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When is a cell not a cell? A theory relating coenocytic structure to the unusual electrophysiology of Ventricaria ventricosa (Valonia ventricosa)

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Ventricaria ventricosa and its relatives have intrigued cell biologists and electrophysiologists for over a hundred years. Historically, electrophysiologists have regarded V. ventricosa as a large single plant cell with unusual characteristics including a small and positive vacuole-to-outside membrane potential difference. However, V. ventricosa has a coenocytic construction, with an alveolate cytoplasm interpenetrated by a complex vacuole containing sulphated polysaccharides. We present a theory relating the coenocytic structure to the unusual electrophysiology of V. ventricosa. The alveolate cytoplasm of V. ventricosa consists of a collective of uninucleate cytoplasmic domains interconnected by fine cytoplasmic strands containing microtubules. The cytoplasm is capable of disassociating into single cytoplasmic domains or aggregations of domains that can regenerate new coenocytes. The cytoplasmic domains are enclosed by outer (apical) and inner (basolateral) faces of a communal membrane with polarised K(+)-transporting functions, stabilised by microtubules and resembling a tissue such as a polarised epithelium. There is evidence for membrane trafficking through endocytosis and exocytosis and so "plasmalemma" and "tonoplast" do not have fixed identities. Intra- and extracellular polysaccharide mucilage has effects on electrophysiology through reducing the activity of water and through ion exchange. The vacuole-to-outside potential difference, at which the cell membrane conductance is maximal, reverses its sign from positive under hypertonic conditions to negative under hypotonic conditions. The marked mirror symmetry of the characteristics of current as a function of voltage and conductance as a function of voltage is interpreted as a feature of the communal membrane with polarised K(+) transport. The complex inhomogeneous structure of the cytoplasm places in doubt previous measurements of cytoplasm-to-outside potential difference.
a, b. Electrical characteristics of small (2–3 mm diameter) mature V. ventricosa cells under different conditions. The internal PDmeasuring electrode was inserted in the cell centre and measured PD vo (the vacuole-to-outside PD). The PD vo was voltage clamped via a Pt-Ir wire terminating near the cell centre, facilitating measurement of the characteristics of current as a function of voltage (I/V) (Beilby 1990). The characteristics of conductance as a function of voltage (G/V) were calculated by differentiation of the I/V profiles (Beilby 1990). a I/V profiles; b G/V profiles. Typical I/V characteristics in seawater (light blue line) were obtained 2 h after electrode insertion; photosynthetically active radiation of 2.02 mol/sm 2 . The " dark " profile (black line) was obtained after 22 min with a photosynthetically active radiation of 0.5 mol/sm 2 from the same cell. I/V characteristics following hypertonic shock (increase of osmotic pressure by 100 mosmol/kg for 16 min; dark blue line) and hypotonic shock (decrease of osmotic pressure by 200 mosmol/kg, 28 min exposure; red line) were obtained from different cells. The effects of [K ] o are included for comparison. The green line shows the average profile from 9 cells stabilized in 100 mM K medium, and the orange line shows the average profile from 6 cells stabilized in 0.1 mM K medium (K data replotted from data in Beilby and Bisson 1999: fig. 1). The data can be categorised into three groups: (1) pumping K in ( " hyper " and " ASW " , dark and light blue lines) with high conductance at positive values of PD vo ; (2) pumping K out ( " hypo " , red line; " 0.1 K " , orange line) with high conductance at negative values of PD vo ; (3) low conductance ( " dark " and " 100 K " , black and green lines) with transporters inactivated or working at low rate. The states 1 and 2 mirror each other. The significance of the similarity between low K and the hypotonic data is yet to be explained
… 
a–e. Diagrammatic and schematic representations of the hypothesised structure of cytoplasmic domains in V. ventricosa cytoplasm. a Outline of a cell traced from Fig. 2 to show relationships between perinuclear microtubules (pm), nuclear centres (nc), and the postulated cytoplasmic domains. The nuclear centres are maximally spaced (McNaughton and Goff 1990). The cytoplasmic domains are represented as circles enclosing the nuclear centres. The actual geometry of the domains is not known, although they maintain a constant ratio between nuclear and cytoplasmic volumes. Most nuclei have either five or six neighbours in whole cells. The nuclei lie beneath the chloroplasts and are interconnected by cytoplasm-coated perinuclear microtubules that span the domain boundaries. Approximate scale bar, on the basis of Fig. 2, is 20 m. The radiating pattern of perinuclear microtubules is based on micrographs of La Claire (1987) and Shihira-Ishikawa and Nawata (1992). b Outline of the same cell showing relationships between cortical microtubules (cm), chloroplasts (white circles in the central cytoplasmic domain), and the postulated cytoplasmic domains. Chloroplasts are situated closest to the cell wall and are surrounded by either five or six neighbours. The parallel cortical microtubules that occupy the thin surface layer of cytoplasm are depicted as straight lines. The pattern of cortical microtubules is based on fluorescence micrographs of La Claire (1987) and Shihira-Ishikawa and Nawata (1992).
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Summary. Ventricaria ventricosa and its relatives have intrigued cell
biologists and electrophysiologists for over a hundred years. Historically,
electrophysiologists have regarded V. ventricosa as a large single plant
cell with unusual characteristics including a small and positive vacuole-
to-outside membrane potential difference. However, V. ventricosa has a
coenocytic construction, with an alveolate cytoplasm interpenetrated by a
complex vacuole containing sulphated polysaccharides. We present a the-
ory relating the coenocytic structure to the unusual electrophysiology of
V. ventricosa. The alveolate cytoplasm of V. ventricosa consists of a col-
lective of uninucleate cytoplasmic domains interconnected by fine cyto-
plasmic strands containing microtubules. The cytoplasm is capable of
disassociating into single cytoplasmic domains or aggregations of do-
mains that can regenerate new coenocytes. The cytoplasmic domains are
enclosed by outer (apical) and inner (basolateral) faces of a communal
membrane with polarised K
-transporting functions, stabilised by micro-
tubules and resembling a tissue such as a polarised epithelium. There is
evidence for membrane trafficking through endocytosis and exocytosis
and so “plasmalemma” and “tonoplast” do not have fixed identities. Intra-
and extracellular polysaccharide mucilage has effects on electrophysiol-
ogy through reducing the activity of water and through ion exchange. The
vacuole-to-outside potential difference, at which the cell membrane con-
ductance is maximal, reverses its sign from positive under hypertonic
conditions to negative under hypotonic conditions. The marked mirror
symmetry of the characteristics of current as a function of voltage and
conductance as a function of voltage is interpreted as a feature of the
communal membrane with polarised K
transport. The complex inhomo-
geneous structure of the cytoplasm places in doubt previous measure-
ments of cytoplasm-to-outside potential difference.
Keywords: Ventricaria ventricosa; Cytoplasmic structure; Vacuolar
structure; Sulphated polysaccharide; Coenocyte; Algal electrophysiology.
Abbreviations: PD
co
cytoplasm-to-outside potential difference; PD
vo
vacuole-to-outside potential difference; PD
vc
vacuole-to-cytoplasm po-
tential difference; I/V current as function of voltage; G/V conductance as
function of voltage.
Cell biology and electrophysiology of Ventricaria
ventricosa: a hundred years of research
The marine alga Ventricaria ventricosa (Olsen and West
1988; formerly Valonia ventricosa) lives in coral rubble in
tropical reef environments such as the Great Barrier Reef.
The giant-celled V. ventricosa and related algae of the order
Cladophorales have fascinated cell biologists and electro-
physiologists alike since the early twentieth century.
Some members of the Cladophorales respond to injury
with the extraordinary process of modified segregative cell
division (for reviews, see La Claire 1982, Menzel 1988).
The entire cytoplasm of a punctured or cut cell contracts
into a network and after about 30 min forms inwardly
swollen cytoplasts connected by thin strands of cytoplasm.
After 1–3 h the cytoplasmic strands are severed leaving nu-
merous separate protoplasts (aplanospores) that then re-
generate cell walls. This process has been studied since the
1930s, in V. ventricosa (Kopac 1933, Doyle 1935, Steward
and Martin 1937, La Claire 1982, Nawata et al. 1993) and
in Boergesenia forbesii (Enomoto and Hirose 1972; La
Claire 1982; O’Neil and La Claire 1984, 1988; Itoh et al.
1984). Protoplast formation in V. ventricosa is illustrated in
Fig.1a and b. The ability to regenerate new cells from frag-
Protoplasma (2004) 223: 79–91
DOI 10.1007/s00709-003-0032-4
PROTOPLASMA
Printed in Austria
When is a cell not a cell? A theory relating coenocytic structure to the unusual
electrophysiology of Ventricaria ventricosa (Valonia ventricosa)
Review article
V. A. Shepherd
1,
*
, M. J. Beilby
1
, and M. A. Bisson
2
1
UNESCO Centre for Membrane Science and Technology, Department of Biophysics, School of Physics,
University of New South Wales, Sydney, New South Wales
2
Department of Biological Sciences, State University of New York at Buffalo, Buffalo, New York
Received July 13, 2003; accepted October 15, 2003; published online June 22, 2004
© Springer-Verlag 2004
* Correspondence and reprints: UNESCO Centre for Membrane Science
and Technology, Department of Biophysics, School of Physics, Univer-
sity of New South Wales, Sydney, NSW 2052, Australia.
ments of cytoplasm distinguishes V. ventricosa and its rela-
tives from most other organisms.
Ventricaria ventricosa has played a key role in plant
cell electrophysiology and continues to do so. Osterhout’s
group made electrical measurements of V. ventricosa in the
1920s that helped establish the concept of a transmem-
brane electrical potential difference (PD) in plant cells
(Hope and Walker 1975). However, the small positive val-
ues measured for the vacuole-to-outside potential differ-
ence (PD
vo
) then and in the 1930s (e.g., 15 mV; Blinks
1930, Steward and Martin 1937), later turned out to be
highly unusual. Most plant cells have a negative PD
vo
,
which is dominated by a highly negative cytoplasm-to-
outside PD (PD
co
) (230 to 280 mV in Chara corallina)
80 V. A. Shepherd et al.: When is a cell not a cell?
and which is only slightly offset by a small vacuole-to-
cytoplasm PD (PD
vc
) (about 20 mV in C. corallina).
There have been many attempts to reconcile the unusual
electrical properties of V. ventricosa with conventional
ideas of the structure of plant cells. Ventricaria ventricosa
is regarded in the electrophysiological literature as a single
spherical cell containing a tonoplast-enclosed central vac-
uole and a thin layer of plasmalemma-bound cytoplasm.
However, the “cells” have a coenocytic construction and
are extraordinarily large, with some up to 10 cm in diame-
ter (Menzel 1988). The possibility that the coenocytic
structure might be coupled to the unusual electrophysiol-
ogy has not previously been considered.
Ventricaria ventricosa, a coenocyte with unusual
cytoplasmic and vacuolar structure
The cytoplasm of V. ventricosa has an unusual and complex
organisation. The electron micrographs of Shihira-Ishikawa
and Nawata (1992) show a remarkable alveolate or “sponge-
like” cytoplasmic topology. Most of the cytoplasmic vol-
ume is occupied by organelles, chloroplasts, nuclei, and
mitochondria, interconnected by a meshwork of fine cyto-
plasmic strands. Interconnected vacuoles occupy the inter-
vening spaces. The cytoplasm does not stream and cortical
and perinuclear microtubules hold the chloroplasts and nu-
clei in a fixed pattern (Shihira-Ishikawa 1987). The perinu-
clear microtubule arrays maintain the multiple nuclei at a
uniform distance apart (McNaughton and Goff 1990) in an
arrangement of optimal packing.
The vacuole of V. ventricosa forms a complex interface
with the alveolate cytoplasm. Theoretical calculations
suggest its membrane surface is “multifolded” by a factor
of nine (Wang et al. 1997, Ryser et al. 1999). The vacuole
is unusual also in that it contains sulphated polysaccharide
mucilages. Sulphated polysaccharides have been identi-
fied as one of several wound “plug precursors” in other
giant algae (Menzel 1988) and V. ventricosa does form
“wound plugs” in response to nondrastic injuries such as
a pinprick with a diameter of 100 m (Nawata et al.
1993). We found that sulphated polysaccharides were inti-
mately involved in the structure of V. ventricosa coeno-
cytes (Shepherd et al. 1998, 2001) (Fig.1c–f).
Protoplasts and young cells both contain and are coated
in sulphated polysaccharide mucilage, and a communal
mucilaginous sheath unites young cells into clusters prior
to development of rhizoids (Fig. 1c–e). Plasmolysis of
young cells reveals bundles of mucilage-coated filaments
spanning the cell wall and connecting intra- to extracellu-
lar compartments (Fig.1f). These filaments resemble the
actin-containing cytoplasmic “microvilli” that are rem-
nants of wall-to-membrane linkages in wounded or plas-
molysed Ernodesmis verticillata cells (Goddard and La
Claire 1993). We obtained a three-dimensional representa-
tion of the relationship between cytoplasm and vacuole by
optically sectioning cells labelled with the fluorochrome
6-carboxyfluorescein (Fig. 2) (Shepherd et al. 2001). We
found that the interconnected vacuoles appearing in thin
sections for electron microscopy are actually projections
from a single, enormously convoluted central vacuole,
V. A. Shepherd et al.: When is a cell not a cell? 81
Fig. 1 a–h. Structure of V. ventricosa protoplasts and cells. The process of wound-induced cytoplasmic segregation and protoplast assembly and its
time-course are highly conservative. 30 min after cutting a mother cell, inward cytoplasmic contraction and segregation produces swollen cytoplasts
connected by fine strands of cytoplasm (about 4 to 7 m in diameter, spanning distances of up to about 200 m). The cytoplasts contract into spherical
protoplasts after 40 min. Most of the cytoplasm is converted to protoplasts after 1 h. The smallest regenerative protoplasts consistently have a diameter
of 10–15 m. Cell walls form after 3 to 8 h. a Contracting cytoplasts (upper arrow) and spherical protoplasts (lower arrow) on a wall segment 40 min
after cutting the mother cell. b A protoplast (long arrow) formed 40 min after cutting the mother cell. Three elongated chloroplasts (short arrow; com-
pare with the isolated chloroplast, bottom left) within a cytoplasmic strand are entering the protoplast. c Sulphated and carboxylated polysaccharides
were identified by staining with Alcian Blue at pH 2.5 (Sheehan and Hrapchak 1980). Alcian Blue stains the sulphated polysaccharide mucilage (arrow)
coating a protoplast. d Sulphated and carboxylated polysaccharides were also identified by staining with 0.2% Toluidine Blue, pH 0.49 (McCully
1970). This 8 h old cell was stained and plasmolysed in glycerol-seawater medium (70 : 30 [w/v]; osmolarity, 3550 mmol/kg). Toluidine Blue
metachromatically stains an extracellular sheath of sulphated polysaccharide mucilage (purple; short arrow). An interior layer orthochromatically
stained (blue, long arrow) may be a membrane-cytoskeleton complex. The inset shows mucilaginous contents of the vacuole of a cell gently crushed
under a coverslip. Sulphated polysaccharide mucilage is found in the vacuole and extracellular sheath of young walled cells. e Toluidine Blue (pH 0.49)
stains sulphated polysaccharide mucilage (purple, arrow) in which a cluster of 12 h old walled cells are embedded. The stained mucilage has a fibrous
appearance. f A 12 h old cell stained with Toluidine Blue at pH 0.49 and plasmolysed. Bundles of stained fine filaments (white arrows, black arrow)
span the cell wall (cw, arrow) and interconnect the plasmolysed protoplast (p, arrows) with the extracellular sulphated polysaccharide sheath (pink).
The purple staining of the filaments suggests that they are coated with sulphated polysaccharides. g The cytoplasm of young cells was fluorescence-la-
belled using 6-carboxyfluorescein diacetate, which is membrane permeant and is cleaved by intracellular esterases to the impermeant, highly fluores-
cent 6-carboxyfluorescein. Fluorescence micrograph (blue exciting light) of a cluster of labelled 3-week-old walled cells. The cytoplasm, including the
multiple nuclei (n), selectively accumulates the fluorochrome. No autofluorescence of the same wavelengths as 6-carboxyfluorescein was detected in
unlabelled cells. h Higher magnification of a cell shown in g. 6-Carboxyfluorescein is accumulated by the cytoplasm, including chloroplasts (c) and nu-
clei (n, overlaid by fluorescing chloroplasts). We interpret the intervening dark (nonfluorescing) regions as protrusions from the central vacuole (v) in-
vaginating the cytoplasm. An extremely thin peripheral layer of cytoplasm probably overlies these regions
which invaginates the alveolate cytoplasm, as do the holes
in a sponge.
Ventricaria ventricosa is capable of regulating its turgor
pressure (Bisson and Beilby 2002 and references therein).
Interestingly the osmotic pressure in the vacuole remains
greater than that in the medium in the early stages of cyto-
plasmic reticulation and segregation (Nawata et al. 1993) –
despite the presence of gaps between retracting cytoplasts.
A recent paper (Heidecker et al. 2003) shows that intracel-
lular sulphated polysaccharide mucilage contributes signif-
82 V. A. Shepherd et al.: When is a cell not a cell?
icantly to resting turgor pressure in Valonia utricularis,
probably by reducing the chemical activity of water. Gel-
associated water with a reduced density becomes K
se-
lective (Wiggins and van Ryn 1990).
The unusual electrophysiology
of the V. ventricosa coenocyte
Aikman and Dainty (1966) speculated that the unusual
PD
vo
of V. ventricosa was dominated by a K
“pump”.
Other researchers reported separate measurements of PD
vc
and PD
co
in V. ventricosa. Gutknecht (1966) measured a
PD
co
of 71 mV in aplanospores, a PD
vo
of 17 mV in
small vacuolate cells, and he estimated PD
vc
as an un-
usual 88 mV. Davis (1981) directly measured PD
co
as
70 mV and PD
vc
as 86 mV in small young cells. In
contrast, Lainson and Field (1976) found the PD
co
of
aplanospores was only 3.6 to 31 mV. They failed to
find negative values for PD
co
in large mature cells, where
PD
vc
appeared to be zero, but noted that the cell walls of
mature cells and aplanospores were significantly negative
(12 mV).
The debate over the electrical properties of V. ventri-
cosa appeared to be resolved by Ryser et al. (1999). They
measured charge-pulse relaxation spectra of plasma-
lemma and tonoplast separately, by adding the pore-
forming antibiotic nystatin to the outside or to the inside
of the cell. The effects of nystatin were transient and vari-
able, and measurements of PD
co
ranged from 15 to
68 mV, but the results supported Gutknecht (1966) and
Davis (1981) rather than Lainson and Field (1976).
Current understanding resolves the small positive PD
vo
of V. ventricosa into a highly unusual PD
vc
of about
90 mV, and a PD
co
of about 70 mV (Ryser et al.
1999). The PD
co
is thought to be a K
diffusion potential,
and the PD
vc
is attributed to an unusual K
pump active-
ly transporting K
into the vacuole, as postulated by
Hastings and Gutknecht (1974).
However, there are other unusual features of the elec-
trophysiology of V. ventricosa that require explanation.
The electrophysiological response of the cells to certain
stimuli is not only unusual but also inverted when com-
pared to other algal cells. The conductance of V. ventri-
cosa cells decreases when the K
concentration in the
medium is increased (Beilby and Bisson 1999), but the
conductance of C. corallina cells increases (Beilby 1985).
The PD
vo
becomes more negative in V. ventricosa cells
experiencing hypotonic conditions and more positive in
hypertonic conditions (Bisson and Beilby 2002). These
stimuli produce I/V (current as a function of voltage) and
G/V (conductance as a function of voltage) characteristics
with a marked mirror symmetry (Fig. 3a, b). However, the
salt-tolerant charophyte Lamprothamnium papulosum ex-
periences PD
vo
shifts in the opposite direction under hypo-
and hypertonic shock, with no obvious symmetry in the
I/V and G/V curves (Beilby and Shepherd 2001, Shepherd
et al. 2002).
Finally, V. ventricosa is not alone in having both un-
usual electrophysiology and cytoplasmic structure. Other
algae of the Cladophorales, Chaetomorpha darwinii and
Valoniopsis pachynema, also have a highly positive PD
vo
and high tonoplast resistance (Findlay et al. 1971, 1978).
V. A. Shepherd et al.: When is a cell not a cell? 83
Fig. 2 a–e. Cytoplasmic and vacuolar structure in young V. ventricosa cells. The figure enables us to demonstrate the wide range of possible locations
of microelectrodes inserted to particular depths in cells of different sizes. A cluster of five 18 h old cells was labelled with 6-carboxyfluorescein and
optically sectioned at 1.8 m intervals using a Leica DMIRB confocal microscope with Leica TCSNT PC-software. A fluorescein isothiocyanate fil-
ter set was used. Unlabelled cells did not autofluoresce in the characteristic yellow-green wavelengths of 6-carboxyfluorescein. The fluorescence of
the fluorochrome was only detected in the cytoplasm. a Reconstruction of twelve serial optical sections. b–e Four optical sections from a representa-
tive cell (asterisk in a). a The reconstruction passes through a range of cell volumes from about 36% (smallest cell, top right-hand side) to about 20%
of the diameter of the cell marked by the asterisk. The cytoplasm of all cells has the characteristic alveolate structure. Cytoplasm forms a topologi-
cally complex interface with the vacuole. The bulk of the cytoplasm consists of chloroplasts (c) and nuclei (n) interconnected by fine fluorescing cyto-
plasmic strands (s) that span the dark regions interpreted as projections from the central vacuole (v) overlaid by a peripheral cytoplasm only about
40 nm thick (La Claire 1987, Heidecker et al. 2003), in which fluorescence is undetectable. The cytoplasm is a mosaic of thicker and thinner regions.
The vacuolar projections (v) are continuous with the central vacuole (seen in the smallest cell). b Optical section of the most superficial layer of a cell
(marked by asterisk in a) 0.2 m from its surface. The interconnected chloroplasts (c) are partially surrounded by vacuolar protrusions (vp) that are
continuous with the central vacuole in subsequent sections. The vacuole invaginates the cytoplasm even at this superficial depth (compare with
epifluorescence image in Fig.1h). c Optical section 7.4 m from the cell surface. Nuclei (n) underlie the chloroplast layer. The vacuolar projection
(vp) is continuous with that indicated in b. Irregular cytoplasmic aggregations associated with nuclei span the vacuole. Thin cytoplasmic strands (s)
interconnect the cytoplasmic organelles and alternate with vacuolar protrusions extending to the periphery of the image. d Optical section 14.6 m
into the cell. The central vacuole (v) occupies a larger percentage of the volume of the cell and is continuous with the vacuolar projection in b and c.
The cytoplasm in the central region is still spongelike, but it is a sponge with larger holes. Nuclei (n) are associated with cytoplasm (asterisk) that
penetrates into the centre of the cell. Fine detail of the spongelike cytoplasm vacuole interface is visible around the edges of the image. e Optical
section 20 m from the cell surface (20% of the diameter of the cell). The vacuole (v) occupies most of the cell interior, continuous with vacuolar
protrusions seen in b–d. Nuclei (n) are still associated with clumps of cytoplasm invaginated by the vacuole. A continuous very fine fluorescent layer
of cytoplasm outlines the cell and this may be the thin peripheral cytoplasm
Valoniopsis pachynema also has stationary cytoplasm and
highly regular spatial geometry of nuclei established by
microtubules (McNaughton and Goff 1990).
A theory relating coenocytic structure
to electrophysiological behaviour
Ventricaria ventricosa is a structurally complex organism
that stretches to their limits the definitions of “cell”, “cy-
toplasm”, “vacuole”, “plasmalemma”, and “tonoplast”.
We present a new interpretation of the coenocytic struc-
ture V. ventricosa (see Fig. 4) on the basis of following
arguments.
1. The cytoplasm of V. ventricosa is structured from
aggregates of uninucleate cytoplasmic domains that can
each reconstitute a whole cell. The cytoplasm of the cells
consists of interconnected cytoplasmic domains, each of
which is a fundamental structural unit, containing a
nucleus, chloroplasts, and smaller organelles (e.g., mito-
chondria, cytoskeleton). Should the organism be damaged
or stressed, the cytoplasm is capable of disassociating into
the fundamental cytoplasmic domains. Single domains
and larger aggregates can regenerate and subsequently re-
constitute whole cells. In whole cells the domains are in-
terconnected by a meshwork of fine cytoplasmic strands
to form a porous communal cytoplasm interpenetrated
by a topologically complex vacuole. The fine dimension
of the cytoplasmic strands approaches that of intercellular
communication channels.
2. The vacuole and extracellular matrix of young cells
contain sulphated polysaccharide mucilage. Both proto-
plasts and young cells have intra- and extracellular com-
partments containing acidic sulphated polysaccharide
mucilage (Fig.1c–f). Extra- and intracellular mucilages
are likely to make significant contributions to ion and
water relations in V. ventricosa. In other marine algae,
sulphated polysaccharide mucilages reduce hydraulic
conductivity, create large “unstirred-layer” effects, and
probably have the capacity for ion exchange (Shepherd
and Beilby 1999).
3. A communal membrane with inwardly and out-
wardly directed polarised functions encloses the cytoplas-
mic domains. The ability of the cytoplasm to segregate,
reaggregate, and regenerate new walled cells suggests a
communal membrane encloses it. The inward- and out-
ward-facing surfaces of the communal membrane could
have polarised K
-transporting functions. In this, the cyto-
plasm would be analogous to a tissue such as the polarised
epithelium. The apical part of the communal membrane
(plasmalemma) is situated close to the cell wall, appears
84 V. A. Shepherd et al.: When is a cell not a cell?
Fig. 3a, b. Electrical characteristics of small (2–3 mm diameter) ma-
ture V. ventricosa cells under different conditions. The internal PD-
measuring electrode was inserted in the cell centre and measured PD
vo
(the vacuole-to-outside PD). The PD
vo
was voltage clamped via a Pt-Ir
wire terminating near the cell centre, facilitating measurement of the
characteristics of current as a function of voltage (I/V) (Beilby 1990).
The characteristics of conductance as a function of voltage (G/V)
were calculated by differentiation of the I/V profiles (Beilby 1990). a
I/V profiles; b G/V profiles. Typical I/V characteristics in seawater
(light blue line) were obtained 2 h after electrode insertion; photo-
synthetically active radiation of 2.02 mol/sm
2
. The “dark” profile
(black line) was obtained after 22 min with a photosynthetically active
radiation of 0.5 mol/sm
2
from the same cell. I/V characteristics
following hypertonic shock (increase of osmotic pressure by
100 mosmol/kg for 16 min; dark blue line) and hypotonic shock (de-
crease of osmotic pressure by 200 mosmol/kg, 28 min exposure; red
line) were obtained from different cells. The effects of [K
]
o
are in-
cluded for comparison. The green line shows the average profile from
9 cells stabilized in 100 mM K
medium, and the orange line shows
the average profile from 6 cells stabilized in 0.1 mM K
medium (K
data replotted from data in Beilby and Bisson 1999: fig.1). The data
can be categorised into three groups: (1) pumping K
in (“hyper” and
ASW”, dark and light blue lines) with high conductance at positive
values of PD
vo
; (2) pumping K
out (“hypo”, red line; “0.1 K”, orange
line) with high conductance at negative values of PD
vo
; (3) low con-
ductance (“dark” and “100 K”, black and green lines) with trans-
porters inactivated or working at low rate. The states 1 and 2 mirror
each other. The significance of the similarity between low K
and the
hypotonic data is yet to be explained
smooth (planar) in electron micrographs, and faces the
cell wall and mucilaginous matrix, as well as a seawater
medium with a high [Na
]/[K
] ratio. The highly con-
voluted basolateral part of the membrane (tonoplast)
separates the communal cytoplasm from the complex
mucilage-containing vacuole with a low [Na
]/[K
] ratio.
The polarity of the communal membrane may be modified
in response to environmental factors. There is evidence
that the maternal basolateral membrane is transformed into
an apical membrane during development of protoplasts,
through endocytosis of the maternal apical membrane and
exocytosis of membrane material to the basolateral surface
(O’Neil and La Claire 1988).
Rationale for arguments
Ventricaria cytoplasm is highly structured
into cytoplasmic domains
The extraordinary capacity for wound-induced cytoplas-
mic regeneration immediately suggests there is something
remarkable about the cytoplasm of V. ventricosa. Cutting
a plant cell open normally results in death, or in the for-
mation of cytoplasmic droplets that are incapable of
regeneration, as occurs in C. corallina. Yet, although both
plasmalemma and tonoplast of a cut Ventricaria cell are
exposed to seawater of high osmolarity and ionic strength,
the cytoplasm responds by contracting into cytoplasts
that then regenerate hundreds of walled cells (Fig.1a).
This response is widespread among the members of the
Cladophorales, and it is very similar to segregative cell di-
vision (La Claire 1982) that occurs in this group of organ-
isms (e.g., in Dictyosphaeria cavernosa; Okuda et al.
1997b). The process unfolds in a conservative pattern
over a similar time-course in our observations, and in
those of others concerning V. ventricosa and related algae
(Kopac 1933; Doyle 1935; Steward and Martin 1937;
La Claire 1982; O’Neil and La Claire 1984, 1988).
This suggests that cytoplasmic segregation and proto-
plast formation are self-organising processes taking place
amongst fundamental units. The smallest protoplast would
contain a single unit and larger protoplasts would be made
up of multiple units.
What is the nature of the fundamental unit? Only those
protoplasts containing at least one nucleus appear to re-
generate (Haberlandt 1928, Tatewaki and Nagata 1970).
Isolated chloroplasts fail to regenerate. The protoplasts
have variable diameters but we find a critical minimum di-
ameter of 10–15 m. We found respective diameters of
8–10 m and 5–8 m for nuclei and chloroplasts, but
Doyle (1935) found some chloroplasts with a diameter of
only 2.5 m. Chloroplasts can also elongate during cyto-
plasmic segregation (Fig.1b). Thus, the smallest regenera-
tive protoplast with a diameter of 10–15 m could contain
a single nucleus and as many as six enwrapping chloro-
plasts, as well as mitochondria and cytoskeletal elements.
We reason that the fundamental regenerative unit is a
single cytoplasmic domain containing a nucleus and chlo-
roplasts as well as smaller organelles. Thus the cytoplas-
mic phase is quantised.
This concept is supported by observations of reproduc-
tive differentiation in the related alga Dictyosphaeria cav-
ernosa, where the multinucleate cytoplasm is cleaved to
form uninucleate zooids through a contractile process that
is comparable to wound-induced cytoplasmic contraction
(Hori and Enomoto 1978). Multinucleate cytoplasmic ag-
gregates are also partitioned into uninucleate gametes by
“protrusions from the vacuole” in Cladophora flexuosa
(Scott and Bullock 1976).
Studies of the relationship between cytoplasm and nu-
cleus further support the concept of interconnected uni-
nucleate cytoplasmic domains. Nonstreaming coenocytic
cells maintain a critical ratio between cytoplasmic and
nuclear volumes (McNaughton and Goff 1990, Kapraun
and Nguyen 1994). Ventricaria cells establish nuclear-
cytoplasmic domains of fixed volume (Doyle 1935,
McNaughton and Goff 1990).
Vacuole and extracellular matrix contain
sulphated polysaccharide mucilage
The cytoplasmic domains within individual cells are embed-
ded in “vacuolar” mucilage, and an extracellular mucilagi-
nous matrix coats the exterior of young cells. Protoplasts
both contain and are coated in sulphated polysaccharide
mucilage. Wall-spanning filaments interconnect intra- and
extracellular compartments, and the cells are clustered and
embedded in a communal mucilage (Fig.1e, f).
Similarly to V. ventricosa, wound-induced protoplast
formation takes 1–1.5 h in B. forbeseii and the primary
cell wall is deposited only 2–3 h after wounding (Itoh
et al. 1984). The outer protoplast membrane quickly be-
comes a functional plasmalemma capable of synthesising
a cell wall. Extracellular sulphated polysaccharide mu-
cilage is present both before and after wall synthesis in V.
ventricosa. Persistent linkages between the plasmalemma
and cell wall tether the contracting cytoplasm to the cell
wall in both wounded Ernodesmis verticillata (Goddard
and La Claire 1993) and plasmolysed Ventricaria cells
(Fig.1f). The membrane-bound cytoplasm is everywhere
V. A. Shepherd et al.: When is a cell not a cell? 85
The preservation of cytoplasmic integrity after cutting
could be explained if the membranes are not actually sev-
ered, but deformed, or if they reseal immediately after
cutting (Menzel 1988). However, the fact that protoplasts
develop cell walls within 2–3 h (Itoh et al. 1984) is diffi-
cult to explain if a separate plasmalemma and tonoplast
initially enclose the maternal cytoplasm. The inner mem-
brane (tonoplast) would have to fuse with the outer
membrane (plasmalemma) to form a single plasmalemma
capable of synthesising a cell wall.
How is the maternal tonoplast transformed into the pro-
toplast plasmalemma? Our argument is twofold. (1) The
polarity of function of apical and basal faces of the com-
munal membrane is stabilised by the microtubule arrays
in whole cells. Environmental factors including wound-
ing, and hyper- or hypoosmotic stress disrupt the micro-
tubule arrays, thus disrupting the polarity between apical
and basolateral membranes. (2) The apical surface could
contract and the basolateral surface expand, through
processes of membrane recycling involving endocytosis
and exocytosis that have been elegantly demonstrated by
O’Neil and La Claire (1988).
Stabilisation of the polarity of the communal
membrane by cytoskeletal arrays
It is clear from the literature that the microtubule cy-
toskeleton is involved in maintaining the cytoplasmic
structure in whole cells. The behaviour of cortical and
perinuclear microtubule arrays is very different from that
of microtubule arrays of higher-plant cells. The perinu-
clear arrays are persistent (La Claire 1987), while they
are present only prior to mitosis in higher-plant cells
(Wasteneys 2002). Unlike those of higher-plant cells, the
cortical arrays are spatially separate from the perinuclear
arrays and are unchanged throughout the nuclear cycle
(La Claire 1987).
The unusual distribution and persistence of microtubule
arrays suggests a role both in maintaining spatial relation-
ships between cytoplasmic domains and in spatially
organising cytoplasmic segregation into regenerative pro-
toplasts. Figure 4a and b shows the arrangement of peri-
nuclear and cortical microtubules, in relation to the array
of nuclear centres, to the chloroplasts closer to the sur-
face, and to the proposed cytoplasmic domains. The struc-
ture of a single cytoplasmic domain is shown in Fig. 4c.
We propose that perinuclear microtubule arrays connect
the domains in whole cells (Fig. 4a), maintain the struc-
tural integrity of individual domains (Fig. 4c), and thereby
maintain the polarity of the communal membrane sur-
86 V. A. Shepherd et al.: When is a cell not a cell?
found in close proximity to sulphated polysaccharide mu-
cilage, even in these fine extensions.
What is the significance of this mucilage? We have pre-
viously shown that such mucilage significantly impacts
upon the cellular response to osmotic shock. Sulphated
polysaccharide mucilage provides a negatively charged
apoplastic barrier that reduces or prevents the opening of
Ca
2
and Ca
2
-activated Cl
ion channels in response to
hypotonic shock (Shepherd and Beilby 1999). Mucilage
provides a significant unstirred layer and reduces hy-
draulic conductivity (Shepherd et al. 1999). Mucilaginous
cells respond to hypotonic shock primarily through
mechanosensitive ion channels (Shepherd et al. 2002).
Sulphated polysaccharide mucilage in the vacuole of
Valonia utricularis significantly contributes to steady-state
turgor pressure, probably by reducing the activity of water
(Heidecker et al. 2003). The mucilage could also con-
tribute to the exclusion of Na
and accumulation of K
by
the vacuole observed by Shihira-Ishikawa and Nawata
(1992). An interesting series of experiments (Wiggins and
van Ryn 1990, Wiggins 1995) shows that gel-associated
water has a lower density than external water, and this
gel-water becomes K
selective. Thus, the mucilage has a
potent effect on ion and water relations that was not con-
sidered by early researchers.
The vacuole fails to accumulate 6-carboxyfluorescein
from the cytoplasm (Figs.1g, h and 2); although, the vac-
uoles of most higher-plant cells (Wright and Oparka 1994),
charophyte internodal cells (Shepherd and Goodwin 1992,
Beilby et al. 1999), and fungal hyphae (Shepherd et al.
1993) rapidly accumulate this fluorochrome. Vacuolar accu-
mulation of xenobiotics in plants and fungi has recently
been attributed to tonoplast-specific ABC transporters (Rea
1999). The lack of characteristic vacuolar transporter sys-
tems, the accumulation of mucilage, the presence of a
highly unusual K
pump (Hastings and Gutknecht 1974),
and the amplified surface area of the membrane (Wang
et al. 1997, Ryser et al. 1999) all make sense if the inner
membrane of V. ventricosa is indeed not a true tonoplast.
We will argue that it is the inner face of a polarised commu-
nal membrane.
Communal membrane with inwardly
and outwardly directed polarised functions
encloses cytoplasmic domains
The remarkable capacity for cytoplasmic segregation and
reassembly into regenerative protoplasts is difficult to un-
derstand if, as for C. corallina, the tonoplast and plasma
membrane are viewed as essentially separate membranes.
rounding the cytoplasmic domains. Perinuclear micro-
tubules are greatly shortened 30 min after wounding (Shi-
hira-Ishikawa and Nawata 1992) when protoplasts are
developing. This would facilitate the separation of the
fundamental cytoplasmic domains so that individual pro-
toplasts containing at least one nucleus could develop.
The polarity of the communal membrane would temporar-
ily disappear as protoplasts develop.
Maternal cortical microtubules persist in cytoplasmic
strands connecting developing protoplasts (La Claire
1987). They could maintain the orientation of the chloro-
plasts. Depolymerisation of cortical microtubules leads
V. A. Shepherd et al.: When is a cell not a cell? 87
Long cortical microtubules are regularly spaced (2 to 4 m apart) in
whole B. forbseii cells (La Claire 1987). This may reflect regular spacing
of the chloroplasts in the theorised domains. c Diagrammatic view of a
transverse section of a single cytoplasmic domain. Extracellular sul-
phated polysaccharide mucilage (m) coats the cell wall (cw). Wall-span-
ning filaments (wf) connect the protoplast to the mucilage; these are
shown in close proximity to cortical microtubules (cm) that occupy the
extremely thin layer of cytoplasm enclosed by plasmalemma (pl) or the
apical portion of the cell membrane. The tonoplast or basolateral portion
of the membrane faces the vacuolar compartment (vac). The vacuole
invaginates the thin cytoplasmic layer (cyt) that coats the organelles
(chloroplasts [chl] and nucleus [n]). The nuclei are interconnected by cy-
toplasm-coated perinuclear microtubules (pm) that span the boundaries
of the cytoplasmic domains. The tonoplast or basolateral membrane has
a greater surface area than the plasmalemma or apical membrane. The
spongelike structure of the cytoplasm–vacuole interface is based on
the electron micrographs and diagram by Shihira-Ishikawa and Nawata
(1992). d Hypothesised mechanism by which cytoplasmic contraction
produces spherical protoplasts. The cytoplasmic domains (cd) are repre-
sented diagrammatically as interconnected solids. Following wounding,
actin filaments (af) in the peripheral cytoplasmic layer contract the apical
but not the basal surfaces of the cytoplasmic domains. This forms a retic-
ulum of actin filaments (seen in La Claire 1989) when viewed from
above. At the same time, the apical-membrane material is endocytosed
and exocytosed to the basolateral membrane (as described by O’Neil and
La Claire 1988). Cortical microtubules persist in cytoplasmic strands
(cs) that connect the cytoplasmic domains. Perinuclear microtubules are
shortened, disconnecting the cytoplasmic domains from one another and
enabling formation of cytoplasts and protoplasts. The process works
through contraction of the upper surface of cytoplasm and endocytosis of
the apical membrane (plasmalemma), without contraction of the basolat-
eral regions of cytoplasm, but with exocytosis of membrane material to
the basal membrane. This would result in the formation of spherical
protoplasts enclosed by a membrane that is essentially a maternal
“tonoplast” transformed by endocytosis and exocytosis into a functional
“plasmalemma”. O’Neil and La Claire (1988) showed that the new
“tonoplast” is probably constructed from recycled maternal “plasma-
lemma”. e Diagrammatic representation of developing protoplasts. For
simplicity, single cytoplasmic domains are shown contracting into the
smallest protoplasts observed (about 10–15 m diameter). The sphere on
the left-hand side shows the interior of such a protoplast. The nucleus (n)
retains short cytoplasm-coated perinuclear microtubules and mucilage
invaginates the cytoplasm as in the postulated domains of whole cells
(drawn and shaded as in c). The sphere on the right-hand side shows the
surface view, where there is space for six flattened chloroplasts (chl).
The thin cytoplasmic strands contain cortical microtubules. These main-
tain the parietal orientation of chloroplasts
Fig. 4 a–e. Diagrammatic and schematic representations of the hypothe-
sised structure of cytoplasmic domains in V. ventricosa cytoplasm.
a Outline of a cell traced from Fig. 2 to show relationships between
perinuclear microtubules (pm), nuclear centres (nc), and the postulated
cytoplasmic domains. The nuclear centres are maximally spaced
(McNaughton and Goff 1990). The cytoplasmic domains are represented
as circles enclosing the nuclear centres. The actual geometry of the
domains is not known, although they maintain a constant ratio between
nuclear and cytoplasmic volumes. Most nuclei have either five or six
neighbours in whole cells. The nuclei lie beneath the chloroplasts and
are interconnected by cytoplasm-coated perinuclear microtubules that
span the domain boundaries. Approximate scale bar, on the basis of
Fig. 2, is 20 m. The radiating pattern of perinuclear microtubules is
based on micrographs of La Claire (1987) and Shihira-Ishikawa and
Nawata (1992). b Outline of the same cell showing relationships be-
tween cortical microtubules (cm), chloroplasts (white circles in the
central cytoplasmic domain), and the postulated cytoplasmic domains.
Chloroplasts are situated closest to the cell wall and are surrounded by
either five or six neighbours. The parallel cortical microtubules that oc-
cupy the thin surface layer of cytoplasm are depicted as straight lines.
The pattern of cortical microtubules is based on fluorescence micro-
graphs of La Claire (1987) and Shihira-Ishikawa and Nawata (1992).
to disordering and clumping of chloroplasts (La Claire
1987). A radial array of cortical microtubules develops as
the first sign of lenticular cell formation in Valonia utricu-
laris and provides positional information for cytoplasmic
aggregation, including chloroplasts, at a specific site
(Okuda et al. 1997a). The wall–membrane tethers found
in contracting Ernodesmis verticillata cytoplasm con-
tained actin microfilaments (Goddard and La Claire 1993)
and similar wall-spanning filaments in plasmolysed V.
ventricosa (Fig.1f) could anchor portions of the contract-
ing apical surface to the cell wall.
Membrane recycling and the reversal
of polarity of the communal membrane
The peripheral cytoplasm in V. ventricosa and related algae
is extremely thin (about 40 nm thick) and it is separated
from the cell wall by a smooth, planar membrane (Hori
and Enomoto 1978, La Claire 1987, Shihira-Ishikawa and
Nawata 1992), the apical portion of the communal mem-
brane (Fig. 4b). The thin peripheral cytoplasm contains the
cortical microtubules (Shihira-Ishikawa and Nawata 1992)
that persist during cytoplasmic segregation (La Claire 1987).
The highly convoluted membrane lining the inner face
of the alveolate cytoplasm is the basolateral membrane
(Fig. 4c). The basolateral membrane separates the periph-
eral and internal cytoplasm from the vacuole and coats the
cytoplasm containing the perinuclear microtubule arrays
that stabilise the cytoplasmic domains.
Cytoplasmic contraction is facilitated by the actin cy-
toskeleton (La Claire 1989). The process does not depend
on microtubules (La Claire 1987). The actin cytoskeleton is
transformed from a punctate pattern to a wide-meshed retic-
ulum of bundles about 45 min after wounding, as protoplasts
develop (La Claire 1989). Fine cytoplasmic strands (La
Claire 1987: fig.12) containing dozens of maternal cortical
microtubules as well as organelles span the gaps between re-
gions of contracting cytoplasm in B. forbseii. We have ob-
served identical strands connecting regions of contracting
Ventricaria cytoplasm 45 min after cutting the mother cell
(not shown). The actin cytoskeleton is associated with
movement of cytoplasm and organelles, whilst the cortical
microtubules provide positional information. Cortical mi-
crotubules control the position but not the movement of
chloroplasts to a specific site during lenticular cell formation
in Valonia utricularis (Okuda et al. 1997a).
We propose that the actin filaments contract the periph-
eral (apical) but not the internal (basolateral) regions of
the cytoplasmic domains (Fig. 4d, e), at the same time as
the perinuclear microtubules shorten and domains sepa-
rate. This would result in inwardly facing cytoplasmic
protrusions connected by cytoplasmic strands that contain
maternal cortical microtubules. The basolateral regions
of the cytoplasmic domains, not being contracted by
actin bundles, would correspond to the inwardly swollen
nascent protoplasts. The nascent protoplast would then be
enclosed largely by the maternal tonoplast (basolateral
membrane; Fig. 4d, e). If this is to occur, the surface area
of the maternal plasmalemma and tonoplast must change
drastically: the former decreasing, the latter increasing.
A fascinating series of papers already shows that this
can occur. First, O’Neil and La Claire (1984) showed that
numerous coated pits and vesicles form in proximity to the
plasmalemma during wound-induced cytoplasmic contrac-
tion in B. forbseii. These organelles are associated with the
endocytosis of large amounts of the plasmalemma. In a
further analysis, O’Neil and La Claire (1988) used cationic
ferritin as a marker of surface membranes and analysed
membrane dynamics during cytoplasmic contraction and
protoplast formation in B. forbseii. They produced power-
ful evidence for membrane recycling. As protoplasts de-
veloped, endocytosis of the maternal plasmalemma was
coupled with exocytosis and insertion of membrane mate-
rial in the maternal tonoplast. After 90 min, the spherical
protoplasts had become distinct entities evenly coated with
ferritin and the former tonoplast and plasmalemma were
indistinguishable. At this point ferritin-containing cyto-
plasmic vesicles began to fuse to form a new central vac-
uole. The time-course of ferritin incorporation (see O’Neil
and La Claire 1988: fig. 5) suggests that the new tonoplast
might form from recycled maternal plasmalemma. The
new surface membrane, a blend of maternal tonoplast and
recycled plasmalemma, then functions as a plasmalemma
and a cell wall forms soon after, at 120 min (O’Neil and
La Claire 1988).
The actin-induced contraction of the superficial cyto-
plasm is thus tightly coupled with endocytosis of the
maternal plasmalemma material and the exocytosis of
membrane material to the former tonoplast, which en-
closes the nascent protoplasts. Plasmalemma can be re-
cycled and merged with the tonoplast to form a new
plasmalemma, and it can be recycled to form a new tono-
plast. This supports our concept of a communal mem-
brane whose basolateral and apical surfaces enclose the
interconnected cytoplasmic domains in whole cells.
Although the cytoskeletal arrays behave differently
from those in higher plants, they have some similarities to
a tissue such as the polarised epithelium. Polarised epithe-
lia (Alberts et al. 1998) also have a system of actin bun-
dles at the apical end of the cell, a system of microtubules
88 V. A. Shepherd et al.: When is a cell not a cell?
running parallel to the cell axis (like the perinuclear mi-
crotubules connecting chloroplasts to nuclei) and a system
of microtubules running parallel to the apex of the cell
(like the cortical microtubules). This cytoskeletal arrange-
ment facilitates the process of epithelial invagination,
which resembles that of protoplast formation.
Nature of the interconnections
between cytoplasmic domains
Electron micrographs (La Claire 1987: fig. 2, Heidecker
et al. 2003) show that cortical microtubules are located in a
peripheral cytoplasmic layer only about 40 nm thick. Since
microtubules have outer and inner diameters of about 25 nm
and about 14 nm, respectively, space permits only lateral
arrangements of single cortical microtubules in the periph-
eral cytoplasm, with only about 15 nm of cytoplasm inter-
posed between microtubule surface and membrane. The
peripheral cytoplasm is so thin that its width approaches the
gross diameter of simple plasmodesmata (see Overall and
Blackman 1996: fig.1). The finest strands of the cytoplas-
mic meshwork interconnecting cytoplasmic domains (esti-
mated from Shihira-Ishikawa and Nawata 1992: figs. 2 and
3) are 80 to 100 nm in diameter, and these strands also con-
tain perinuclear microtubules. The strands have a diameter
similar to that of the pores of the intercellular communica-
tion channels in a basidiomycete fungus (about 70 nm;
Shepherd et al. 1993). As an indicator of how fine these
interconnecting strands actually are, the diameter of the
nuclear-pore complex is 83.9 nm (Jaggi et al. 2003). Com-
munication between cytoplasmic domains through the
fine interconnecting cytoplasmic strands might have some
properties in common with communication through plas-
modesmata, nuclear pores, and other intercellular channels.
Communication between domains is potentially “gated” and
could have a molecular-size exclusion limit.
Meaning of electrophysiological data
obtained from a coenocyte
How can the structural analysis described above be recon-
ciled with the electrophysiology of V. ventricosa? The
very concept of PD
co
relies on the cytoplasm being a rela-
tively homogeneous phase, which is clearly not the case
for V. ventricosa (Fig. 2). The lateral conductivity of the
interconnections between the cytoplasmic domains has
not been studied, but if it is low, or variable, PD
co
will not
have a single unique value. Furthermore, the membrane
trafficking (O’Neil and LaClaire 1988) blurs the distinc-
tion between the outer and the inner membranes. The
mosaic of thicker and thinner regions of cytoplasm
interpenetrated by the convoluted vacuole (Fig. 2) proba-
bly explains the wide range of PD measurements obtained
by different researchers. It is highly unlikely that a micro-
electrode with tip diameter of about 1m will impale
the 40 nm thick peripheral cytoplasm or the about 80
to 300 nm diameter interconnecting cytoplasmic strands.
Such a microelectrode will miss the fine cytoplasmic
meshwork and peripheral cytoplasm and enter nuclei
and/or chloroplasts (Fig. 2). The negative values for PD
co
found by Davis (1981) and Gutknecht (1966) could be the
PD of cytoplasmic organelles. The transience of negative
PDs measured by Ryser et al. (1999) could be due to re-
pair of the nystatin-treated membrane through membrane
endocytosis and exocytosis (O’Neil and La Claire 1988).
However, if the microelectrode is inserted into the centre
of the cell, it is reasonably certain that it crosses the com-
plex cytoplasm into a vacuolar region (Fig. 2). The centre
of the smallest cell in Fig. 2a is occupied by vacuolar mate-
rial. This microelectrode placement would give PD
vo
mea-
surements that are akin to a tissue potential and integrate
over all of the cytoplasmic phases and domains. Using a
central microelectrode placement, we found that small cells
gave consistent vacuole-to-outside I/V behaviour, in sea-
water and under a range of different conditions (Fig. 3)
(Beilby and Bisson 1999, Bisson and Beilby 2002).
The cells are thought to regulate turgor pressure
through ATP and pressure-dependent K
and Cl
trans-
porters in the plasmalemma (Heidecker et al. 2003). The
vacuole accumulates K
and Cl
(K
vac
and Cl
vac
are re-
spectively 21 times and 1.2 times their concentrations in
seawater) but excludes Na
(Na
vac
is only about 15.6%
of the seawater concentration; Shihira-Ishikawa and
Nawata 1992). The putative K
pump is expected to
maintain this concentration imbalance in seawater. The
I/V and G/V curves (Fig. 3a, b; pale blue line) show that a
cell in seawater is more conductive at positive PD
vo
. In
hypertonic medium (Fig. 3a, b; dark blue line) the K
pump is expected to work harder to increase the internal
K
concentration, and the cell conductance does indeed
increase, with a positive PD
vo
generated by the import of
K
. This is analogous to the negative PD
vo
in charophytes
and higher-plant cells resulting from export of H
by the
proton pump. On the other hand, we expect K
to be ex-
ported in hypotonic medium, and, as expected, the con-
ductance increases, with a negative PD
vo
generated by K
export (Fig. 3a, b; red line). These results could poten-
tially be explained by a reversal of the polarity of the K
pump in situ or by its migration to the other membrane
surface through endo- or exocytosis.
V. A. Shepherd et al.: When is a cell not a cell? 89
In the dark (Fig. 3a, b; black line) the cell has a PD
vo
close to zero and low conductance at the resting PD. Simi-
lar I/V profiles are obtained when the cell is exposed to
metabolic inhibitors that result in depletion of ATP (M. J.
Beilby and M. A. Bisson unpubl.) suggesting that the po-
larised ion transport is ATP dependent. When exposed to a
medium with high K
concentration, the cell conductance
declines, but PD
vo
remains positive at about 50 mV
(Fig. 3a, b; green line). Conversely, the PD
vo
becomes neg-
ative and the conductance slightly increases in a medium
with low K
concentration (Fig. 3a, b; orange line). We
initially interpreted the response to changes of [K
]
o
in
terms of a K
channel-dominated plasmalemma, with a
large positive offset due to a K
pump on the tonoplast
membrane (Beilby and Bisson 1999). However, the E
K
was calculated using the cytoplasmic K
concentration of
434 mM (Gutknecht 1966), which is likely to be an overes-
timate due to the complex cytoplasmic structure. These re-
sults could also be interpreted in terms of a reversal of
polarity between inner and outer membrane and/or move-
ment of the K
pump between basolateral and apical
membranes through endo- or exocytosis.
The I/V and G/V curves in seawater, low K
, and hypo-
tonic and hypertonic conditions show a marked mirror
symmetry across the PD spectrum. The hyper- and hypo-
tonic curves show a mirror symmetry in comparison with
each other. These are features we might expect of a com-
munal membrane that is dominated by polar K
transport
and which can reverse its polarity or even interchange its
surfaces following environmental stress.
Conclusion
There are key features of the V. ventricosa coenocytic struc-
ture that are essential for understanding its electrophysi-
ology. First, the cytoplasm is structured into uninucleate
domains that can separate to regenerate new cells and which
behave collectively as a tissue. Second, the cytoplasmic do-
mains are bounded by a communal membrane with basolat-
eral and apical faces whose functions are polarised in whole
cells. Third, the basolateral and apical membranes can recy-
cle components and transform into one another in response
to stress. Finally, interior and exterior mucilages profoundly
influence ion and water transport.
At first glance the ability of V. ventricosa to form new
cells from fragments of cytoplasm seems to contradict
Virchow’s dictum that all cells must come from a preexist-
ing cell. However, a coenocyte can be viewed as a gestalt.
According to Esau (1965), a coenocyte is an aggregation
of units, each consisting of a nucleus and adjacent cyto-
plasm and each representing a cell. Electrophysiological
measurements of PD
co
in such a coenocyte are unreliable,
but measurements of PD
vo
constitute a tissue potential that
reflects polarised transcytoplasmic K
transport from api-
cal to basolateral membranes. This interpretation will open
new directions for electrophysiological research.
Note added in proof. The “spongy” structure of the Valo-
nia utricularis cytoplasm was also described in a recent pa-
per by S. Mimietz et al. (Protoplasma 222: 117–128, 2003).
Acknowledgments
We thank an anonymous reviewer for constructive comments and knowl-
edge of the literature that enabled us to improve this paper. We thank
Chris Cherry-Gaedt for her contribution to early stages of this work,
Dave Logan of Heron Island Research Station for collecting the
Ventricaria ventricosa algae and Jan Latham for printing the pictures.
We are grateful to Teruo Shimmen for supplying copies of papers from
Memoirs of the Faculty of Science, Kochi University. This research was
funded by an Australian Research Council Small Grant to M.J.B.
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V. A. Shepherd et al.: When is a cell not a cell? 91
... Our finding in C. coliformis of calcium oxalate crystals within vacuoles separate from the large central vacuole suggests a separation of content and function among distinct vacuolar compartments. This possibility has consequences for studies of ion accumulation in giant algal cells, and it is also pertinent to the concept of a single system of connected vacuoles proposed for some related coenocytic algae (Mimietz et al. 2003;Shepherd et al. 2004). The parietal localization of calcium oxalate crystals in C. coliformis demonstrates another interesting variation in the packaging of calcium oxalate by the algae. ...
... An unconventional, highly dynamic concept of vacuolar structure has been described for the coenocytic algae Valonia utricularis and Ventricaria ventricosa (Ryser et al. 1999;Mimietz et al. 2003;Shepherd et al. 2004). The vacuole is viewed as a highly fenestrated system interdigitated with the cytoplasm to such an extent that the cytoplasm appears spongy, yet the vacuole is considered to be a single structure. ...
... Whether or not the cytoplasmic vacuoles are part of a highly fenestrated, spongy architecture, the precipitation of calcium oxalate crystals must occur in ionically distinct compartments separate from the larger vacuolar system. The localization of calcium oxalate crystals to the layer of small vacuoles provides a visible marker that these mineralizing vacuoles are separate from the large central vacuole, and it shows partitioning of content and function among separate vacuolar compartments, a situation differing from the proposed model of a single system of connected vacuoles in the related coenocytic alga, Valonia (Mimietz et al. 2003;Shepherd et al. 2004). ...
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Living cells of field-collected specimens of the giant-celled marine green alga Chaetomorpha coliformis (Montagne) Kutzing were found to have birefringent cellular inclusions whose composition was determined to be calcium oxalate on the basis of their reactions to diagnostic chemical solubility tests and the Yasue cytochemical staining procedure. The inclusions consisted of individual bipyramidal crystals up to 50 mu m in greatest dimension and variously sized aggregates of much smaller crystals. Some aggregates consisted of rosettes or spheres of radiating elements that resemble embryophyte druses more than any other calcium oxalate deposit yet reported in the algae. Both single crystals and aggregates occurred in the same cells with no discernible pattern to their distributions. The calcium oxalate crystals were anchored on the vacuolar face of the thin cytoplasmic layer rather than being dispersed within the voluminous central vacuole. Light and transmission electron microscopy demonstrated the presence of a nearly continuous layer of small vacuoles between the organelle-rich parietal cytoplasm and the large central vacuole, and the calcium oxalate crystals were associated with this layer of vacuoles. The occurrence of mineralization in the parietal vacuoles, but not the large central vacuole, indicates that differentiation of vacuoles according to ionic contents may be occurring.
... There have been a number of measurements made on algae, initially because some have very large cells (up to 1 cm in length) where measurements of the cytoplasmic and vacuolar ion concentrations could be more easily made than in most vascular plant cells -although the complex nature of the cytoplasm of the Cladophorales may confound some of these data (Shepherd et al., 2004). In general, freshwater algae growing in the absence of external salt contain between 1 and 50 mM Na þ (Raven, 1976;Cameron et al., 1986;Okihara and Kiyosawa, 1988;Whittington and Bisson, 1994). ...
... Techniques as different as flux analysis, measurements of slightly vacuolated tissues by atomic absorption spectroscopy and, for cells, by X-ray microanalysis all tell the same story (a conclusion borne out for Na þ and K þ , but not for Cl À , in a comparison of techniques using a non-halophyte, maize; Hajibagheri et al., 1988). However, there remains uncertainty in the distribution of ions within the cytoplasm; for example, the cytoplasm of the coenocytic algae is not homogeneous (Shepherd et al., 2004). Moreover, there has long been discussion of vesicular transport of ions in higher plants (see Lazof and Cheeseman, 1986, and references therein;Shabala and Mackay, 2011). ...
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Halophytes are the flora of saline soils. They adjust osmotically to soil salinity by accumulating ions and sequestering the vast majority of these (generally Na(+) and Cl(-)) in vacuoles, while in the cytoplasm organic solutes are accumulated to prevent adverse effects on metabolism. At high salinities, however, growth is inhibited. Possible causes are: toxicity to metabolism of Na(+) and/or Cl(-) in the cytoplasm; insufficient osmotic adjustment resulting in reduced net photosynthesis because of stomatal closure; reduced turgor for expansion growth; adverse cellular water relations if ions build up in the apoplast (cell walls) of leaves; diversion of energy needed to maintain solute homeostasis; sub-optimal levels of K(+) (or other mineral nutrients) required for maintaining enzyme activities; possible damage from reactive oxygen species; or changes in hormonal concentrations. This review discusses the evidence for Na(+) and Cl(-) toxicity and the concept of tissue tolerance in relation to halophytes. The data reviewed here suggest that halophytes tolerate cytoplasmic Na(+) and Cl(-) concentrations of 100-200 mm, but whether these ions ever reach toxic concentrations that inhibit metabolism in the cytoplasm or cause death is unknown. Measurements of ion concentrations in the cytosol of various cell types for contrasting species and growth conditions are needed. Future work should also focus on the properties of the tonoplast that enable ion accumulation and prevent ion leakage, such as the special properties of ion transporters and of the lipids that determine membrane permeability. © The Author 2014. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
... In particular, examples of endocytosis in algae are difficult to find. Membrane recycling occurs in response to mechanical wounding in the giant unicellular alga Ventricaria ventricosa where endocytosis involving coated pits allows transformation of maternal tonoplast into protoplast plasma membrane (Shepherd et al., 2004) and in Fucus distichus zygotes membrane recycling occurs during polar growth of the rhizoid (Belanger & Quatrano, 2000). Marcote et al. (2000)). ...
... Endocytosis involving coated pits has been shown to occur in the giant unicellular alga Ventricaria ventricosa in response to mechanical wounding (Shepherd et al., 2004). Recent molecular studies of sporulating tissue of Ulva linza have revealed expressed sequence tags for a clathrin vesicle coat protein . ...
... It is often ignored or forgotten that eukaryotic cells can, in fact, be viewed as multicellular ecosystems, in effect "cells within cells" [6,7]. Their organelles, such as mitochondria and plastids, are semi-autonomous endosymbiotic cells [8][9][10][11][12][13]. The symbiotic origin of the nucleus is emerging as a highly plausible scenario [9,[14][15][16][17]. Cell theory has been an important concept unifying the whole of biology and has played a central role in our understanding of life [1,[18][19][20]. ...
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Cells emerged at the very beginning of life on Earth and, in fact, are coterminous with life. They are enclosed within an excitable plasma membrane, which defines the outside and inside domains via their specific biophysical properties. Unicellular organisms, such as diverse protists and algae, still live a cellular life. However, fungi, plants, and animals evolved a multicellular existence. Recently, we have developed the cellular basis of consciousness (CBC) model, which proposes that all biological awareness, sentience and consciousness are grounded in general cell biology. Here we discuss the biomolecular structures and processes that allow for and maintain this cellular consciousness from an evolutionary perspective. Motto: Within their insulating membranes, cells can establish order …they display a sense of purpose. Nurse, P. What is Life? (2020)
... Even before completely contracting to a compact mass, protoplasts began the accumulation of amorphous β-glucan residues which polymerized into a membranous structure containing sulfated and carboxylated polysaccharides, confirming earlier observations (Shepherd et al. 2004). By the time the protoplast attained a nearly round configuration these acidic polysaccharides had collected around the protoplast, polymerized and served as its initial cover for the next 24 hours as reconstruction of the cell wall started. ...
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Ultrastructure and cytoskeletal properties of the coenocytic green alga, Valonia, were described using light field, immunofluorescence and electron microscopy to investigate the dynamics among cell wall, cell membrane, and protoplasm during cell regeneration. Protoplasts were artificially induced in three species by cutting thalli and extruding the protoplasm. Protoplasts contracted and formed irregularly shaped masses within 30 minutes concomitant with bundling of actin filaments (AFs), convolution of cortical microtubules (CMTs) and formation of a thin enveloping membrane composed of polysaccha-rides. Size affected survival rates: protoplasts less than 10 μm in diameter displayed lower viability than larger protoplasts. A new cell wall was produced within 24 hours simultaneous with CMT and AF depolymerization. AFs were reduced to granular structures and aggregates that repolymerized by 48 hours. Concurrently, new CMTs polymerized and attained a parallel arrangement. Actin-and microtubule-destabilizing agents had variable effects on protoplast contraction indicating a minor role of intact cytoskeletons in this process; however, resulting cells exhibited abnormal protoplasm distri-bution and cell deformation after three days. Rhizoids began to form after 7 days on untreated cells which subsequently produced lateral branch cells that eventually developed into mature thalli.
... Commonly used models for plant cells are large unicellular green algae [21]. They are easy to work with and the area of disturbance by a probing device, e.g. an AFM tip, is insignificant in size compared with the cell [22][23][24]. ...
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A substantial proportion of the architecture of the plant cell wall remains unknown with a few cell wall models being proposed. Moreover, even less is known about the green algal cell wall. Techniques that allow direct visualization of the cell wall in as near to its native state are of importance in unravelling the spatial arrangement of cell wall structures and hence in the development of cell wall models. Atomic force microscopy (AFM) was used to image the native cell wall of living cells of Ventricaria ventricosa (V. ventricosa) at high resolution under physiological conditions. The cell wall polymers were identified mainly qualitatively via their structural appearance. The cellulose microfibrils (CMFs) were easily recognizable and the imaging results indicate that the V. ventricosa cell wall has a cross-fibrillar structure throughout. We found the native wall to be abundant in matrix polysaccharides existing in different curing states. The soft phase matrix polysaccharides susceptible by the AFM scanning tip existed as a glutinous fibrillar meshwork, possibly incorporating both the pectic- and hemicellulosic-type substances. The hard phase matrix producing clearer images, revealed coiled fibrillar structures associated with CMFs, sometimes being resolved as globular structures by the AFM tip. The coiling fibrillar structures were also seen in the images of isolated cell wall fragments. The mucilaginous component of the wall was discernible from the gelatinous cell wall matrix as it formed microstructural domains over the surface. AFM has been successful in imaging the native cell wall and revealing novel findings such as the 'coiling fibrillar structures' and cell wall components which have previously not been seen, that is, the gelatinous matrix phase.
Chapter
Actin cytoskeleton was discovered some 70 years ago, and it is well known to be responsible for cellular transport phenomena and contractilities, with animal muscles representing the most obvious example. This ancient cytoskeletal system is present in all eukaryotic cells, responsible for all kinds of intracellular motilities. For example, the synaptic vesicle recycling also relies on the actin cytoskeleton, which supports all types of membranes structurally and functionally. Action potentials are fundamental for the long-distance signaling in both animals and plants. Although it is not generally appreciated, action potentials are mechanistically and functionally interlinked with the actin cytoskeleton associated with membranes. In both animals and plants, the inherent bioelectricity of membranes is closely linked with the actin cytoskeleton. Despite the fundamental importance of this phenomenon, it remains to be under-investigated, and future studies will be needed to illuminate the elusive electrochemical and bioelectric nature of cellular life.
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Cells and organelles are surrounded by at least one membrane that controls the exchange of energy and matter between the cytoplasm and its environment. Ion transport through membranes is an essential process for life. For instance, algae nutrition, osmotic and hydrostatic pressure regulation and cell signaling are typical cellular functions that are directly controlled by the transport of ions through the plasma membrane. The quantitative description of membrane transport through algae (and plants in general) is usually restricted to the uniform steady state case. However, spatial and temporal dynamics arising from the nonlinear properties of ion transport have recently been revealed to be of prime importance for cell signaling and developmental axis emergence. We will review the mechanisms of ion transport (active and passive transport) in marine algae and their implication in cell physiology, morphology and homeostasis. The basic principles of ion transport will be explain and we will show how the nonlinear coupling between different ion transport systems such as ATPases, channels and co-transports can give rise to self-organized spatiotemporal events such as action potentials or stationary patterns of transcellular currents observed in marine algae.
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The protoplasm of giant-celled Bryopsis species is totipotent. When protoplasm is extruded from the fresh alga into seawater, the organelles - including protoplasts, nucleus and other cell contents - can aggregate and reconstitute a whole cell, even regenerate into a mature individual. It is believed that a lectin-carbohydrate complementary system is involved in these surprising processes. To address this issue, a polyclonal antibody against the lectin in Bryopsis hypnoides was prepared and used to evaluate the lectin's role in organellar agglutination. Results showed that the agglutination of cell organelles was blocked completely by the antibody. Real-time quantitative polymerase chain reaction of this lectin gene (EU410470.1) demonstrated that it was down-regulated, with expression reaching the lowest level at 6 h and returning to maximal level at 12 h after protoplasm release. Western blotting revealed that a 43-kDa protein appeared at 3 h after protoplasm release, and then disappeared at 6 h. Immunogold localization indicated that the lectin was located in both chloroplast and cytoplasm. These results indicate that lectin plays an indispensable role in agglutination of the cell organelles and the regeneration of this giant-celled alga.
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Wounding Boergesenia forbesii cells initiates contraction and separation of the cytoplasm into numerous protoplasts, which later form cell walls. Incubating wounded cells with cationized ferritin results in differential binding to surface membranes. Initially, ferritin binding is restricted to the vacuolar side of contracting cytoplasm. During contraction, bound ferritin becomes distributed evenly around the spherating protoplasts. Pulse-labelling experiments suggest that spreading occurs by lateral flow, within the membrane plane. As lateral flow continues, the marker contacts the cytoplasmic surface nearest the parental wall, and only then does ferritin become internalized via coated membranes. Endocytic ferritin is evident in partially-coated and smooth vesicles, eventually being deposited in the central vacuole developing within each protoplast. Results indicate that localized endocytosis and exocytosis simultaneously replace or modify the surface membranes of developing protoplasts prior to cell wall formation, and internalized surface membranes or components thereof become incorporated into the vacuolar membrane within each protoplast.
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Phylogenetic relationships were investigated among species of Valonia as part of a larger generic-level study of 28 taxa in the Siphonocladales-Cladophorales complex (Chlorophyta). Data were obtained from comparisons of morphology, details of cell division, and immunological distance measurements among species. The results show that Valonia ventricosa is more closely related to Siphonocladus and Dictyosphaeria than to Valonia aegagropila, V. jastigiata, and V. utricularis. A new genus, Ventricaria, is therefore proposed to accommodate it.
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When the vegetative thallus of the gigantic unicellular marine alga, Boergesenia forbesii, is either placed in concentrated sea-water (×2.0) or subjected to mechanical stimulation (pinching with a pincette or probing with a fine glass needle), its protoplasm changes swiftly into numerous aplanospores. Most of these aplanospores develop into normal new plants in culture.
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Chromosome complements of 1 N = 12, 24 and 16, 32 are reported for four species of Siphonocladales. Spatial arrangement of nuclei through the division cycle is described. Microspectrophotometry with the DNA-localizing fluorochrome DAPI is used to estimate nuclear genome sizes in twelve species. Nuclear genome sizes estimated from nuclear volumes (NV) obtained with image analysis measurements were found to be highly correlated with estimates from fluorescence intensity (If) values. Nuclear genome sizes in these taxa range from 1.1 to 19.2 pg, with 2 C nuclei having DNA contents of 2.6–4.9 pg. Homologous haploid and diploid cells appear to maintain a constant nuclear genome content by regulating the number and/or ploidy level of nuclei. Interspecific comparisons suggest an inverse relationship between genome size and ‘area of cytoplasmic domain’ per standardized nuclear DNA unit. The possibility is discussed that the very large nuclear genomes in these algae may have two functions in addition to a genic role: (1) maintaining NY: cytoplasm ratios in large coenocytic cells, and (2) balancing increased extra-nuclear plastid DNA concentrations.
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In the large coenocytic cell of the marine green alga Chaetomorpha darwin ii, the electric potential difference, .pvo, between the vacuole and the outside seawater can have either of two distinct states, a positive, and more usual state, with .pvo = +5 mY, and a negative state with .pvo = - 29 m V. The p.d. across the plasmalemma of the cell was approximately -72 mY, and the difference between the positive and negative states occurred at the tonoplast with .pvc = + 77 m V or + 43 m V respectively. In the change from the positive state to the negative state, the electrical resistance of the plasmalemma increased from 510 to 750 n cm2 , and the resistance of the tonoplast increased from 4900 to 7100 n cm2
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Microtubule (MT) arrangements were investigated, with immunofluorescence and electron microscopy, in two related species of coenocytic green algae. Intact cells of both Ernodesmis verticillata (Kützing) Boergesen and Boergesenia forbesii (Harvey) Feldmann have two morphologically distinct populations of MTs: a highly regular cortical array consisting of a single layer of parallel, longitudinal MTs; and perinuclear MTs radiating from the surface of the envelope of each interphase nucleus. In both algae, mitotic figures lack perinuclear MTs around them. Pre-incubation with taxol does not alter the appearance of these arrays. The cortical and nuclear MTs appear to coexist throughout the nuclear cycle, unlike the condition in most plant cells. At the cut/contracting ends of wounded Ernodesmis cells, cortical MTs exhibit bundling and marked convolution, with some curvature and slight bundling of MTs throughout the cell cortices. In Boergesenia, wound-induced reticulation and separation of the protoplasm into numerous spheres also involves a fasciation of MTs within the attenuating regions of the cytoplasm. Although some cortical MTs are fairly resistant to cold and amiprophos-methyl-induced depolymerization, the perinuclear ones are very labile, depolymerizing in 5-10 min in the cold. The MT cytoskeleton is not believed to be directly involved in wound-induced motility in these plants because amiprophos-methyl and cold depolymerize most cortical MTs without inhibiting motility. Also, the identical MT distributions in intact cells of these two algae belie the very different patterns of cytoplasmic motility. Although certain roles of the MT arrays may be ruled out, their exact functions in these plants are not known.
Article
The subcellular distribution of actin was investigated in two related species of coenocytic green algae, with immunofluorescence microscopy. Either no, or fine punctate fluorescence was detected in intact cells of Ernodesmis verticillata (Kützing) Børgesen and Boergesenia forbesii (Harvey) Feldmann. A reticulate pattern of fluorescence appears throughout the cortical cytoplasm of Ernodesmis cells shortly after wounding; this silhouettes chloroplasts and small vacuoles. Slender, longitudinal bundles of actin become evident in contracting regions of the cell, superimposed over the reticulum. Thicker portions of the bundles were observed in well-contracted regions, and the highly-convoluted appearance of nearby cortical microtubules indicates contraction of the bundles in these thicker areas. Bundles are no longer evident after healing; only the reticulum remains. In Boergesenia, a wider-mesh reticulum of actin develops in the cortex of wounded cells, which widens further as contractions continue. Cells wounded in Ca(2+)-free medium do not contract, and although the actin reticulum is apparent, no actin bundles were ever observed in these cells. Exogenously applied cytochalasins have no effect on contractions of cut cells or extruded cytoplasm, and normal actin-bundle formation occurs in treated cells. In contrast, erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA) completely inhibits longitudinal contractions in wounded cells, and few uniformly slender actin bundles develop in inhibited cells. These results indicate that wounding stimulates a Ca(2+)-dependent, hierarchical assembly of actin into bundles, whose assembly and functioning are inhibited by EHNA. Contraction of the bundles and concomitant wound healing are followed by cessation of motility and disassembly of the bundles. The spatial and temporal association of the bundles with regions of cytoplasmic contraction, indicates that the actin bundles are directly involved in wound-induced cytoplasmic motility in these algae.
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Plasmodesmata are minute channels that traverse the plant cell wall to provide a cytoplasmic pathway for communication between neighbouring cells. Recently, these connections have been shown to transport molecules much larger than previously thought possible. Indeed, plasmodesmata now appear to be highly dynamic structures that can actively and selectively transport very large molecules between cells. Emerging physiological and molecular data must now be incorporated with information from electron microscopy to generate a dynamic model for their structure.