Download Actin in plants

COMMENTARY
Actin in plants
CLIVE LLOYD
Department of Cell Biology, John Innes Institute, Colney Lane, Nonvich NR4 7UH, UK
It has been known for some time that actin exists in
lower plants, the most familiar examples being provided by certain algae in which actin constitutes the
cytoplasmic cables along which particles and organelles
stream. The choice of material was an important part of
determining the identity of the filaments because these
giant filamentous cells were capable of being broken
open to expose cytoplasmic strands, which could then
be decorated with heavy meromyosin (HMM)
(reviewed by Williamson, 1986).
This approach was effectively closed to higher plants
where most cells occur in complex, non-filamentous
tissue, which excludes the large HMM polypeptide.
Nevertheless, by analogy with algal particle movement,
it was assumed that cytoplasmic streaming in higher
plant cells would be actin-dependent. The development of fluorescent phallotoxins (such as phalloidin
and phallacidin) as diagnostic probes for filamentous
actin has vindicated this view (Wulf et al. 1979; Barak
el al. 1980). The very small size of phallotoxins
(approx. \2Q0Mr) enables them to enter walled cells
and tissues pre-treated with fixative or detergent, and
there are now several examples that demonstrate that
thick cytoplasmic cables in higher plants are composed
of actin filaments (reviewed by Lloyd, 1987).
Are actin filaments absent from dividing cells?
These cables are detected during interphase but what is
the function, if any, of actin in dividing plant cells?
Cytoplasmic streaming appears to cease at the onset of
prophase and so the absence of F-actin during mitosis
and cytokinesis would be consistent with the idea that
its sole function is to stir the cytoplasmic contents. The
ability to isolate meristematic cells and label them with
antibodies enabled the microtubule cycle to be followed, throughout division, by immunofluorescence
microscopy (Wick et al. 1981). Subsequent staining of
such cells by rhodamine-phalloidin indicated that Factin was not restricted to cells during interphase but
also occurred in the cytokinetic apparatus (Clayton &
Lloyd, 1985). This device, the phragmoplast, contains
Journal of Cell Science 90, 185-188 (1988)
Printed in Great Britain © The Company of Biologists Limited 1988
two circular opposing, picket fences of short microtubules, which (at their mid-line) mark the perimeter
of the new, discoid, cell plate. Unlike the centripetal
(purse-string) cytokinesis of animal cells, the cell plate
expands centrifugally until it contacts the maternal side
walls. It has been suggested that actin filaments
amongst the double ring of phragmoplast microtubules
propel to the mid-line those vesicles that coalesce to
form the floating cell plate.
In 1987 a succession of papers appeared that showed
that F-actin is not absent between interphase and
cytokinesis. A common underlying theme concerns the
conditions necessary to preserve actin filaments during
division, for it now seems that the thick interphase
cables represent only the most stable class of dynamic
arrays of actin.
Using low (1-5%, w/v) formaldehyde concentrations in a phosphate buffer, fine arrays of actin could
be seen at the cortex of alfalfa and Vicia cell suspensions, in addition to the thicker sub-cortical cables
(Seagull et al. 1987). Although these interphase arrays
seemed to disappear at prophase, actin filaments were
seen in the mitotic spindle as well as in the phragmoplast.
In another paper (Palevitz, 1987), rhodamine-phalloidin was used to stain fixed onion root-tip cells and to
show a ring of fluorescence around the cortex at preprophase. This ring of actin was observed in formaldehyde-fixed cells whether Pipes or phosphate was
used in the medium. Subdividing prophase with a
prefix reflects the added importance attached to premitotic events by plant cell biologists. The term preprophase was applied to the period during which the
evenly distributed cortical microtubules of interphase
gave way to a narrow, dense microtubule band, which
(still at the cortex) encircled the nucleus and predicted
the future division plane (Pickett-Heaps & Northcote,
1966). Actin and microtubules therefore form a ring at
the perimeter of the division plane. But the disappearance of this pre-prophase band (PPB) prior to mitosis
makes it difficult to account for the way in which the
cytokinetic apparatus will be directed out to that pre185
ordained ring (often for tens of micrometres) when the
cytoskeletal components marking the boundary of the
division plane have already depolymerized.
Avoiding or minimizing the effects of aldehydes
Mild extraction with detergent and dimethyl sulphoxide (DMSO) enables cytoplasm to be stained with
rhodamine-phalloidin without the requirement for
pre-fixation (Traas et al. 1987). This has shown that
extremely fine filaments of actin run transversely
around the cortex of carrot suspension cells, parallel to
the interphase microtubules. It has also shown that Factin exists in the PPB, but more importantly it has
revealed a class of actin that does not disappear with the
onset of mitosis and may therefore be involved in the
spatial control of division. These actin filaments cage
the nucleus and, importantly for cellular morphogenesis, run between both mitotic and cytokinetic apparatus and the cortex. There is something comforting
about the use of aldehyde for preserving cytoplasmic
structure, so that its omission casts doubt on the view
obtained by extraction methods alone. On the other
hand, it is clear that fixation obliterates much of the
fine cytoplasmic structure visible in living cells (Mersey
& McCully, 1978). Electroporation is a technique that
bypasses both aldehyde fixation and detergent extraction; pulses of direct current transiently open pores in
the plasma membrane that allow rhodamine-phalloidin to label F-actin directly. This alternative method
(Traas et al. 1987) confirms the view obtained by mild
extraction: that there is a class of F-actin associated
with the nucleus, connecting it to the cortex throughout division.
Reflecting this concern that aldehyde has not revealed the true distribution of actin in plants, Kakimoto & Shibaoka (1987a) reported two methods for
minimizing the harmful effects of fixation. In the first
method, tobacco suspension cells were pre-labelled
with rhodamine-phalloidin using 0-1 % Triton X-100
to permeabilize the cells. Lysine at 1-5 % was added to
the extraction mixture. Subsequently fixed with formaldehyde, the cells could be processed for doublestaining with anti-tubulin, since the lysine treatment
apparently protected the pre-labelled actin filaments
during the rigours of antibody labelling. In this way,
fine actin filaments were seen: at the cell cortex;
paralleling the pre-prophase band microtubules; and
lying between the cell cortex and the phragmoplast.
Actin filaments within the phragmoplast itself were not
well preserved by this method. Another report from
these authors (Kakimoto & Shibaoka, 19876) outlines
the use of heavy meromyosin/tropomyosin as a protectant during aldehyde fixation, although results are
stated to be generally superior using lysine.
186
C. Uoyd
Actin in the spindle
A curious aspect of some of these studies is that F-actin
is detected not only in the cytokinetic apparatus but
also in the mitotic spindle (Seagull et al. 1987; Traas et
al. 1987). The existence of F-actin in the spindle has
long been debated for animal cells where, if it were
present, it might represent an elastic element for
chromatid separation. The recent demonstration
(Koshland et al. 1988) that isolated chromosomes move
towards the minus end of microtubules in vitiv, as the
plus end disassembles, argues against the need for an
external force (as might be supplied by actomyosin)
during anaphase movement. So what is F-actin doing
in the plant spindle?
Whether cells are fixed prior to phallotoxin labelling
(Seagull et al. 1987), or electroporated or extracted
unfixed (Traas et al. 1987), the spindle of suspension
cells can be stained. These combined observations
argue against a single unusual set of circumstances
causing actin oligomers to decorate spindle microtubules. That it is not due to some stabilizing property
of labelled phallotoxin can be derived from the report
that actin antibodies also decorate filaments, which not
only cage the nucleus of Haemanthus endosperm cells,
but pass between the spindle poles, parallel to microtubules (Schmit & Lambert, 1987). Fluorescent antiactin labelling coincided with rhodamine-phalloidin,
and immunogold labelling identified thin filaments
amongst the spindle microtubules. Despite the close
proximity of these two elements, perfusion with cytochalasin B or D at 10/igmP 1 did not affect chromosome transport.
Microtubules and actin filaments are now recognized
as running parallel to one another during interphase,
pre-prophase and cytokinesis. The parallelism at mitosis may simply reflect this propensity for interaction
shown during other phases of the cell cycle; the
ineffectiveness of cytochalasin and the cessation of
streaming during mitosis tending to suggest that actinbased contractility might, anyway, be inhibited during
this period.
Another explanation for the existence of F-actin in
the plant spindle is suggested by the manner in which
the phragmoplast appears to be formed. Phragmoplast
microtubules share the same polarity as spindle microtubules (Euteneuer & Mclntosh, 1980) and it often
appears (although it is not proven) that the phragmoplast arises out of the post-anaphase spindle. As the
double ring of phragmoplast microtubules expands
outwards, actin filaments can clearly be seen parallel to
the tubules and are likely to function in cell plate
deposition. Actin filaments amongst the spindle microtubules may be an early indicator of a function expressed at cytokinesis.
Animal cells relinquish shape control during division
whereas plant cells do not: they retain their asymmetry
and one of the key shaping processes (alignment and
deposition of the cross wall) actually occurs over this
period.
Perhaps further clues for the basis of these differences between higher plants and animals can be found
in organisms, such as the filamentous alga Spirogyra, in
which cytokinesis occurs by a mixture of in-furrowing
and cell plate deposition. As Pickett-Heaps (1975) has
observed, cytokinesis initially occurs by the in-growth
of an annular septum, but where this contacts the
central cytoplasmic cylinder separating the telophase
nuclei, a small phragmoplast develops and completes
division by forming a cell plate. In the most recent
paper, Gotto & Ueda (1988) have now used rhodamine-phalloidin to map the distribution of actin filaments in dividing Spirogyra. At prophase, actin
bundles dispersed in the cytoplasm come together, just
beneath the plasma membrane, to form a ring around
the middle of the cell (cf. the preprophase band of actin
in higher plant cells). The diameter of this ring
narrows as in-furrowing proceeds - clearly inviting
comparison with the contractile ring of animal cytokinesis. No specific mention is made of how the ring of
actin relates to the central algal phragmoplast. However, since F-actin is reportedly present in a ring "until
the cell is finally divided into two daughter cells" it is
possible that actin is involved in both phases of
cytokinesis. In view of the position Spirogyra may hold
in the evolution of the phragmoplast/cell plate from a
primitive mechanism of cytokinesis (cleavage) (PickettHeaps, 1975) it seems important to clarify the fine
distribution of F-actin during the transition from
furrowing to phragmoplast formation, and to correlate
this with the microtubule staining pattern.
The role of actin in establishing the division plane
Apart from actin in the spindle, the actin filaments that
connect the dividing nucleus to the side walls explain
two features of division plane enactment not accounted
for in microtubule-only models. The division plane can
be pictured from above as two concentric rings: one,
the pre-prophase band, which anticipates the perimeter
of the plane in which the cell plate will be deposited;
the other the phragmoplast, which will spread out
within that plane, from an initially central location,
until its perimeter contacts the former pre-prophase
band site. Because these devices are separated in time,
it has not been clear why they should occupy the same
plane. Why doesn't the expanding edge of the phragmoplast wander off into another plane? What guides
the expanding disc out to the former band site? The
discovery that cytoplasmic actin filaments remain
throughout division (Traas e/ al. 1987) and, in particular, bridge the leading edge of the phragmoplast to the
cortex (Lloyd & Traas, 1988), offers an explanation.
Sinnott & Bloch (1941) long ago drew attention to the
fact that cytoplasm re-arranged itself prior to division.
Working on large, vacuolated cells induced to divide as
a result of tissue wounding, they reported that cytoplasmic strands that radiated from the nucleus during
interphase adopted a much simpler arrangement at
prophase. Instead of radiating to all points of the cell,
these transvacuolar strands coalesced at this stage to
form a transverse baffle across the cell, within which
the nucleus migrated and division occurred. Apart
from this transverse phragmosome, the only other
cytoplasmic strands run from each spindle pole to the
opposing end wall, thereby forming a Maltese cross.
This pre-cytoskeletal image now appears to be reflected
in the distribution of actin filaments, suggesting a
mechanism in which F-actin and microtubules act in
concert to set up the division plane (Lloyd & Traas,
1988).
It is not known in detail how the pre-prophase band
of microtubules forms (from old microtubules or from
new tubulin) but one conclusion (Doonan et al. 1987)
is that the interphase microtubules 'bunch up'. Concentrating microtubules in the PPB would not only
sweep the parallel cortical F-actin into the band but
also draw the thicker nucleus-to-cortex bundles into the
plane circumscribed by the PPB. Even though the rim
of this device (both PPB microtubules and circumferential F-actin) disappears by metaphase, radial spokes
of actin remain connected to the nucleus at its hub
(Lloyd & Traas, 1988). These spokes form a temporal
and spatial connection between cortical division site
and central division apparatus, remaining to guide the
phragmoplast outwards within that pre-determined
plane.
What actually brings the PPB to a particular plane in
the first instance is another question. Strain has often
been suggested to be an epigenetic device for aligning
division planes across tissues (Green, 1987). Nucleusassociated actin filaments would seem to be likely
candidates for the tension strands.
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