Plant Cell, Vol. 12, 2425-2440, December 2000, Copyright © 2000, American Society of Plant Physiologists
Plant Nuclei Can Contain Extensive Grooves and
Invaginations
David A. Collings1,a,
Crystal N. Cartera,
Jochen C. Rink2,a,
Amie
C. Scotta,
Sarah E. Wyatt3,a, and
Nina Strömgren Allena
a Department of Botany, North Carolina State University, Raleigh,
North Carolina 27695-7612
Correspondence to:
Nina Strömgren Allen, nina_allen{at}ncsu.edu (E-mail), 919-515-3436 (fax)
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ABSTRACT |
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Plant cells can exhibit highly complex nuclear organization. Through dye-labeling
experiments in untransformed onion epidermal and tobacco culture cells and through the expression of
green fluorescent protein targeted to either the nucleus or the lumen of the endoplasmic
reticulum/nuclear envelope in these cells, we have visualized deep grooves and invaginations into the
large nuclei of these cells. In onion, these structures, which are similar to invaginations seen in
some animal cells, form tubular or planelike infoldings of the nuclear envelope. Both grooves and
invaginations are stable structures, and both have cytoplasmic cores containing actin bundles that can
support cytoplasmic streaming. In dividing tobacco cells, invaginations seem to form during cell
division, possibly from strands of the endoplasmic reticulum trapped in the reforming nucleus. The
substantial increase in nuclear surface area resulting from these grooves and invaginations, their
apparent preference for association with nucleoli, and the presence in them of actin bundles that
support vesicle motility suggest that the structures might function both in mRNA export from the
nucleus and in protein import from the cytoplasm to the
nucleus.
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INTRODUCTION |
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Although cellular function often requires maximization of
surface area relative to volume, notably in organelles such as the endoplasmic reticulum (ER) and
Golgi apparatus, traditional representations of the nucleus depict a rounded structure with little
internal organization. Recently, however, the nuclei of animal cells have been found to show
considerable spatial and structural organization at the chromosomal level. The discrete and
comparatively stable territories that chromosomes occupy within the nucleus are separated by
interchromosomal domains through which transcribed RNA and other macromolecules can diffuse (reviewed
in Lamond and Earnshaw 1998 
). Animal nuclei also sometimes deviate from the characteristic
rounded shape. Deviations include a folded, grooved, or notched surface (Majno et al. 1969 ) and tubular invaginations. Electron microscopy has shown that these nuclear invaginations can
penetrate deep into the nucleus and confirmed the presence of the double membrane of the nuclear
envelope (Ellenberg et al. 1997 ; Fricker et al. 1997 ; Clubb and Locke 1998 ). However, revelation of the full nature of nuclear invaginations in living
animal cells has required confocal microscopy of fluorescent dyes (Fricker et al. 1997 ; Lui et al. 1998a , Lui et al. 1998b ) or green fluorescent protein
(GFP) fusion proteins targeted to the nuclear envelope (Ellenberg et al. 1997 ; Broers et al. 1999 ). The number and nature of invaginations vary from cell type to cell type,
ranging from simple invaginations to intricate branched structures that can penetrate into and through
the nucleus. Invaginations often associate with nucleoli (Fricker et al. 1997 ) and appear
to be stable (Ellenberg et al. 1997 ; Broers et al. 1999 ). Nuclear grooves
and invaginations substantially increase the surface area of the nucleus and have been suggested to
function in signaling from the cytoplasm to the nucleus (Lui et al. 1998a , Lui et al. 1998b ) or to coordinate transport processes between the nucleus and the cytoplasm (Fricker et al. 1997 ). However, whether any relationship exists between the distribution of
nuclear invaginations and chromosome domains within the animal nucleus remains to be
determined.
Plant nuclei show numerous structural and organizational features that are similar
to those of animal cells. Chromosomes occupy specific territories within the nucleus (Heslop-Harrison et al. 1990 ; Abranches et al. 1998 ; Gonzalez-Melendi et al. 2000 ; reviewed in Heslop-Harrison and Bennett 1990 ; Franklin and Cande 1999 ) and can be dynamic, having the ability to change
shape (Heslop-Harrison and Heslop-Harrison 1989 ; Chytilova et al. 2000 ).
Plant nuclei, too, can deviate from the traditional rounded structures shown in
textbooks in various ways. For example, placental cells in Lilium ovaries contain a nuclear reticulum
in which the inner nuclear membrane forms extensive tubular invaginations that penetrate through the
center of the nucleus, the lumen of this reticulum being contiguous with the lumen of the ER (Singh and Walles 1995 ; Singh et al. 1998 ). This type of structure differs from
the shallow and narrow invaginations of both membranes of the nuclear envelope found in developing
microspores of various species (Aldrich and Vasil 1970 ; Dickinson and Bell 1970 , Dickinson and Bell 1972 ; Li and Dickinson 1987 ) and also differs
from nuclear vacuoles, membrane-bound inclusions within the nucleus that are also generally found in
meiotic cells (Sheffield et al. 1979 ; Karasawa and Ueda 1983 ; Sangwan 1986 ; Yi et al. 1994 ). Additional published examples document deep
cytoplasmic channels penetrating into the nuclei of nonreproductive cells of various species,
including Narcissus (Gunning and Steer 1996 ), Pisum (Bowes 1996 ), and onion
epidermis (Kartusch et al. 2000 ). In fact, the large nuclei of onion epidermal cells show
marked irregularities in nuclear shape, having large groovelike infoldings that contain rapid
cytoplasmic movement (Lichtscheidl and Url 1988 ; N.S. Allen, personal observation).
However, whether these nuclear infoldings represent invaginations or slices through nuclear grooves
cannot be determined without optical sectioning techniques. Furthermore, the function (if any) of
these structures remains unknown; as with animal cells, however, the great increase in the
contact area between the nucleus and the cytoplasm may be important.
The current study evolved
from confocal investigations with fluorescently labeled probes that nonspecifically labeled the
interior membranes of onion epidermal cells, revealing not only ER organization and dynamics but also
extensive grooves that scored the nucleus. Subsequently, we have transiently expressed GFP targeted
either to the nucleus (N-GFP) or to the ER (ER-GFP) to characterize these grooves, along with tubular
nuclear invaginations, in living onion nuclei. We have furthermore observed similar structures in the
nuclei of suspension-cultured tobacco cells. These structures, similar to the grooves and
invaginations described by Ellenberg et al. 1997 , Fricker et al. 1997 , Lui et al. 1998a , Lui et al. 1998b , and Broers et al. 1999 ,
substantially increase the surface area of the nucleus. Importantly, investigating nuclear grooves and
invaginations in GFP-expressing cells should further our understanding of communication between the
nucleus and the cytoplasm.
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RESULTS |
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Untransformed Onion Epidermal Nuclei
Are Highly Convoluted and Contain Deep Grooves and Invaginations
Untransformed onion epidermal
nuclei are lens-shaped, are as large as 30 µm in diameter, and as shown by
4',6-diamidino-2-phenylindole (DAPI) labeling of nuclear DNA in Fig 1 (and the QuickTime movie Fig 1included in the
online version of this article), contain deep grooves that score their surface and from which DAPI
labeling was absent (G in Fig 1A [and in Fig 1sequence 1]). These grooves penetrated as much as 6 µm into the nucleus,
deeper than previous differential interference contrast (DIC) video microscopy might suggest (Lichtscheidl and Url 1988 ). DAPI labeling was also absent from tubular invaginations that
projected as far as 8 µm into the center of the nucleus (Fig 1A,
arrow [ Fig 1sequence 1, arrow]). Furthermore, image reconstructions in
orthogonal planes (Fig 1A, planes XZ and YZ) confirmed that DAPI-labeled
DNA surrounded these nuclear invaginations. DAPI labeling was likewise absent from nucleoli (Nc in
Fig 1A and Fig 1B [and in Fig 1sequence 1]), which in this case were separated by a nuclear groove.
Interestingly, one nucleolus lay close to the nuclear invagination (Fig 1A, arrow). Three-dimensional image reconstructions with a surface shadowing
algorithm emphasized the highly convoluted shape of this onion nucleus, showing dramatically how the
deep grooves score the nuclear surface and how the invagination penetrates through the nucleus (Fig 1C [ Fig 1sequence 2]). Concurrent DIC
imaging clearly visualized nuclear grooves and invaginations (G and arrow in Fig 1B) and demonstrated that these structures lay outside the nucleus and were not
simply areas within the nucleus that lacked DNA.
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Figure 1.
Untransformed Onion Epidermal Nuclei Are Highly Lobed and
Grooved.
(A) and (B) Optical sections shown at 2.0-µm intervals from the
surface of an untransformed onion nucleus, as indicated by numbers in the lower right of sections in
(A). (A) Confocal fluorescence images of DAPI-labeled DNA, revealing numerous nuclear
grooves (G), two nucleoli (Nc) in separate nuclear lobes, and an invagination (black arrows) that
could be followed through multiple focal planes. White bars in the section at 6 µm from the
nuclear surface show the locations at which Metamorph software was used to reconstruct orthogonal
slices in the XZ and YZ planes from the entire data set. These reconstructed slices are shown in the
corresponding inset, where white arrows indicate the plane of the original optical section. These
reconstructions confirm that the nuclear invagination is surrounded entirely by DNA. (B)
Concurrent DIC images showing that grooves (G), nucleoli (Nc), and the nuclear invagination (black
arrow) are all visible with transmitted light.
(C) Three-dimensional reconstruction with
surface shadowing of DAPI fluorescence from (A), revealing the convoluted shape of the nucleus.
The nucleus was rotated 27° in the direction of the arrow between the left and central projections
and shows the grooves (G) that lay across the nuclear surface. Rotation through a further 153°
shows the nucleus in cross-section roughly at its center, where two nucleoli (Nc) and the nuclear
invagination (arrows) are seen.
(D) Polarization modulation DIC images (Holzwarth et al. 1997 ) of an onion nucleus showing the nuclear envelope (arrowheads), nucleolus
(Nc), and two nuclear grooves (G). A single vesicle (arrows) moved rapidly through one of these
grooves at  3 µm sec-1. Times in seconds are shown at the lower right of each
image.
(E) and (F) Optical sections shown at 6-µm intervals from the surface
of an untransformed onion nucleus, as indicated by numbers in the lower right of sections in
(F). The nucleus is seen in cross-section and lies adjacent to the cell wall. (E)
Confocal fluorescence images of DIOC6(3) labeling of internal membranes show bright
mitochondria (Mt) adjacent to the nucleus and extensive nuclear invaginations (arrow) and grooves (G)
inside the nucleus. Note that the nuclear groove lies very close to the position of the nucleolus.
(F) Concurrent DIC images showing that nucleoli (Nc) and the nuclear invagination (arrow) are
visible with transmitted light. CW, cell wall.
Bar in(B) = 10 µm for (A)and (B); bar in
(C) = 10 µm; bar in(D) = 2 µm; bar in(F) = 10 µm for (E)and (F).
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That nuclear grooves and invaginations lie
outside the onion nucleus was further confirmed by high-resolution polarization-modulation DIC video
microscopy (Holzwarth et al. 1997 ). Two grooves that penetrated several micrometers into
the nucleus were visible, one of which lay adjacent to a nucleolus (Fig 1D, Nc) and showed rapid cytoplasmic streaming (3 µm
sec-1) of spherosomes and other small vesicles (Fig 1D,
arrow). Vesicle movement was also seen in nuclear invaginations (data not shown).
Confocal
imaging of 3,3'-dihexyloxacarbocyanine iodide (DIOC6(3)), a membrane-permeant
carbocyanine dye that labels both ER and mitochondria, further demonstrated the presence in onion
nuclei of deep grooves and invaginations (Fig 1E), although this
technique presented several problems, including rapid fading of signal and labeling of other particles
such as mitochondria. In both fluorescence (Fig 1E) and DIC images
(Fig 1F), extensive nuclear grooves and invaginations (arrow) were
visible, often close to the nucleoli.
Nuclear-Targeted GFP Confirms the Irregular
Shape of Onion Nuclei
Confocal optical sections through the nuclei of living onion cells
transiently expressing N-GFP confirmed observations made with DAPI labeling. Because the N-GFP is not
a DNA stain and is free to diffuse throughout the nucleus (Chytilova et al. 2000 ), the
extensive nuclear grooves (Fig 2A, Fig G)
that slice deeply into the nuclear surface near to nucleoli (Fig 2A, Nc)
must lie outside the nucleus, rather than being DNA-free compartments inside the nucleus. The
resulting highly convoluted shape of the nucleus is visible in three-dimensional reconstructions
(Fig 2B).
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Figure 2.
N-GFP Confirms the Presence of Nuclear Grooves in Onion
Cells.
(A) Optical sections at different depths into the nucleus of an
N-GFP–expressing onion cell (relative distances in micrometers are shown at the lower right of
each image) reveal the complex nuclear organization typical of onion epidermal cells. Nuclear grooves
(G) exclude N-GFP, which lie outside the nucleus, although nucleoli (Nc) within the nucleus also
exclude the N-GFP.
(B) A three-dimensional reconstruction of N-GFP in the nucleus from
(A) confirms the convoluted shape of the nucleus. The nucleus was rotated 22° in the
direction of the arrow between projections.
Bars in(A) and (B) = 5
µm.
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ER-GFP Localizes to the Nuclear Envelope
That Bounds Invaginations and Grooves
Imaging of ER-GFP demonstrates dynamic subcortical ER and
stable polygonal arrays of cortical ER (Haseloff et al. 1997 ; Ridge et al. 1999 ; Scott et al. 1999 ), which we used, along with the presence of cytoplasmic
streaming, to confirm that the cells remained healthy during the experiments. However, as shown in
Fig 3, ER-GFP also reveals nuclear structure because the lumen of the ER
is continuous with the nuclear envelope (Herman et al. 1990 ).
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Figure 3.
Three-Dimensional Reconstructions of ER-GFP Fluorescence Reveal the Highly
Complex Organization of Onion Nuclear Grooves and Invaginations.
Confocal optical sections were
taken at 0.4-µm intervals through an ER-GFP–expressing onion cell but are presented here at
1.6-µm intervals in sections labeled XY, with relative depths in micrometers given at the lower
right of each section. Orthogonal slices in the XZ and YZ planes were calculated from the entire data
set at the locations marked with bars in the XY sections and are presented in the insets labeled XZ
and YZ, above and at the left of the XY images. Arrows in the corresponding XZ and YZ orthogonal
sections indicate the planes of the original optical sections. The lens-shaped nucleus in this cell is
typical of nuclei in the onion epidermis. It is squeezed between the central vacuole (V) and the cell
wall (CW). A thin layer of cortical ER (CER) is present between the plasma membrane and the nucleus.
Well-defined but branched nuclear grooves (G) wind through the nucleus, whereas invaginations are
visible as projections in longitudinal section or as rings in cross-section (asterisks). Such rings
appear as projections in the orthogonal reconstructions (arrowheads). Grooves and invaginations often
show nonfluorescent centers, which suggests that they contain cytoplasmic cores. Bar = 10
µm for all sections and reconstructions.
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Confocal optical
sections (Fig 3, XY planes) through an ER-GFP–expressing onion
cell, and three-dimensional reconstructions of the orthogonal planes derived from these sections (XZ
and YZ), show how the lens-shaped nucleus was squeezed between the central vacuole and the cortical ER
layer adjacent to the cell wall. Consistent with DAPI and DIOC6(3) observations, two
distinct variations from a rounded nuclear structure were apparent. First, the nucleus contained
numerous grooves—planes of cytoplasm, often narrow (one to several micrometers wide) and
branched—that extended many micrometers into the core of the nucleus and split the nucleus into
several lobes. The nucleus shown in Fig 3 also contained nuclear
invaginations, linear projections of ER that also can extend several micrometers into the nucleus.
When seen in longitudinal section, the invaginations looked similar to nuclear grooves, but when
cross-sectioned they were visualized as rings. However, invaginations seen as rings in the XY plane
appeared as linear or curved structures in the reconstructed perpendicular XZ and YZ planes
(arrowheads). Neither nuclear grooves nor nuclear invaginations were found in any preferred
orientation.
As shown in Fig 4 (and the online movie Fig 4some nuclei showed variations in nuclear invaginations. Faint and branched
channels of ER-GFP traversed the entire nucleus (Fig 4A, arrow [ Fig 4arrows]) but were not visible in transmitted light (data not shown), nor
were they visible with DAPI counterstaining. However, DAPI staining confirmed that the location of
these ER channels was entirely surrounded by DNA, indicating that they were tubular in nature rather
than deep grooves (Fig 4B). ER-GFP was also prominent in the nuclear
grooves found between nuclear lobes (Fig 4A, Fig G). The saturation of the fluorescence signal apparent in these locations, and
the lack of discernable structure because of this, resulted from the need to detect faint fluorescent
signals from the nuclear channels.
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Figure 4.
ER-GFP Confirms That Invaginations Can Pass Entirely
through the Onion Nucleus.
(A) ER-GFP accumulates in nuclear grooves (G) with the
fluorescence signal here becoming saturated so that several fainter ER strands can be imaged. These
strands (arrow) represent an invagination that passes completely through the nucleus.
(B)
DAPI labeling confirms that the location of the ER strands, marked by the arrow in the identical
location as that shown in (A), occurs in the center of the nucleus.
Shown are
simultaneous confocal optical sections of ER-GFP and DAPI-labeled DNA in a lens-shaped onion nucleus,
seen in cross-section. Relative depths from the approximate center of the nucleus are indicated in
micrometers at the lower right side of each section in (A). Bar in(B) = 10 µm for (A)and (B).
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Actin Is Present in Grooves and
Invaginations
The presence of actin bundles around the nucleus of permeabilized onion epidermal
cells is demonstrated in Fig 5. Actin is also prominent in nuclear
grooves visualized with DAPI-labeling of the DNA (Fig 5A, arrows) and
DIC optics (Fig 5D, arrows). Actin bundles are also present in the
highly convoluted nucleus, within invagination-like structures (Fig 5B,
Fig 5C, and Fig 5F). These observations
are consistent with the presence of cytoplasmic streaming within grooves (Fig 1D).
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Figure 5.
F-Actin Is Present in Onion Nuclear Grooves and Surrounds the
Nucleus.
Optical sectioning of a glycerol-permeabilized onion epidermal cell. Relative depths
from the top of the nucleus are given in micrometers and are indicated at the bottom right of sections
shown in (A). Arrows placed in identical locations in (A) to (D) show the
locations of actin-containing nuclear grooves.
(A) A highly convoluted nuclear shape is
defined by DAPI labeling of DNA.
(B) Rhodamine–phalloidin—labeled actin is
present in several nuclear grooves and also apparently within the nucleus (F).
(C) False
color images showing both DAPI (cyan) and actin (red) indicate the presence of actin in grooves and in
other sites within the nucleus (Nc) that are presumed to be invaginations.
(D) Nuclear
grooves (arrows) and nucleoli (Nc) were visible by transmitted light in DIC optics.
Bar
in (D) = 10 µm for(A) to(D).
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Onion Nuclear Grooves and Invaginations Are Stable
Structures
The plant nucleus is a motile structure, showing actin-dependent rotation and
translocation within the cell (Chytilova et al. 2000 ). Although this dynamism partially
confounded our analyses, time-lapse studies of ER-GFP–expressing and DAPI-labeled cells
confirmed that the nuclear lobes, grooves, and invaginations of onion epidermal cells were stable, as
shown in Fig 6 and Fig 7 (online movie
Fig 6A DAPI-labeled nucleus of an ER-GFP–expressing cell having a
deep groove between two lobes, with each lobe containing a single nucleolus, was visualized at a
single focal plane by time-lapse confocal microscopy for 1 hr. Although the distribution of
perinuclear ER showed rapid changes, groove structure as visualized with DAPI, ER-GFP, and DIC optics
(Fig 6A to 6C, respectively) remained constant. Rendering of the full
time-lapse data set into a movie sequence confirmed the dynamism of the perinuclear ER and the
stability of the prominent nuclear groove; moreover, it demonstrated that the nucleus slowly
rocked backward and forward (Fig 6Observations of N-GFP–expressing
onion epidermal cells demonstrated similar stability (data not shown).
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Figure 6.
Structures of Invaginations and Grooves in Onion Nuclei Are
Stable.
This confocal time-lapse sequence shows an ER-GFP–expressing cell at a single
focal plane. Times in minutes are given at the lower left of time-lapse images shown in
(A).
(A) DAPI fluorescence revealed the presence of two nucleoli (Nc) that were
present in different nuclear lobes separated by one of several prominent nuclear grooves
(G).
(B) ER-GFP fluorescence showed little change of nuclear-associated ER but
substantial changes in ER patterns around the nucleus.
(C) Concurrent DIC images showed
that the location of the two nucleoli (Nc) remained constant, demonstrating that the nucleus moved
little through the focal plane.
Bar in (C) = 10
µm for (A) to(C).
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Figure 7.
Onion Nuclear Structure Is Stable, Even after Disruption of the
Actin Cytoskeleton.
(A) Optical sections through an ER-GFP–expressing onion cell
demonstrate numerous nuclear grooves (G). Relative depths from the top of the nucleus are shown in
micrometers at the lower right of sections.
(B) Time-lapsed confocal sections show that
grooves remain unchanged both before and after actin disruption. Times in minutes before (minus sign)
and after the addition of 1 µM cytochalasin D are shown at the lower right of the time-lapse
images. Because the nucleus moved slightly within the cell, and because of movement of the onion
epidermal layer on the microscope stage, this sequence comprises images from optical stacks that were
manually aligned.
Bar in (B) = 10 µm for(A) and(B).
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Disruption of actin with
cytochalasin D (1.0 µM) does not destabilize the nuclear grooves, although such treatment inhibits
cytoplasmic streaming (Fig 7). As Fig 7A
shows, confocal optical sectioning revealed the presence of extensive grooving within a nucleus. The
addition to that nucleus of 1 µM cytochalasin D (at t = 0 min), a concentration
that disrupts actin organization in onion epidermal cells (data not shown), did not affect nuclear
shape or organization of the nuclear grooves (Fig 7B); the
cytochalasin did, however, stop cytoplasmic streaming (data not shown). Similarly, microtubule
depolymerization with oryzalin (10 µM) did not modify nuclear structure (data not
shown).
Tobacco NT1 Nuclei Contain Invaginations and Grooves That May Originate
during Cell Division
The nuclei of tobacco NT1 suspension-cultured cells are large, as large as
25 µm in diameter; appear to have a smooth surface, unlike the convoluted shape of onion
nuclei; and contain a single, prominent nucleolus. DIOC6(3) labeling of NT1 cells
showed the dynamic arrays of cortical and subcortical ER normally found in plant cells but also
revealed membranous inclusions into many nuclei in the form of narrow invaginations or channels
through the nucleus (data not shown). However, these inclusions were difficult to image because of
image fading and a high background.
Stable transformants of the NT1 cell line that express
ER-GFP have proved ideal for investigating nuclear invaginations, because at least 20% of the cells
from cultures undergoing logarithmic growth and a high percentage of cells from a stationary-phase
culture demonstrate various forms of nuclear channels and invaginations (S.L. Gupton, D.A. Collings,
and N.S. Allen, unpublished data). As shown in Fig 8,
ER-GFP–expressing cells showed normal patterns of dynamic ER organization in the cell cortex
(Fig 8A, 0 µm). Fig 8A also shows two
different types of ER-bounded nuclear inclusions: a long ER strand arched through the nucleus in
multiple optical sections (arrows), whereas several smaller invaginations were present in the nearly
spherical nuclear surface (arrowheads). When the fluorescence image was overlaid on the DIC image, the
ER strand was clearly shown to be wrapped around the nucleolus (Fig 8C,
arrows), and many of the invaginations projected in toward the nucleolus (arrowheads). However,
neither channels nor invaginations were visible with transmitted light (Fig 8B). Furthermore, in most tobacco NT1 cells in which nuclear invaginations and
channels were observed, they clearly associated with the nucleoli.
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Figure 8.
Nuclear Grooves and Invaginations Occur in Tobacco NT1
Suspension-Cultured Cells.
Selected optical sections of a tobacco NT1 cell stably expressing
ER-GFP are shown at various intervals from the cell surface. The depths in micrometers are indicated
at the lower right of sections in (C).
(A) ER-GFP demonstrates a single
groove-like structure (arrows) and several invaginations (arrowheads) within the nucleus
(N).
(B) Concurrent DIC images show a well-defined nucleolus (Nc) at the center of the
nucleus (N). Grooves and invaginations could not be visualized in transmitted light.
(C)
A composite image showing GFP (red) and transmitted light (cyan) demonstrates that the nuclear groove
(arrows) wraps around the nucleolus.
Bar in (B) = 10
µm for (A) to(C).
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The presence of nuclear
invaginations and channels in tobacco NT1 cells immediately after cytokinesis suggests that such
structures form during cell division. Fig 9 shows that nuclear channels
and invaginations occurred in cells immediately after reformation of the nuclear envelope, even before
the cell plate had fully fused to the parent cell wall. Importantly, in both daughter nuclei, there
was a close association of the nuclear channels and invaginations with the redeveloping nucleoli that
had yet to return to their normal spherical shape (Fig 9A and Fig 9C). Furthermore, in the majority of cases observed, the nuclear channels
ran parallel to the presumed orientation of the spindle axis, as judged by the position of the
expanding cell plate (Fig 9B).
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Figure 9.
Tobacco NT1 Nuclear Invaginations and Grooves May Form during
Cytokinesis.
Two tobacco cells undergoing cytokinesis demonstrate that transnuclear ER strands
may form during cell division.
(A) ER-GFP fluorescence shows that cytokinesis is
incomplete, because the cell plate has yet to fuse completely with the parent cell walls (arrowhead)
and remains wavy (arrow), although the nuclear envelopes have reformed around the daughter nuclei. Two
types of irregularities occur in these nuclei, with the upper nucleus showing an invagination (I),
whereas the lower nucleus shows a channel that passes completely through the nucleus and is surrounded
both above and below by the nucleus (C). Note that this channel lies parallel to the presumed spindle
axis.
(B) Concurrent DIC images confirm that the cell plate has yet to fuse with the
parent cell wall (arrow and arrowhead) and that neither channels nor invaginations are visible by
transmitted light. However, comparison of fluorescence and transmitted light images indicates that
both the channel and the invagination show close association with the redeveloping nucleoli (Nc) of
the daughter cells.
Optical sections at different depths from the surface of the upper cell are
shown in micrometers at the lower right of sections in (B). Bar in(B) = 10 µm for (A)and (B).
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As found with onion epidermal
nuclear grooves and invaginations, the channels and invaginations present in tobacco NT1 cells,
including the cell shown in Fig 8, were stable and remained unchanged
for as long as 1 hr (data not shown).
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DISCUSSION |
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Through dye-labeling
experiments in untransformed cells, and through the expression of GFP targeted to either the nucleus
or the lumen of the ER/nuclear envelope, we have demonstrated that the nuclei of several plants
contain various structures such as grooves, invaginations, and channels that greatly increase the
surface area of the nucleus. Although no function can yet be ascribed to these structures, their
apparent association with nucleoli suggests that they could function in increasing nucleocytoplasmic
trafficking of proteins and RNA.
Transient Expression of GFP Visualizes Grooves and
Invaginations in Onion Nuclei
The nuclei of onion epidermal cells deviate from a lenslike shape
in two ways—through the formation of nuclear grooves and nuclear invaginations. Invaginations
are linear projections of cytoplasm into the nucleus, surrounded on all sides by the nucleus, whereas
grooves are sheets of cytoplasm folded or trapped between lobes of the nucleus. (Nuclear channels were
also observed on occasions, but we consider these a special case of nuclear invagination that extends
entirely through the nucleus.) These structures share several common features, including being bounded
by the nuclear envelope/ER, and three pieces of evidence confirming that they both contain cytoplasmic
cores. First, the centers of both grooves and invaginations lack fluorescence when seen in
cross-section; second, both grooves and invaginations contain actin, which supports vesicle
motility and cytoplasmic streaming; and third, ER dynamics sometimes are observed within grooves
and invaginations.
Overnight incubation of epidermal peels on agar plates, along with the
ensuing transient expression of GFP constructs, does not induce the formation of nuclear grooves and
invaginations in onion epidermal cells. These structures were also found in untransformed tissue and
could be observed by DIC video microscopy, by DAPI and DIOC6(3) labeling, and by the
microinjection of fluorescently labeled 70-kD dextrans, the molecular mass of which exceeded the
exclusion limit for passive diffusion through nuclear pores (data not shown). Furthermore, we can see
no immediately apparent way in which N-GFP, a ß-glucuronidase (GUS)–GFP fusion protein with
a nuclear localization sequence (Grebenok et al. 1997 ), or ER-GFP, a fusion of GFP with ER
targeting and retention sequences (Haseloff et al. 1997 ), might induce the formation of
nuclear grooves and invaginations. This is not necessarily the case for all GFP constructs. For
example, although Ellenberg et al. 1997 could observe nuclear invaginations in
untransformed cells, the frequency of invaginations was greater in cells expressing GFP fused to the
lamin B receptor, a protein normally found on the nuclear face of the nuclear envelope, indicating
that expression of this transgene might induce the formation of invaginations.
Nuclear grooves
and invaginations appear to associate preferentially with nucleoli in onion epidermal cells. This was
apparent with transmitted light (Fig 1D), DAPI-labeling of DNA (Fig 1A, Fig 1B, Fig 5C, and Fig 6A), transmitted light in combination
with DIOC6(3) labeling of internal membrane (Fig 1E and Fig 1F), N-GFP localized to the nucleus (Fig 2A), and transmitted light in combination with ER-GFP (Fig 6B and Fig 6C). Detailed measurements of this
phenomenon, and an accompanying statistical analysis to determine whether the apparent association is
indeed significant, lie beyond the scope of this article. However, because of the functional
implications of such a localization (see below), further investigation is required.
If nuclear
grooves and invaginations occur in untransformed onion epidermal cells, why have they not been
characterized previously? Previous research has demonstrated the presence of groovelike structures in
onion epidermal cells by DIC (Lichtscheidl and Url 1988 ; N.S. Allen, personal
observation) and electron microscopy (Kartusch et al. 2000 ). These methods, however, are
limited in their ability to resolve structures in three dimensions. Confocal microscopy of
DIOC6(3) labeling partially overcame such problems but was limited by rapid fading, the
labeling of other organelles (including mitochondria), and the possibility that long-term treatment
with DIOC6(3) might itself induce changes in ER organization. GFP is thus an ideal way to
visualize invaginations and grooves, but even the approach used in this study has its limitations.
Unlike the studies of Ellenberg et al. 1997 and Broers et al. 1999 , in which
fusion to specific proteins (lamin B receptor or lamin A, respectively) restricted GFP to the nuclear
envelope, our ER-GFP construct diffused freely throughout the ER. This made the imaging of faint
intranuclear structures against a background of ER accumulation around the nucleus difficult and often
required the fluorescence signal from the ER to be saturated, as is apparent in Fig 4.
Nuclear Invaginations in Onion Are Similar to Structures
Seen in Animal Cells
The nuclear invaginations found in onion epidermal cells are unlike the
various small nuclear invaginations and nuclear vacuoles primarily associated with meiotic plant cells
(Sassen 1964 ; Aldrich and Vasil 1970 ; Dickinson and Bell 1970 , Dickinson and Bell 1972 ; Sheffield et al. 1979 ; Karasawa and Ueda 1983 ; Sangwan 1986 ; Li and Dickinson 1987 ).
Furthermore, because the structures in onion contain a cytoplasmic core, they are unlike the nuclear
reticulum found in Lilium ovary placental cells, in which only the inner membrane of the nuclear
envelope invaginates, so that the centers of these invaginations are continuous with the lumen of the
ER rather than the cytoplasm (Singh and Walles 1995 ; Singh et al. 1998 ).
Grooves and invaginations in onion may be similar to the deep cytoplasmic grooves
occasionally reported in electron micrographs of various different plant nuclei, including
Narcissus (Gunning and Steer 1996 , see plate 29) and Pisum (Bowes 1996 , see plate 2.23). Interestingly, extensive infolding has also been shown in onion
epidermal nuclei by electron (Kartusch et al. 2000 , Figure 3B) and DIC video microscopy
(Lichtscheidl and Url 1988 ). However, the true nature of these structures, and whether
they represent nuclear grooves or invaginations, has not been determined.
Instead, the
invaginations seen in onion nuclei seem most similar to those found in living nuclei of various
animals. Animal nuclear invaginations are stable structures as large as 1 or 2 µm in diameter and
can branch or traverse the nucleus, although the number and form of the invaginations vary from cell
type to cell type (Ellenberg et al. 1997 ; Fricker et al. 1997 ; Lui et al. 1998a , Lui et al. 1998b ; Broers et al. 1999 ). Electron
microscopy has shown these invaginations to be bounded by the double nuclear membrane and to contain
nuclear pores (Fricker et al. 1997 ). Importantly, animal nuclear invaginations often show
a preferred orientation—perpendicular to the substrate—although once again the degree of
orientation varies from cell type to cell type (Fricker et al. 1997 ; Broers et al. 1999 ). Animal nuclear invaginations can also associate with nucleoli, which is believed to
have functional implications (Fricker et al. 1997 ).
Nuclear invaginations visualized
with ER-GFP in plant cells were stable for extended times (as long as 1 hr) in both onion and tobacco,
consistent with observations in various animal cell types (Ellenberg et al. 1997 ;
Broers et al. 1999 ), although these observations stand in contrast to the apparently
dynamic invaginations reported in various animal cells by Fricker et al. 1997 . The factors
controlling the stability of invaginations remain unknown. However, stability appears to reside within
the nucleus itself, or more likely, within the nuclear envelope and the accompanying nuclear matrix
(Yu and de la Espina 1999 ; reviewed in de la Espina 1995 ; Smith 1999 ). This is because the cytoplasmic cytoskeleton is highly unlikely to contribute to
nuclear invagination stability in either plant or animal nuclei. Although onion nuclear grooves and
invaginations contain actin bundles, actin destabilization with cytochalasin neither modifies nor
destabilizes the nuclear groove or invagination structure. Depolymerization of microtubules with
oryzalin also did not modify grooves or invaginations, although this is less surprising because the
interphase plant microtubules are primarily cortical rather than associated with the nucleus (Hepler and Hush 1996 ). Similarly, although nuclear invaginations in animal cells have been
observed to contain actin bundles (Suarez-Quian and Dym 1992 ; Clubb and Locke 1998 ), they also remain stable when either actin microfilaments or microtubules are
depolymerized (Suarez-Quian and Dym 1992 ).
Transient Expression of GFP
Visualizes Grooves, Channels, and Invaginations in Tobacco Nuclei
The nuclei of tobacco NT1
cells deviate from spherical through the formation of nuclear grooves and nuclear invaginations,
structures similar to those seen in onion nuclei. Although nuclear grooves were rare, extensive
invaginations were seen that, when extending fully through the nucleus, formed nuclear channels.
Nuclear grooves and invaginations were best studied in stably transformed cell lines expressing
ER-GFP; however, the fact that these structures were also visible in untransformed cells labeled
with DIOC6(3) indicates that they were not induced by the transformation process or by GFP
expression.
The nuclear grooves and invaginations present in tobacco NT1 cells have considerable
similarities to the grooves and invaginations seen in onion epidermal nuclei. For example, the
association of channels and invaginations with the nucleoli of tobacco NT1 cells was pronounced and
persisted throughout interphase, consistent with our observations of grooves and invaginations in
onion and with others' observations of invaginations in various animal cells (Fricker et al. 1997 ). Rhodamine–phalloidin labeling visualizes actin bundles traversing the nucleus
(Collings et al. 1998 ) and present in nuclear inclusions (Kengen et al. 1993 )
in tobacco BY-2 cells, which are closely related to the NT1 cell line. This not only suggests the
presence of channels and invaginations but also indicates that such structures have cytoplasmic
centers, similar to those found in onion nuclei. However, several differences exist between tobacco
and onion cells. Tobacco NT1 nuclei are rounded, lacking the extensive grooves found in onion nuclei,
and the invaginations, grooves, and channels seen with confocal microscopy were not visible by
transmitted light. Such differences may explain why these nuclear structures have not previously been
visualized in tobacco NT1 cells.
Nuclear invaginations and channels were regularly observed to
have a preferred orientation in recently divided cells—adjacent to the reformed nucleoli and
running parallel to the presumed axis of the spindle—although no preferred orientation was
observed for the channels and invaginations at later stages of interphase. On the basis of dye
studies, Fricker et al. 1997 proposed that the nuclear invaginations found in various
living animal cells form during mitosis and remain in the nucleus through interphase. Subsequent
experiments by Ellenberg et al. 1997 and Broers et al. 1999 , using GFP fused
to the lamin B receptor and lamin A, respectively, confirmed this by showing nuclear invaginations
reappearing as the nuclear envelope reformed after mitosis. We are currently investigating the nature
of invagination formation in synchronized NT1 cell cultures and determining whether their formation
occurs through ER strands being trapped in the nucleus during chromatin condensation, as seems likely.
However, the origin of grooves and invaginations in onion bulb epidermal cells, a storage tissue where
cells remain undivided for many months, remains unknown.
Are Grooves and
Invaginations Present in All Plant Nuclei?
The distribution of nuclear grooves and
invaginations, and related structures, among different higher plants has not been determined. Using
ER-targeted GFP, we visualized these structures in tobacco NT1 and onion epidermal cells and observed
similar structures in the epidermal nuclei of cells in whole Nicotiana benthamiana plants
(seed lines courtesy of Dr. David Baulcombe, John Innes Institute, Norwich, UK). Other studies have
shown similar nuclear infoldings in electron micrographs of petals from Narcissus (Gunning and Steer 1996 ), root cells from Pisum (Bowes 1996 ), and radical
and hyphal cells from the parasitic plant Cuscuta (dodder) (K. Vaughn, personal
communication). Given the many examples of cells that lack nuclear invaginations, presumably grooves
and invaginations are limited to specific cell types, possibly those with larger nuclei or those that
have undergone multiple rounds of nuclear endoreduplication. Such specialization strongly hints at a
functional significance for the presence of these structures in particular
cells.
The Functions of Grooves and Invaginations
What, if any, functional
significance do grooves and invaginations have in plant nuclei? Various roles have been proposed for
the invaginations present in animal nuclei, including suggestions that increased nuclear surface area
resulting from grooves and invaginations leads to increases in rates of nucleocytoplasmic transport
and signaling. Fricker et al. 1997 suggested, partially on the preferential distribution
of invaginations near nucleoli, that these structures might facilitate mRNA export to the cytoplasm.
Alternatively, Lui et al. (1998a, 1998b) proposed that nuclear invaginations might contribute to the
transmission of calcium signals from the cytoplasm to the nucleus. Calcium within the nuclear envelope
can also regulate the functioning of nuclear pores (Perez-Terzic et al. 1997 ).
The
structural properties of nuclear invaginations in plant cells and their similarities to the structures
seen in animal cells suggest that similar functions might occur. A role in calcium signaling is
possible: the plant ER acts as a calcium store (Malho et al. 1998 ), and plant
nuclei respond to calcium signals (Tahtiharju et al. 1997 ). However, the extensive
DNA endoreduplication present in differentiated plant tissues, notably in epidermal cells, where
grooves and invaginations are often found, and the accompanying increase in nuclear volume (Traas et al. 1999 ) make it more likely that grooves and invaginations are involved in the
efficient trafficking of RNA out of the nucleus and of proteins into the nucleus. Although such
transport could occur by diffusion, the movement of some mRNA in animal cells is an actin-dependent
process (Bassell and Singer 1997 ). Evidence also indicates that mRNA localization in plant
cells is cytoskeleton dependent (Muench et al. 1998 ), as is the import of proteins and
other signaling molecules from the cytoplasm into the plant nucleus (Smith and Raikhel 1998 , Smith and Raikhel 1999 ). Thus, the presence of cytoplasmic streaming and actin
bundles within grooves and invaginations would be consistent with a role in mRNA transport and protein
import. Consistent with this hypothesis, nuclear pores are seen in the infoldings present in
Narcissus nuclei (Gunning and Steer 1996 ), and we are now determining the
distribution of nuclear pores in onion epidermal cells to determine how grooves might function in
trafficking between the nucleus and the cytoplasm.
Although the processes described above might
be aided by the presence of grooves and invaginations, these structures might not be necessary, for
mRNA export or protein/signal import. However, a function for these structures in nucleocytoplasmic
trafficking would take on further significance if a relationship between chromosome positioning and
the orientation of nuclear grooves could be established. In animal cells, fluorescent in situ
hybridization of fixed nuclei (Lamond and Earnshaw 1998 ) and GFP techniques in living
cells (Marshall et al. 1997 ) have demonstrated that chromosomes localize into discrete and
reproducible zones that remain constant throughout interphase and within different cells. Although the
application of such chromosome painting methods to plant nuclei has been limited to systems in which
chromosomes derive from different parent species, a similar extent of chromosome organization is
apparent (Heslop-Harrison et al. 1990 ; Abranches et al. 1998 ; Gonzalez-Melendi et al. 2000 ; reviewed in Heslop-Harrison and Bennett 1990 ; Franklin and Cande 1999 ). Because onion epidermal cells show a consistent
distribution of nucleoli with respect to nuclear lobes and grooves, with nucleoli generally occurring
in separate lobes, and because invaginations in tobacco nuclei show a marked preference for localizing
near nucleoli, some regular chromosome zonation might exist relative to the position of nuclear
grooves and invaginations. The potential for the functional significance of such organization, with
respect to the export of mRNA or signaling, suggests that further investigation of chromosome
organization in onion and tobacco nuclei is
warranted.
|
|
METHODS |
|---|
Labeling of Onion and Tobacco NT1
Cells
To label their DNA, onion (Allium cepa cv Tango; Martin Produce, Greeley,
CO) and tobacco (Nicotiana tabacum NT1 suspension culture line) cells were incubated for 60
min with 1.0 µg mL-1 4',6-diamidino-2-phenylindole (DAPI), rinsed briefly,
and viewed immediately. Internal membranes of onion and tobacco cells were labeled for 1 to 2 min with
20 to 40 µg mL-1 3,3'-dihexyloxacarbocyanine iodide (DIOC6(3))
and viewed immediately.
Rhodamine–Phalloidin Labeling of Actin in Onion 24
Epidermal Cells
Actin was labeled in permeabilized epidermal peels by the methods of Olyslaegers and Verbelen 1998 . Inner epidermal peels were floated for 20 min on cytoskeleton
stabilization buffer (100 mM Pipes, pH 6.9, 10 mM EGTA, 4 mM MgSO4, and 0.2 M mannitol)
containing 2% (w/v) glycerol and 0.2 µM rhodamine–phalloidin (Molecular Probes, Eugene, OR),
then washed with and mounted in cytoskeleton stabilization buffer containing 0.1%
p-phenylenediamine and 1 µg mL-1 DAPI. Attempts to colocalize actin and
endoplasmic reticulum (ER)–targeted green fluorescent protein (GFP) in permeabilized cells
failed because the glycerol treatment compromised the integrity of the ER.
Transient
Expression of GFP in Onion Epidermal Cells
Epidermal cells of onion bulbs were transformed
according to Scott et al. 1999 . Epidermal peels from the inner epidermis of onion scales
were placed mesophyll-side down on 2.0% (w/v) agar plates (Murashige and Skoog 1962 media
supplemented with 3% sucrose) and bombarded under vacuum at 1100 mm Hg (Biolistic PDS-1000/He;
Bio-Rad) with 1-µm-diameter gold particles coated with DNA. Two constructs were used.
Nuclear-targeted GFP (N-GFP) consisted of a nuclear localization
signal–GFP–ß-glucuronidase (GUS) fusion protein, behind the cauliflower mosaic virus
35S promoter inserted into a pUCAP vector (Grebenok et al. 1997 ); GFP targeted to the
ER (ER-GFP) used a signal sequence coupled to GFP and the HDEL ER-retention sequence (mGFP5;
Haseloff et al., 1997 ) behind the 35S promoter inserted into a pUCAP vector. Before
observation, peels were incubated for 18 to 24 hr at 22°C in the dark. Some preparations were also
counterstained with DAPI, as described above.
Tobacco NT1 Cell Lines Expressing
ER-Targeted GFP
Protoplasts of the tobacco NT1 suspension culture line were transformed by
electroporation with a pUC-based vector containing the cauliflower mosaic virus 35S promoter, signal
sequence, mGFP5 construct, HDEL retention sequence, and a nopaline synthase terminator. Callus cells
were regenerated, and after 3 weeks, a stable transformant was selected on the basis of high GFP
expression. The selected cell line was grown in suspension culture with weekly subculturing in
Murashige and Skoog medium supplemented with 3% sucrose, 1 µg mL-1 thiamine, 100
µg mL-1 inositol, 0.2 µg mL-1 2,4-dichlorophenoxy acetic
acid, and 255 µg mL-1 KH2PO4, in full darkness at 24°C,
with shaking at 120 rpm.
Microscopy
Confocal fluorescence and concurrent
differential interference contrast (DIC) images were recorded in optical section and time series modes
with Leica TCS NT and SP confocal systems with 20 x NA 0.8 dry, 40 x NA 1.25
oil-immersion, and 63 x NA 1.2 water-immersion lenses (Leica, Wetzlar, Germany). Excitation
sources were 351 and 361 nm for DAPI, 488 nm for GFP and DIOC6(3), and 568 nm for
rhodamine–phalloidin. Fluorescence was recorded either with band-pass filters (420 to 460 nm for
DAPI, 515 to 545 nm for GFP and DIOC6(3), and 585 to 615 nm for rhodamine–phalloidin)
or with the SP-scanning head set to similar wavelengths. DIC images were recorded concurrently with a
red helium/neon laser (633 nm) and a red filter placed in front of the transmitted light detector,
taking advantage of the stable helium/neon laser. For a single experiment (see Fig 3), a Zeiss (Carl Zeiss, Inc., Thornwood, NY) confocal microscope with a 40
x NA 1.2 water-immersion lens was used, with excitation from an argon/krypton laser at 488 nm
and emission recorded with a 515- to 545-nm band-pass filter. Confocal optical stacks were transformed
into three-dimensional reconstructions with a surface shadowing algorithm in the Leica software;
orthogonal slices were produced by using Metamorph (Universal Imaging, West Chester, PA). All images
were processed with Adobe Photoshop (Grand Prairie, TX).
|
|
FOOTNOTES |
|---|
1 Current address: Department of Biological Sciences, Purdue University, West Lafayette, IN
47907-1392. 
2 Current address: Max Planck Institute for Molecular Cell Biology and
Genetics, 01307 Dresden, Germany.
3 Current address: Department of Environmental
and Plant Biology, Ohio University, Athens, OH 45701.
Online version contains Web-only data.
|
|
ACKNOWLEDGMENTS |
|---|
We thank George
Allen, Eric Davies, and Timothy Oliver (North Carolina State University [NCSU]) for technical
assistance and comments; Dana Moxley (NCSU) for culturing tobacco NT1 cells and conducting the
DIOC6(3) experiments; and Kristi de Courcy (Virginia Tech, Blacksburg, VA) for
assistance with the Zeiss confocal microscope. We also thank George Allen (NCSU) for his generosity in
providing the tobacco NT1 cell line transformed with mGFP5 (originally constructed by Jim Haseloff,
Medical Research Council, Cambridge, UK), Jim Haseloff (MRC) for the ER-GFP (mGFP5) clone, and David
Galbraith (University of Arizona, Tucson, AZ) for the N-GFP construct. Ingeborg Pauluzzi (University
of Vienna, Austria) kindly provided a video copy of Lichtscheidl and Url's 1988 film. Early
studies were conducted at the Marine Biological Laboratory, Woods Hole, MA. This work was supported by
the North Carolina State University (NCSU) NASA Specialized Center of Research and Training Grant No.
NAGW-4984 and by NCSU North Carolina Agricultural Research Service
grants.
Received July 7, 2000; accepted October 13, 2000.
|
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ABSTRACT
INTRODUCTION
