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Plant Physiol, January 2001, Vol. 125, pp. 119-122
Trekking along the Cytoskeleton
Chris R.
Hawes* and
Béatrice
Satiat-Jeunemaitre
Research School of Biological and Molecular Sciences, Oxford
Brookes University, Headington, Oxford OX1 3RA, United Kingdom
(C.R.H.); and Institut des Sciences Végétales, Centre
National de la Recherche Scientifique, Unité Propre de Recherche
40, 91198 Gif-sur-Yvette, France (B.S.-J.)
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MOVEMENTS IN PLANT CELLS |
For centuries, amateur botanists
with access to microscopes, and latterly plant scientists, have
marveled at the dynamic nature of the cytoplasm that is apparent in
many diverse cell types. Many teachers have relied, and still do, on
the dramatic cytoplasmic streaming displayed by the internodal cells of
various members of the Characeae family to stimulate their students'
interest in the plant world (9). Likewise, where would the study of
mitosis and cytokinesis be without the numerous films and videos of
nuclear division in Tradescantia virginiana
stamen hairs and Haemanthus (Scadoxus) liquid endosperm cells? Until the 1950s,
the light microscope was the only instrument available for such
studies. However, once the major problems associated with the
preservation of biological material by fixation were overcome, the
unprecedented resolution offered by the electron microscope resulted in
it dominating the field of microscopy for the next two decades. It is
not surprising that it was not long before all this dynamic
intracellular activity was attributed to systems of filaments and
tubules within the cytoplasm (10), the now well-characterized actin and
microtubule cytoskeletons.
Of course every major scientific advance has its Achilles' heel and
with the advent of electron microscopy it was the loss of the ability
to study living material. It is fortunate that the light microscope was
soon to be complemented by technological advances, such as the ability
to record high-resolution images generated by UV microscopy (11, 12)
and the improvement in differential interference contrast by the
application of video and computer enhancement techniques (2). This
permitted high-resolution imaging of living cytoplasm and an insight
into the dynamics of the organelles previously characterized by
biochemistry and electron microscopy. However, it has been the advances
in fluorescence technology, including development of vital stains for
organelles (17), the introduction of fluorescent proteins into cells
(fluorescent analog cytochemistry; 8), and most recently fluorescent
protein expression in cells (20) that has transformed the cell into a
miniature laboratory.
Although cytoplasmic streaming in its many forms and nuclear
division are the most obvious cytoplasmic movements, various other
cytoskeletal-controlled movements, including chloroplast alignment and rotation and nuclear positioning, are well
documented (15, 21). However, far more subtle and less-understood
organelle movements have been reported from the application of
high-resolution light microscopy.
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UV- AND VIDEO-ENHANCED MICROSCOPY |
The potential advantages of UV microscopy over conventional light
microscopy have been known for 100 years. The shorter wavelengths used
offer extremely high resolution (approximately 0.1 µm) and contrast
is easily realized due to the absorption of UV by biological material.
Back in the early 1950s, Irene Manton (14) was resolving substructure
in fern gamete flagella with such an instrument. However, there are
problems associated with the need for specialized quartz lenses and in
viewing the UV images. As a result, the technique was generally ignored
by plant microscopists until UV-sensitive video cameras became
available. With such an instrument Lichtscheidl and Url (12)
published stunning images of onion epidermal cell cytoplasm detailing
fine strands of the cortical endoplasmic reticulum (ER)
meshwork, putative actin bundles, and a range of organelles tentatively
identified as leucoplasts, mitochondria, Golgi bodies, and various
"spherosomes." Similar reports on the structure of living onion
epidermal cell cytoplasm came from the application of video-enhanced
differential interference contrast microscopy, a technique developed by
the late Robert Allen (2). It became clear that the cortical network of
ER was reasonably static and consisted of a polygonal organization of
lamellae and cisternae which was connected to a dynamic layer of ER and
the more actively streaming ER in transvacuolar strands of cytoplasm
(1). Movement of organelles and particles over this dynamic layer of ER
was reported and these were suggested to be associated with actin filaments. Treatment of the material with cytochalasin-B inhibited all
movement but appeared not to perturb the ER network. Of course, to a
certain extent the interpretation of these images, astonishing though
they are, is a matter of faith. Although the cortical ER can be
relatively easily related to electron microscope section and
negative-stained images (7), the identification of the numerous motile
particles associated with it and the cytoskeleton is more problematical.
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FLUORESCENCE TECHNOLOGY |
The development of a range of vital fluorescent probes in
combination with confocal microscopy made a major contribution to our
understanding of this putative relationship between moving organelles
and the cytoskeleton. Quader has confirmed that the onion bulb
epidermal ER, when stained with the carbocyanine dye DiOC6, is dependent on the actin cytoskeleton for
its organization, with the notable exception of the static cortical
network which appears to be associated with the plasma membrane (18).
This and other dyes (17) can also be used to image mitochondria in
streaming cytoplasm, but unfortunately for those of us interested in
the secretory pathway and the dynamics of the Golgi apparatus, the ceramide dyes so popular with the mammalian community failed to work in plants.
JELLYFISH TO THE RESCUE
It is fortunate that one of the major recent advances in cell
biology, the cloning of the gene encoding a green fluorescent protein
(GFP) from the jellyfish (Aequoria victoria), the now-famous GFP, has come to our rescue (20). It was used originally as a marker
for gene expression but it was not long before GFP had been tagged to
proteins or had short peptide-targeting sequences spliced on to it.
Some of the first reports of the use of GFP in plants used the protein
targeted to the ER by addition of a signal peptide to the N terminus
and the H/KDEL peptide retrieval signal to the C terminus. In
Arabidopsis roots the ER could be seen to be both motile (4, 6)
and labile (4) and associated with it were what appeared to be small
oval-shaped organelles. These structures, first reported by
Haseloff (6), appear to move rapidly over the ER
(http://www.plantsci.cam.ac.uk/Haseloff/IndexMOVs.html) and one
would assume that this movement is driven by the cytoskeleton. In our
laboratory we have confirmed the suggestion of Gunning that these
bodies are in fact inclusions in the ER (5; H. Zheng, I. Moore, and C. Hawes, unpublished data) and thus they demonstrate a novel level
of motility. Because they are confined by the membrane of the ER,
they must presumably interact, via yet-to-be characterized transmembrane linker proteins, with the cytoskeleton.
The structure of the cortical ER as reported by high-resolution light
microscopy and fluorescent dye staining has now been confirmed by GFP
expression in leaves (4). However, the cortical network, although less
motile than ER deeper in the cytoplasm, is in fact labile in that the
polygonal network continually changes shape and small islands of
lamellae can grow and shrink at the vertices of the polygons. Tubules
were also observed growing out of the network and fusing to other
tubules of ER (4). It is not surprising that disruption of the actin
cytoskeleton with pharmacological agents stops all such movement, but
does not radically alter the shape of the network, an indication of the
involvement of other factors, perhaps plasma membrane linkers, in ER
organization (7).
The work on tobacco (Nicotiana clevelandii) leaf ER
also reported the movement over ER tubules of small GFP-containing
structures, tentatively interpreted to be Golgi bodies (4). However, it
was not until direct labeling of Golgi with GFP was achieved that the
remarkable behavior of the individual stacks was revealed (3, 16). In
both tobacco leaf and suspension culture cells, Golgi stacks displayed
organized and rapid streaming along cytoplasmic strands. In tobacco cv
Bright Yellow-2 cells these stacks demonstrated stop-and-go
movement. By using GFP spliced to the Arabidopsis HDEL receptor
homolog (aERD2), Boevink et al. (3) were able to visualize both the ER
and Golgi in tobacco leaves. The individual stacks were also observed
to be associated with and moving over the surface of the more
stationary cortical ER tubules. Such Golgi movement is best observed in
time lapse confocal movies
(http://mcdb.colorado.edu/~nebenfue/golgi/and http://www.brookes.ac.uk/schools/bms/research/molcell/hawes/gfp/gfp.html). Actin and myosin inhibitors were reported to stop this movement, and
staining of the actin cytoskeleton in leaves showed a remarkable co-alignment of ER tubules over cortical actin bundles, with individual Golgi stacks apparently attached to the actin cables (Fig.
1). Golgi positioning in plant cells had
previously been attributed to the actin cytoskeleton (19) and this work
on living cells demonstrated that Golgi movement is also attributable
to the actin cytoskeleton (Fig. 2) and is most likely myosin mediated.
The biological significance of the Golgi movements has yet to be
determined but it has been suggested that they are traveling between
vesicle pick-up points on the ER (16) or are actively collecting
ER-derived products for secretion as they move along the tubules
(3).
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Figure 1.
Single-leaf epidermal cell of Nicotiana
clevelandii expressing ER-/Golgi-targeted GFP (aERD2-GFP) and
stained with rhodamine phalloidin to highlight the actin cytoskeleton.
This image shows that Golgi stacks are closely associated with actin
cables. (Micrograph courtesy of P. Boevink [S.C.R.I., Dundee, UK],
reproduced with permission from Plant Journal 3).
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View larger version (44K):
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Figure 2.
Simplified model of the organization of the
cortical cytoplasm. A polygonal network of ER overlies an actin
cytoskeleton over which both Golgi (G) and mitochondria (M) trek. Note
that ER tubules can grow along actin cables (arrows) and homotypically
fuse with other tubules. Omitted from this diagram are the cortical
microtubule cytoskeleton and endocytic structures such as
clathrin-coated pits and vesicles.
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It is surprising that GFP-tagged mitochondria also appear to exhibit
very similar patterns of movement in GFP-transformed Bright Yellow-2
cells (K. Van Gestel and J.-P. Verbelen, personal communication;
Fig. 2). Mitochondria have also been shown to have associated myosin,
as has the ER (13). However, this motor protein has yet to be
associated with the Golgi.
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THE FUTURE IS BRIGHT: THE FUTURE IS GREEN, YELLOW, CYAN, AND
RED! |
And so to the future. Expressing GFP constructs in cells by a
number of methods can now be considered to be a routine procedure. With
the spectral variants of GFP and the newly introduced DsRed fluorescent
proteins, multiple expression will soon become the norm. Thus, dynamic
events and interactions between a variety of organelles will be imaged
concurrently. Green Golgi will be visualized moving over red actin and
blue ER while secreting yellow fluorescent protein. The interactions of
proteins involved in the molecular machinery of such organelle movement
or even in vesicle budding and fusion will be analyzed by fluorescence
resonance energy transfer technology and the dynamics of the
trafficking of proteins around the cell will be revealed by the use of
fluorescence recovery after photobleaching. At the same time the
fluorescent proteins will have been engineered to continually monitor
physiological changes in the cytoplasm. In these self-reporting cells
we will be able to introduce or express components of, or inhibitors
of, the molecular machinery which regulates the cytoskeleton (i.e. the
Rho proteins) or membrane and vesicle transport (the Rab proteins and
various vesicle coat proteins) and motor proteins such as myosin. Such
technical advances will be invaluable aids in the task of unraveling
and understanding the three-dimensional organization and regulation of
plant cell cytoplasm.
In conclusion, it is a sobering thought that it took 25 years
after the discovery of fluorescent proteins before they were utilized
as cellular reporters. Whoknows what technology lies around the corner
and what surprise development will once more "revolutionize" the
study of cellular dynamics?
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ACKNOWLEDGMENTS |
We would like to acknowledge the work of all the excellent plant
microscopists, who over the years have made major contributions to our
understanding of the complexity of plant cell cytoplasm but whom we
have not been able to cite in a short article such as this.
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FOOTNOTES |
*
Corresponding author; e-mail chawes{at}brookes.ac.uk; fax
44-0-1865-483955.
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MOVEMENTS IN PLANT CELLS
UV- AND VIDEO-ENHANCED...