exceeding 80 µm we were unable to
effectively trap a microsphere with the available energy of our laser.
The microspheres were optically damaged at energies exceeding 250 µJ
when the trapping depth was less than 40 µm.
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Figure 2.
Calibration of average trapping force versus
energy in the focal plane for 1.5-µm diameter Con A-coated
polystyrene microspheres using Stokes' Law following the method of
Wright et al. (1994). Error bars are the SE of the mean of
at least 20 separate trials in 0.4 M NaCl, 10 mM Tris, pH 7.2, 0.01% (w/v) calcofluor, 10 mM
CaCl2, and 10 mM MnCl2.  , 20 µm depth;  , 40 µm depth;  , 60 µm depth; ×, 80 µm depth.
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The microscope objective in our apparatus (Fig. 1) serves two purposes:
to form the optical trap and to image microscopic features onto a video
camera. The video images of a microsphere attached to a Hechtian strand
(Fig. 3), therefore, shows the
perspective in the reference frame of the trapped microsphere. The
values of displacement and strand length, 
x and
L, respectively, were determined after calibrating image
distances with 10-µm cross-hairs on the microscope objective. For
example, a distance of 1 mm on the television monitor corresponded to
0.2 µm at high magnification. A precision of approximately ±0.1 µm
was achieved for transverse distances. A video cassette recorder (model
SLV 660HF, Sony, Park Ridge, NJ) was used to store the images on
videotape at the standard rate of 30 frames s1,
and video play-back allowed us to perform frame-by-frame analyses. Measurements were made at chamber temperatures of approximately 20°C
and approximately 10°C for normal and hardened cells. To prevent
water condensation on the trapping chamber, the coverslip was wiped
with immersion oil, and the room temperature was lowered to
approximately 15°C. All trapping measurements were repeated at least
five times to obtain reliable average values.
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Figure 3.
Videotape frames showing a Hechtian strand (with
an attached 1.5-µm diameter polystyrene microsphere) between the cell
wall (CW) and plasmalemma (PM) of a plasmolyzed Ginkgo
biloba cell. A, Equilibrium position (no displacement) showing
the relative position of the strand (dashed black line) with a
convenient chloroplast (c). Strand displaced by a
distance, x (B, dashed white line) compared with the
equilibrium position (A). The ablated hole though which the microsphere
was manipulated is marked in B (arrow). Bar is 5 µm.
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Number Density Determination
The selected cells used in all parts of this study were: (a) cells
that were adequately isolated from the callus mass (to provide
sufficient back-lighting, allow unobstructed ablation, and facilitate
insertion of microspheres), (b) cells that could be plasmolyzed to
approximately 70% of the original volume (at 0.4 M NaCl),
and (c) cells that remained 
1 h in the plasmolyzed state (assumed to
have reached a stable condition).
The mean number density was determined by counting the number of
observed strands in an optical section under high magnification, and
dividing by the arc length of the observed region of the cell wall and
the apparent depth of field (estimated to be 1 µm). We averaged over
strands of any length and over repeated measurements at different focal
depths. Unresolved strands probably did not exist, otherwise we would
have encountered invisible structures blocking the motion of the
optically trapped microspheres as they were moved throughout the
plasmolysis-induced void space (PVS).
Assays of Strands for Cytoplasm, ER, and F-Actin
Prior to all assays, callus cells of N. tabacum and
G. biloba were plasmolyzed to 0.4 M
NaCl using the normal step gradient described above to yield
well-defined Hechtian strands. FDA (Fluka Biochemika, Buchs,
Switzerland) at the same concentrations used for cell viability
(Widholm, 1972) was used to test for cytoplasm in Hechtian strands.
Cells were illuminated with the 488-nm line of an
Ar+ laser. Blue light was blocked at the CCD
camera (Fig. 1) with a 3-69 glass filter (Corning Glass, Corning, NY)
to allow the detection of fluorescence from the cells.
DiOC6(3) (1 µg mL1,
Sigma) was used to determine the presence of ER in Hechtian strands
according to the method of Quader and Schnepf (1986). Cells were
illuminated with 488 nm of light from the Ar+
laser and viewed at ×2,000 magnification. Cells were assayed 0.5 h
following the introduction of DiOC6(3).
N-7-nitrobenz-2-oxa-1,3-diazol-4-yl phallacidin (NBD-ph)
(Molecular Probes, Eugene, OR) was used to test for the presence of
F-actin in Hechtian strands following the protocol of Barak et al.
(1980) for living cells; 10 mg mL1
p-phenylenediamine was added to reduce photobleaching
(Heath, 1987). After incubation (1 h), fluorescence was initiated
with the 488-nm line and blue light was blocked as previously described.
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RESULTS AND DISCUSSION |
Tension in the Hechtian Strands
To determine the elastic properties of a Hechtian strand, we
performed a classic measurement similar to plucking a string (Fig. 3).
A microsphere was attached at the center of the strand and slowly
translated perpendicular to the strand axis. At the position where the
tension in the strand equals the trapping force, the microsphere is
pulled out of the optical trap. We assume the strand obeys Hooke's
Law, F = k x, where F is the
restoring force of the strand and is equal in magnitude to the maximum
transverse trapping force when x = xmax, and where
xmax is the displacement from the
equilibrium position when the microsphere is pulled out of the trap,
i.e. Ftrap = k
xmax. Once released from the trap, the strand
retracts to its equilibrium position without oscillating. This maximum
displacement was measured over a range of laser energies to
determine the average value of the spring constant (k) for a
given strand of length, L (measured from a center point on
the cell wall to a center point on the plasmalemma). Experimentally, we
find xmax 
L, and
thus Hooke's Law is expected to be valid. Since the strand is in
equilibrium, the tension (T) must balance the applied force
and, thus, we find T = 1/4kL.
For simple elastic bodies, k may be expressed in terms of
the elastic modulus (E) k = 12A2E/L3, where
A is the cross-sectional area of the strand (Landau and Lifshitz, 1959; Kelly, 1993). The longer the strand of a given elasticity, the easier it is displaced from equilibrium. Hechtian strands, however, are not simple bodies. For example, the strand may
lengthen by adding more mass or by stretching as the cell plasmolyzes.
We assume the main source of strand tension is the differential
pressure across the plasmalemma (P) resulting from the
cell's effort to make osmotic adjustments. Although we recognize that plant cells have irregular shapes and do not exhibit uniform
plasmolysis, the essential physics may be understood by developing a
model based on a spherical callus cell. The tension may be expressed as
T = 
1 P
(1 L/R0)2,
where R0 is the radius of the cell and
is the number of strands per unit area along the cell
wall. As the value of L approaches R0, one may expect that strands will
break, and thus the tension in each remaining strand will increase to
balance the pressure. This will also occur if the strands merge
together. Assuming a simple linear decrease in the strand
population, = 0(1 L/Lcr), where
0 is the initial strand density and
Lcr is a material-dependent parameter
that characterizes the critical distance at which no strands remain
(due to breakage or coalescing), we find that the tension,
may either increase or decrease with L, depending on
the value of Lcr. When
Lcr < R0
the tension increases to infinity at L =
Lcr, and the tension increases
monotonically (i.e. dT/dL > 0) if
Lcr < 1/2R0; otherwise, the tension
initially decreases for small values of L, and then
increases monotonically. Thus, one may expect the observed tension to:
(a) increase rapidly with strand length in cells containing easily
broken or merged strands; (b) remain relatively constant in cells with
moderate resistance to coalescing and breakage; and (c) slowly decrease
for short strand lengths and rapidly increase for long strand lengths
in cells having strong strands that do not merge.
Experimentally, the tension is determined by evaluating the expression,
T = 1/4kL, where k = Ftrap/xmax,
Ftrap is known from the beam energy
and the calibration curves (Fig. 2), and
xmax and L are measured
from the microscope images. At the upper range of values of the applied
trapping force, the microsphere would sometimes detach from the strand.
Furthermore, attached microspheres easily detach if displaced parallel
to the strand, and thus direct measurements of the Young's modulus
were not possible using optical tweezers.
The determined values of k and T are shown in
Figures 4 and
5, respectively, for normal and
cold-hardened cells. Surprisingly, the average values of the spring
constants for G. biloba and N. tabacum, shown in
Figure 4A, were statistically indistinguishable, and the spring
constants did not change significantly after cold hardening.
Measurements taken at low (10°C) and room (20°C) temperature were
also indistinguishable. Even within the same cell, significant differences in tension were found for different strands (data not
shown). We attribute this variation to: (a) naturally occurring differences between strands (such as the strand length and diameter), and (b) relaxation of the tension over time due to natural
deplasmolysis. Averaging the mean spring constants gave a value
kavg = 2.7 × 107 N m1 and a
SD of ±10%.
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Figure 4.
Measured values of the spring constant
(k) when a Hechtian strand of length L is
transversely displaced from equilibrium. Measurements were made at room
temperature (RT) at approximately 20°C (circles) and low temperature
(LT) at approximately 10°C (diamonds). Values of k for
normal (Norm) RT,  ; cold-hardened (CH), LT,  , and Norm LT,  ,
for tobacco, (B); Norm G. biloba (GB) RT, , and Norm
GB LT, , (C); and cold-hardened G. biloba, RT, ,
and LT, (D).
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Figure 5.
Measured values of the tension in Hechtian
strands for normal G. biloba (A), cold-hardened
G. biloba (B), normal N. tabacum (C), and
cold-hardened N. tabacum (D).
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In all cases, the tension (Fig. 5) was T 1 2 pN when L = 20 µm. These plots suggest that in
cold-hardened cells, the tension increases with strand length. This
trend was not evident in normal cultured cells because either the
effect is weaker or the range of strand lengths was too small. In
G. biloba, the ranges of length for the normal and
cold-hardened data overlap and, thus, based on our discussion above, we
conclude that strands in normal cells are less prone to breaking or
coalescing than those in hardened cells. The population of strands in a
given cell (or equivalently the population density, )
decreases more readily in cold-hardened cells. Indeed, this was
qualitatively verified by our observation that strands sometimes
appeared thicker (maybe due to finer strands joining together to yield
thicker strands, Fig. 6) when the cells were cold-hardened or when the strand length was very long.
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Figure 6.
Sequence from videotape of a normal cultured
N. tabacum callus cell showing Hechtian strand formation
during plasmolysis. A, Incipient plasmolysis at 0.3 M NaCl.
B through G, Plasmolysis with the gradual introduction of 0.4 M NaCl. Time of sequence is indicated in the upper right
corners, and strands are at small arrows. Photograph of the same cell
about an hour later showing changes in the strands (H, arrows). An
apparent strong attachment point (B-G, large arrow) has disappeared in
H. These strands were not used for tension measurements. The apparent
depth of field is approximately 1 µm. CYT, Cytoplasm; PM, plasma
membrane; and CW, cell wall. Bar is 10 µm.
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Since rapid changes can occur in cells during cold acclimation (Gilmore
et al., 1988; Guy, 1990; Zabotin et al., 1998), we also measured
transitory effects by testing G. biloba and N. tabacum strand tensions at room temperature and cold temperatures
for normal cultured and cold-hardened cells. We were unable to insert microspheres into the PVS and perform measurements on normal cultured N. tabacum at cold temperatures. This was attributed to an
unexpected phenomenon: a strong flow of liquid from the ablated hole
that persisted for hours. Normally, after ablating a hole roughly the size of the microsphere, this flow subsided after a few minutes. We
were still unable to introduce microspheres after doubling the width of
the hole. Apparently, the decreasing temperature triggers a mechanism
that boosts the pressure within the cell. Perhaps this activity is
related to our inability to maintain N. tabacum callus
cultures for more than 2 weeks at 5°C.
Strand Population Dynamics and Number Density
Hechtian strands were investigated in G. biloba,
N. tabacum, and Arabidopsis grown under normal and
cold-hardening conditions. Roughly 50% of the normal callus cells of
G. biloba and N. tabacum had detectable Hechtian
strands (Table I). Statistical analysis was performed using the Student's t test by comparing the
means of data collected and assuming a normal distribution. Neither the
normal nor cold-hardened Arabidopsis callus cells produced observable
strands at magnifications up to ×4,000 (although they were observed in
leaf epidermal cells). Remarkably, the population of cells producing
strands in cold-hardened G. biloba dropped in half to
roughly 25% (P < 0.006), whereas the decrease in
N. tabacum was not significant. Compared with normal callus,
we also found a 2.5-fold increase in the mean number density of strands in cold-hardened G. biloba (Table I), thus confirming the
work of others (Scarth, 1941; Johnson-Flanagan and Singh, 1986).
According to our discussion of strand tension above, one may expect
less tension in cold-hardened G. biloba for short strand
lengths due to the increased number of strands. Indeed, a comparison of
Figure 5, A and B, supports this expectation at L 15 µm.
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Table I.
Comparisons of the relative population, number
density, and binding force for Hechtian strands in normal and
cold-hardened cells
The total number of observed cells is shown in parentheses. Different
superscript letters indicate that a significant difference exists
between individual conditions within columns. The strand frequency
(P < 2.2 × 107) and
maximum binding force (P < 0.01) in cold
G. biloba were significantly higher compared with the other
categories. The statistical comparisons were by the Student's
t test.
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Hechtian strands became visible with the onset of plasmolysis (Fig. 6),
in contrast to some previous reports. Bower (1883) mentioned several
plant species in which no strands were visible until 30 min or more had
passed after plasmolysis. He hypothesized that individual strands
coalesced to form thicker strands, thereby making them visible. During
plasmolysis we did not observe the formation of additional strands, nor
did we observe strands merging to produce thicker strands. However,
thick, slow-moving plasmalemma sheets persisting across the PVS after
plasmolysis in cold-hardened cells were seen to merge; the mass of
these particular structures precluded their use in our measurements.
After a prolonged state of plasmolysis (>1 h), we discovered that some
strands disappeared (Fig. 6), as reported by others (Bower, 1883;
Plowe, 1931; De Boer et al., 1994). We suspect fine Hechtian strands
may merge, perhaps accounting for the apparent disappearance of thin
strands and the appearance of thick strands during prolonged
plasmolysis (Fig. 6). Strand breakage and absorption could also account
for strand disappearance. However, strands present after more than 1 h were observed to remain intact for as long as 12 h in the plasmolyzed state, suggesting that the strands had reached a stabilized state. Only stabilized strands were used in our experiments.
Earlier, Johnson-Flanagan and Singh (1986) reported using 0.2 M increments of NaCl:CaCl2 (9:1) to
minimize strand breakage in Brassica napus. We also found
that the use of a gradual step gradient minimized strand breakage,
allowing strands to remain intact in >95% of strand-producing cells.
Consequently, we used slow plasmolysis to provide the most consistent
strands for our experiments. In contrast, rapid plasmolysis (directly
increasing from 0.1-0.4 M
NaCl or a rapidly applied step gradient) caused the strands to break or
fail to be observed in most tested cells.
These results suggest that the number of Hechtian strands in a given
cell is determined before the onset of plasmolysis. This number may
decrease due to breakage or coalescence; however, to our knowledge, the
formation of additional strands has only been observed after
plasmolysis (Plowe, 1931) and then only by re-contacting the protoplast
to the cell wall with microdissection needles. Thus, the hypothesis
that Hechtian strands conserve excess plasmalemma during plasmolysis
(Oparka, 1994) is not supported by this finding. Kell and Glaser (1993)
describe an alternative mechanism for plasmalemma conservation,
endocytotic vesiculation that has been shown to occur in isolated
protoplasts. Upon deplasmolysis, incorporation of excess plasmalemma
into the expanding membrane is necessary to prevent lysis. Lysis was
never observed in our work.
Microsphere to Strand Binding Strength
Carboxylated polystyrene microspheres bind the plant lectin Con A
through hydrophobic interactions at neutral or higher pH (Interfacial
Dynamics catalog, 1997-1998). Furthermore, Con A selectively binds
sugars with the following order of affinity: Man 
-1,2-Man
-1,2-Man > Man -1,2-Man > -Man > -Gluc > GlcNAc (Goldstein and Poretz, 1986). Therefore, Con A
should bind to glycoproteins associated with the plasmalemma. Indeed,
such binding was observed, whereas negative controls (uncoated and
BSA-coated microspheres) did not adhere to the Hechtian strands or to
the plasmalemma.
To attach optically trapped microspheres to a Hechtian strand, we
positioned the microsphere above the strand and lowered the focal plane
of the beam. Once attached, the microsphere became defocused when the
trapping point was displaced from the strand. A period of approximately
20 s was often required to secure the microsphere, although on
some occasions, binding occurred immediately.
The binding force between the microsphere and the strand was determined
by increasing the optical trapping force until it had sufficient
strength to detach the sphere. This measurement was made while pulling
the microsphere perpendicular to the length of the strand. We noticed
that the microsphere could be more easily detached by translating it
along the length of the strand. However, the physical effects of a
shear force on the connection between the microsphere and the strand
are unknown. On the average, we found that cold-hardened cells had
higher binding strengths than normal cells (Table I). This difference
was particularly evident for G. biloba, in which the binding
forces increased by a factor of 1.45 ± 0.11; for N. tabacum the enhancement factor was less than the relative error.
This increase suggests a greater density of glyco-bearing binding
substances on the strands, as reported by Castonguay et al. (1998). In
some cases the sphere would not detach from the strand when the largest
optical trapping force was applied. We never observed a strand breaking
when perturbed with the optical tweezers, nor did we observe a
displacement of the cell wall or protoplast from forced displacements
of the strand.
Deplasmolysis Experiments
To explore possible relationships between the plasmalemma and
Hechtian strands, we monitored the motion of microspheres attached to
the strands during deplasmolysis/replasmolysis cycles. A video sequence
of such a plasmolysis cycle is shown in Figure
7 with two microspheres attached to the
same strand. The distance (along the strand) between the protoplast and
the stationary cell wall, as well as the distance between the centers
of the microspheres, is plotted as a function of time in Figure
8. In the latter case, a separation less
than the sphere diameter indicates that one sphere moves behind the
other. Within the first minute of deplasmolysis, the sphere distance
initially increases from 3.5 to 5.0 µm, and then decreases to an
average value of 2.7 ± 0.7 µm over the 10-min deplasmolysis
period. During replasmolysis, little relative motion of the spheres was
observed, and their separation was roughly constant, averaging 2.0 ± 0.4 µm.
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Figure 7.
Sequence from a videotape of a typical
deplasmolysis/replasmolysis experiment with 1.5 µm-diameter Con
A-coated microspheres attached to the same Hechtian strand (A, arrows).
A plot of the microsphere movement for this sequence is shown in Figure
8. Time of the sequence is in the upper right-hand corner of each
frame. A through C, 0.4 0.35 M NaCl step-gradient; D
and E, 0.35 0.3 M NaCl; F, 0.3 M, just
before introduction of 0.35 M NaCl; G and H, 0.35 0.4 M NaCl gradient. Bar is 10 µm.
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Figure 8.
Measured distances between the centers of the
attached microspheres () and the position of the protoplast (with
respect to the cell wall) as the cell first deplasmolyzes (0-610 s)
and then replasmolyzes (660-1,120 s), corresponding to Figure 7 ( ).
Varying the concentration of NaCl in steps controlled the osmotic
pressure. Distance measurements have an uncertainty of ±0.5 µm. The
mean distance between the microspheres is 2.7 ± 0.7 µm and
2.0 ± 0.4 µm during deplasmolysis and replasmolysis,
respectively.
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Individual glycoproteins are thought to move independently on the fluid
mosaic lipid bilayer of the plasmalemma (Singer and Nicolson, 1972).
This is supported by our observations in Figures 7 and 8, which
indicate that the microspheres can move independently.
Based on a series of such experiments (Table
II), we observed that the microspheres
migrated and adhered to the protoplast 43% of the time and to the cell
wall 57% of the time. In about half of the experiments
(n = 18) the strands broke before deplasmolysis was
completed. After the strand broke, the microspheres were just as likely
to move to the cell wall as toward the protoplast. In about 10% of the
cells having attached microspheres, deplasmolysis did not occur due to
cell death.
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Table II.
Microsphere movements while attached to Hechtian
strands during deplasmolysis/replasmolysis cycling experiments (n = 32)
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These results suggest that the strand contents may be absorbed by the
protoplast during deplasmolysis. In cases when deplasmolysis rates are
sufficiently slow, tension remains in the strand as it is apparently
absorbed and, thus, the protoplast eventually meets the microsphere.
When the deplasmolysis rate is too fast, the strand loses tension and
the microsphere may drift toward either the protoplast or the cell wall.
Interestingly, on one occasion we filmed the breakage of a strand
having two attached microspheres as the cell replasmolyzed. Using the
Law of Conservation of Energy, this observation allowed us to measure
the elastic energy (
) stored in the strand, since = (1/2)mv02,
where v0 = 13 µm s1 is the
initial velocity, m = (4/3)
Rp3 
is
the mass, and = 1.1 g cm3
is the density of a microsphere. The energy of this strand of length,
L = 9.8 µm, is calculated to be remarkably small:
= 3.2 × 1025 J. The magnitude of
the stored energy may provide some indication of underlying mechanical
and chemical processes. During plasmolysis, work is done (due to the
differential osmotic pressure) to lengthen the strand; however, during
deplasmolysis, the strand does not appear to experience osmotic
re-compression. It therefore requires more energy to plasmolyze
than to deplasmolyze the cell, and, thus, the deplasmolyzed state is
energetically more favorable, as expected. This suggests that the small
elastic potential energy that we measured in the broken strand serves
only to return a given area of the plasmalemma to its original location
adjacent to the cell wall.
Assays for Cytoplasm, ER, and F-Actin in Hechtian Strands
We observed DiOC6(3) fluorescence in some,
but not all, Hechtian strands of G. biloba and N. tabacum, indicating the presence of ER (data not shown). We did
not detect fluorescence in strands using either FDA or phalloidin,
indicators of cytoplasm and F-actin, respectively (data not shown).
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CONCLUSIONS |
We observed Hechtian strands in the callus cells of normal and
cold-hardened G. biloba and N. tabacum cells, as
well as in leaf epidermal cells of Arabidopsis. They were not observed
in the callus cells of either normal or cold-hardened Arabidopsis, and
only about half the cells in the other species had resolvable strands
that remained intact at least 1 h after plasmolysis. In cold-hardened G. biloba, only a quarter of the cells had
strands, although the number density was at least twice as large in
those cells (compared with normal G. biloba or either normal
or cold-hardened N. tabacum).
Using laser microsurgery, we ablated a small hole within the cell wall
of G. biloba and N. tabacum. Optical tweezers
were then used to insert 1.5-µm diameter microspheres coated with Con A into the PVS of the plasmolyzed cells. The microspheres were brought
into contact with Hechtian strands, and binding occurred within 20 s. The binding strength increased by 14% after cold-hardening in N. tabacum and by 31% after cold-hardening in G. biloba (Table I).
By pulling on a bound microsphere and measuring the displacement of the
strand from equilibrium, we determined the strand tension (Figs. 4 and
5). A theoretical model was developed to relate the tension to the
number density of strands. Comparing the data for G. biloba
with the model, we concluded that the number density of cold-hardened
strands was more likely to decrease with increasing strand length, due
to either breaking or merging, than normal strands. While the data for
cold-hardened N. tabacum also suggested a decreasing number
density with strand length, insufficient data were available to make a
comparison with normal strands. We propose that strand coalescence
provides a mechanism to maintain the integrity of the Hechtian strands
during plasmolysis (which may naturally occur when the plant
dehydrates). Given the evidence of enhanced combining of neighboring
strands and enhanced binding of microspheres to glyco-bearing
structures on the strand, we suggest further explorations to determine
whether the glyco structures are responsible for the merger.
Surprisingly, we found little difference between the spring constants
of G. biloba (an ancient gymnosperm that survives winters to
30°C) and N. tabacum (an annual non-hardy angiosperm).
According to our model, this indicates that the initial number density
of strands is roughly the same in both species. Indeed, the measured number densities were nearly identical in normal and hardened N. tabacum and normal G. biloba; however, the density was
approximately 2.3 times higher in cold-hardened G. biloba.
Due to the large variation in the data, it was not possible to
correlate this increased number density with either the strand tension
or the spring constant.
We also found that the Hechtian strands break easily under rapid
plasmolysis, and concluded that the strands are not effective at
conserving the excess plasmalemma. We never observed strands coalescing
while the cell was plasmolyzed, only plasmalemma sheets in
cold-hardened cells were observed to merge. We also did not observe new
strands being formed to replace broken ones. We observed many cells
after prolonged plasmolysis (>60 min) that had fewer strands, and
sometimes these remaining strands appeared thicker, suggesting that
merging occurs (Fig. 6).
The ability to attach microspheres to Hechtian strands adds a new
dimension to the study of plant physiology
events can be monitored
virtually in situ. Typical microinjection methods would require boring
a larger hole to allow a micropipette with a microsphere to enter a
cell without having the microsphere scraped off as the micropipette is
pushed through the wall. With optical tools, a microsphere can be
maneuvered onto a 50-nm-thick Hechtian strand that is barely resolvable
with visible optics. Using the microsphere as a physical handle, forces
of piconewton magnitudes were measured, allowing us to conclude that
there are very specific alterations that occur with cold acclimation in
the connections between the plasmalemma and cell wall.
We thank a former graduate student, K. Gahagan, now at Los
Alamos National Labs (Los Alamos, NM), for helping to develop the optical system. We also are grateful to D. Walcerz (Applied Sciences Department, York College, York, PA) for the flow chamber; G. Li (Worcester Polytechnic Institute [WPI] Physics Department) for assistance with calibration experiments; A. Walther (WPI Physics Department) for suggestions on microscopy; J. Petruccelli (WPI Math
Department) for assistance with the statistical analysis; and K. Wobbe
(WPI Chemistry and Biochemistry Department) for donating Arabidopsis
seeds and technical suggestions.
Received September 30, 1999; accepted November 15, 1999.
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ABSTRACT
INTRODUCTION