Optical tweezers for the micromanipulation of plant cytoplasm and organelles

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Optical tweezers for the micromanipulation of plant cytoplasmand organellesChris Hawes1, Anne Osterrieder1, Imogen A Sparkes1 and Tijs Ketelaar2

Laser trapping of micron-sized particles can be achieved

utilizing the radiation pressure generated by a focused infrared

laser beam. Thus, it is theoretically possible to trap and

manipulate organelles within the cytoplasm and remodel the

architecture of the cytoplasm and membrane systems. Here we

describe recent progress, using this under utilized technology,

in the manipulation of cytoplasmic strands and organelles in

plant cells.

Addresses1 School of Life Sciences, Oxford Brookes University, Oxford,

OX3 0BP, UK2 Laboratory of Plant Cell Biology, Wageningen University,

Droevendaalsesteeg 1, Wageningen, 6708PB, The Netherlands

Corresponding author: Hawes, Chris ([email protected])

Current Opinion in Plant Biology 2010, 13:731–735

This review comes from a themed issue on

Cell biology

Edited by Christian Luschnig and Claire Grierson

1369-5266/$ – see front matter

# 2010 Elsevier Ltd. All rights reserved.

DOI 10.1016/j.pbi.2010.10.004

IntroductionLaser tweezers, often known as optical tweezers or optical

traps, permit the capturing and micromanipulation of

microscopic particles along X, Y and Z axes using the

radiation pressure generated by a focused laser beam,

normally in the infrared region of the spectrum. For

trapping to be successful, the object to be captured must

have a higher refractive index than that of its surrounding

medium and forces generated by individual traps must be

in the piconewton range [1]. Single optical traps are

generally used to capture particles in the micron range,

although multiple traps have been developed that can be

utilized to move larger objects.

Optical traps are commonly used in single-molecule

techniques where, for example, they can be used to

measure forces exerted on polymer beads coated with

motor proteins. Thus, for instance, the mechanism of

kinesin walking on microtubules or myosin on actin

can be probed [2,3]. At the other end of the spectrum

the optical traps are often used for the mechanical stimu-

lation of cells [4] or manipulation of whole cells such as

germinating fungal conidia [5].

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What is, however, very apparent is that the full potential

for using optical tweezers to manipulate organelles within

living cells has yet to be fully exploited, with the majority

of reports working at the whole-cell or single-molecule

levels. Ashkin and Dziedzic [6�] were perhaps the first to

show that cytoplasmic particles can be manipulated invivo, when they demonstrated the pulling cytoplasm

strands across vacuoles of onion epidermal cells and

displacement of Spirogyra chloroplasts. Redirection of

the growth of fungal hyphae by manipulation of the

Spitzenkorper has been elegantly demonstrated through

lateral displacement of this tip organelle [7].

Recently, and often in combination with confocal micro-

scopy, it has been demonstrated that optical traps can be a

very powerful tool in unravelling cytoplasmic dynamics.

Here we discuss some of the few applications that have

revealed new physical aspects of cytoplasmic and orga-

nelle dynamics in plant cells.

Revealing cytoskeletal interactionsMature plant cells generally possess large central vacuoles

that are surrounded by a cortical layer of cytoplasm.

Strands of cytoplasm penetrate the vacuole to intercon-

nect different regions of the cortical cytoplasm and that

surrounding the nucleus. Bundles of actin filaments, over

which cytoplasmic streaming takes place, are the struc-

tural basis around which the cytoplasm is organized [8–13,14�]. Cytoplasmic organization is extremely dynamic

and cytoplasmic strands constantly reorganize, branch and

fuse with other strands. The actin filament bundling

protein villin [15] and myosin motor proteins [16] play

a role in maintaining and modifying the organization of

the cytoplasm. However, besides knowing the molecular

players, physical parameters also need to be considered in

order to fully understand how the cytoplasm is configured

[14�]. To gain insight into forces that are involved in

intracellular organization, optical tweezers are an ideal

tool and have been used to investigate the physical

properties of the plant cytoplasm in a number of studies

[6�,14�,17�,18–20].

Grabski et al. [17�] determined the tension of cytoplasmic

strands in soybean cells by measuring the success rate of

their lateral displacement at increasing tweezer intensi-

ties. Tension in these strands markedly decreased during

the application of 20 mM cytochalasin D, which depoly-

merizes actin filaments. Using the displacement assay,

Grabski et al. [17�,18–20] tested the effect of different

signalling molecules on tension in cytoplasmic strands


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732 Cell biology

and concluded that this tension in the strands, and thus of

the actin cytoskeleton, is affected by changes in cyto-

plasmic pH and Ca2+ concentration. In addition, the

phytohormones auxin and cytokinin modulate the tension

in cytoplasmic strands [18]. This clearly shows that the

actin-mediated tension in the cytoplasm is affected by

different types of signalling.

Van der Honing et al. [14�]) used optical tweezers to

manipulate the architecture of the cytoplasm by trapping

an unknown organelle in the perinuclear cytoplasm and

dragging it into the vacuolar lumen of tobacco BY-2

suspension cultured cells. During this displacement,

the tonoplast membrane deforms such that a cytoplasmic

connection with the trapped organelle remains. These

cytoplasmic connections were termed ‘tweezer-formed

cytoplasmic protrusions’. By creating protrusions in

GFP:FABD2 (F imbrin Actin Binding Domain 2) [21]

expressing cells, Van der Honing et al. [14�] determined

that actin filaments enter tweezer-formed cytoplasmic

protrusions within minutes after their formation

(Figure 1 and supplementary movie 1). Application of

the myosin inhibitor 2,3-butanedione monoxime (BDM)

showed that myosin motor activity is essential for the

entry of actin filaments into tweezer-formed protrusions

[14�]. During treatment with low concentrations

(100 nM) of the actin-depolymerizing drug latrunculin

B, conditions during which actin polymerization is prob-

ably reduced but actin filaments are still present, entry of

actin filaments into tweezer-formed protrusions was not

inhibited (Van der Honing et al., unpublished result).

Thus, myosin-motor-based relocation of existing actin

filaments, and not polymerization of actin filaments, is

likely to be the mechanism by which actin filaments enter

Figure 1

Pulling a cytoplasmic protrusion into the vacuole. Actin filaments appear with

in a tobacco BY-2 suspension cultured cell. This cell stably expresses GFP:FA

channel, and the bottom image the corresponding transmission image. The

protrusion. Bar = 5 mm.


tweezer-formed protrusions, implying that the existing

actin cytoskeleton can be reorganized by myosin motors.

In contrast to actin filament bundles that support naturally

occurring cytoplasmic strands, the entry of actin filaments

into a tweezer-formed cytoplasmic protrusion did not

stabilize the protrusion: the retraction of a tweezer-formed

strand after switching off the tweezers was not delayed after

the entry of actin filaments [14�]. When actin filaments

were depolymerized, retraction of a tweezer-formed strand

was slightly slower than in untreated cells, suggesting that

the retraction velocity does depend on the stiffness of the

cytoplasm, and not on the presence of actin filaments in

tweezer-formed cytoplasmic protrusions.

The stiffness of the cytoplasm is negatively correlated to its

deformability. To investigate the role of the actin cytos-

keleton in regulating the stiffness of the cytoplasm, Van der

Honing et al. [14�] produced tweezer-formed protrusions

using a range of trapping forces. The formation of tweezer-

formed strands was significantly easier after complete

depolymerization of the actin cytoskeleton and was more

difficult in cells treated with BDM than in untreated cells.

This suggests that the actin cytoskeleton is responsible for

maintaining the stiffness of the cytoplasm, and that myo-

sin-based sliding of actin filaments over each other is

responsible for reduction of the stiffness after tweezer-

mediated manipulation of the cytoplasmic organization.

The actin cytoskeleton not only determines the cyto-

plasmic organization, but is also involved in positioning

organelles in the cytoplasm. In growing root hairs of

Arabidopsis, the nucleus trails the growing tip at a fixed

distance of 77 � 15 mm. The position of the nucleus is

in minutes after pulling a tweezer-formed protrusion with optical tweezers

BD2, which decorates filamentous actin. The top image shows the GFP-

arrow points to actin filaments that have entered the tweezer-formed


Optical tweezers for the micromanipulation of plant cytoplasm and organelles Hawes et al. 733

maintained by the actin cytoskeleton [22]. Using optical

tweezers, Ketelaar et al. [22] investigated the importance

of nuclear positioning in root hair growth. By placing a

series of time-shared traps around the nucleolus, the

position of the nucleus could be fixed. During the first

10–15 min, tip growth continued at approximately

1 mm min�1, increasing the distance between nucleus

and tip by approximately 10–15 mm. After 10–15 min

of trapping, tip growth was arrested. This shows that

actin-based nuclear positioning is essential for tip growth.

Together, the above-mentioned data show that optical

tweezers are an excellent tool to study physical aspects of

the organization of the plant cytoplasm.

Probing secretory pathway dynamicsLaser tweezers have been used to demonstrate potential

membrane contact sites between the endoplasmic reti-

culum (ER) and chloroplasts [23]. In this study, Arabi-

dopsis leaf protoplasts expressing GFP in the ER were

ruptured by a laser scalpel. Fragments of ER that were

attached to chloroplasts could be stretched out by micro-

manipulating the optically trapped chloroplasts and forces

up to 400 pN could not detach chloroplasts from ER

tubules, suggesting the presence of physical contact sites

between the two organelles. However, such experiments

have yet to be performed within intact cells.

Attachment of the ER to another organelle has been

shown by the trapping of Golgi bodies within Arabidopsis

leaf epidermal cells [24�]. Because of the thin cortical

layer of cytoplasm, cells of the leaf epidermis are ideal

specimens for relatively high resolution confocal micro-

scopy of endomembrane organelles. Leaf epidermal cells

of Arabidopsis plants expressing both an ER targeted

construct (GFP-HDEL) and a Golgi targeted construct

(the signal anchor sequence of a rat sialyltransferase fused

to mRFP – ST-mRFP) were used for trapping exper-

iments and trapping was carried out with a Molecular

Machines and Instruments (MMI) optical trap using a

Figure 2

Optical trapping of a Golgi body in an Arabidopsis leaf epidermal cell. Part of

body (red, arrowhead) attached to the cortical ER network (green). The Golg

trailing ER tubule fuses with the anchor point, and further manipulation of the

Sparkes et al. [24�]. Bar = 2 mm.


1064 nm 3000 mW Nd:YAG laser attached to a Zeiss

LSM 510 META confocal microscope.

Both the ER and Golgi bodies in leaf epidermal cells have

been reported to have an acto-myosin-based motility [25–30]. Golgi bodies, in particular, are extremely motile and

move with, or over, the cortical network of ER tubules, and

were not successfully trapped until movement was inhib-

ited due to disruption of the actin cytoskeleton by treat-

ment with latrunculin B. This is likely to represent a

technical limitation in trapping highly motile Golgi stacks

rather than a direct effect of intact microfilaments. Remark-

ably, on lateral displacement of individual Golgi bodies by

X, Y manipulation of the trapping laser, in the majority of

cases ER tubules remained attached to the Golgi and the

tubular ER network could be remodelled at the cortex of

the cell (Figure 2 and supplementary movie 2). ER remo-

delling was not a result of trapped ER in the system. It has

to be assumed that the new ER tubule formed behind the

trapped Golgi body would be composed of existing mem-

brane components that can easily flow around the ER

network as opposed to de novo synthesis of new membrane.

This dramatic lability of the cortical ER network is well

documented, with tubule growth and streaming being

mediated by myosin XIK [31,32]. The ability of ER mem-

brane proteins to rapidly diffuse in a vectorial manner has

also been described from photoactivation experiments

[31,33]. From the trapping experiments it was concluded

that in the leaf system Golgi bodies are indeed in direct

contact with the ER, although from such experiments it

could not be concluded whether there was direct mem-

brane continuity between the two organelles.

Using a new technique termed persistency mapping,

Sparkes et al. [31] have identified motile and non-motile

subsets of the cortical ER network and from the maps

they extracted data revealing a population of non-motile

punctae that were suggested to be anchoring points of the

cortical ER tubules. Optical trapping has shown that the

manipulation of ER tubules towards such anchor points

a movie sequence showing the trapping and manipulation of a leaf Golgi

i body is pulled towards a putative ER anchor point (white arrow) and the

Golgi body results in the formation of a three-way ER branch. Image from


734 Cell biology

through Golgi manipulation (Figure 2 arrowhead) can

result in the adhesion and stabilization of the tubule at

the anchor site, permitting a change in direction to be

made to a tubule trailing behind a trapped Golgi body

(Figure 2 arrow, supplementary movie 2). To date there is

no information regarding the protein complement of the

anchor sites or on the structures to which they anchor the

cortical ER, although the best candidate is the plasma

membrane. It has, however, been suggested that ATM1, a

myosin VIII, is a good candidate for an anchor site

proteins, as fluorescent protein fusions have been shown

to locate to punctae distributed over the ER surface

[24�,34]. Manipulation of the ER network in this manner

also revealed two other features, in that three-way junc-

tions could be formed by pulling ER tubules away from

anchor points (Figure 2) and that homotypic fusion of ER

tubules can easily be induced by dragging tubules to and

across existing tubules.

Finally the Golgi trapping work demonstrated that, on

rare occasions, individual Golgi bodies could be pulled

free from their attached ER tubules and be manipulated

in the cortical cytosol [24�]. Such Golgi bodies could be

used to ‘pick up’ free ends of ER tubules, indicating that

there must be interacting molecules on the organelles,

such as the predicted ER – Golgi tethers that form part of

the complex of proteins know as the Golgi matrix [35–37].

ConclusionsAlthough the full potential of laser tweezers has yet to be

realized, what is becoming clear is that it is possible to use

laser traps in vivo within the plant cell cytoplasm. Com-

bined with the use of fluorescent protein fusions to label

individual organelles and the Z resolution offered by con-

focal microscopy, new information on cellular dynamics

can readily be obtained from relatively straightforward

experiments. Hopefully the work discussed here will help

pave the way for a whole range of investigations on orga-

nelle and organelle cytoskeleton interactions in plants.

AcknowledgementsPart of the work discussed here was supported by a grant to CH from theBiotechnology and Biological Sciences Research Council (BB/F008147/1).TK thanks Hannie van der Honing (Wageningen University) for providingFigure 1.

Appendix A. Supplementary dataSupplementary data associated with this article can be

found, in the online version, at doi:10.1016/j.pbi.2010.

10.004.

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Optical tweezers for the micromanipulation of plant cytoplasm and organelles