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Vol. 14, Issue 5, 1808-1817, May 2003
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* Laboratory of Neurobiology, NINDS, National Institutes of Health, Bethesda,
Maryland 20892;
Department of Physiology, University of Connecticut Health Center, Farmington,
Connecticut 06032; and
Marine Biological Laboratory, Woods Hole, Massachusetts 02543
Submitted March 25, 2002;
Revised December 9, 2002;
Accepted December 27, 2002
Monitoring Editor: Ted Salmon
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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1 h
after wounding, indicating that loss of compartmentation barrier makes the
structure unstable; surprisingly, the oocyte still completed meiotic divisions
when exposed to maturation hormone, indicating that the compartmentalization
and translocation of cdk1 and its regulators is not required for this
process. Multiphoton excitation provides a new means for producing controlled
damage deep within tissues or living organisms. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). It is possible to photodynamically destroy individual
structures or cells labeled with fluorescent dyes (e.g.,
Tamm, 1978), or specific
proteins that have been targeted with fluorescent antibodies (CALI;
Buchstaller and Jay, 2000).
With the appropriate fluorescent label, the plasma membrane can be disrupted
using a laser in a confocal microscope (Bi
et al., 1995), and it has been shown that neurons loaded
with specific dyes are susceptible to ablation
(Liu and Fetcho, 1999). It is
also possible to damage unlabeled structures. UV light from a mercury arc lamp
has been used to cut microtubules (Walker
et al., 1989), whereas individual Caenorhabditis
elegans cells can be killed with energy from a dye laser
(Bargmann and Avery, 1995), and
centrosomes in cultured cells have been ablated with pulses from an Nd-YAG
laser (Khodjakov et al.,
1997).
We investigated the use of multiphoton excitation as a potential new way of
producing a highly localized disruption at greater depths than has been
possible. Under conventional continuous illumination conditions, a single
photon causes the transition of an electron to a higher energy state within a
molecule. A mode-locked Ti-Sapphire laser can provide high-density photon
pulses such that two or more photons combine to cause transitions, and because
the probability of excitation depends on the square of photon density,
excitation occurs only in the focal plane rather than all along the light path
(Denk et al., 1990,
1995). This small excitation
volume has proven effective at reducing photodamage during live cell imaging
(e.g., Jontes et al.,
2000). Additional benefits of multiphoton microscopes are the
longer wavelengths that provide deeper specimen penetration and the ability of
commercial multiphoton microscopes to precisely vary the intensity and
x-y position of this light. By taking advantage of the high degree of
spatial localization, depth penetration, and the ability to precisely vary the
laser's intensity and position, we have produced site-specific controlled
disruption in unstained cells and tissues at greater depths than previously
possible.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). At the two-cell stage, the chamber was brought to the
multiphoton microscope for wounding. To monitor development after wounding, a
Bio-Rad MRC 600 (Cambridge, MA) coupled to a Zeiss Axioskop was used, with a
Zeiss 20x N.A. 0.5 lens (Carl Zeiss Inc., Thornwood, NY). Centrifugation
of starfish oocytes was done as previously described
(Kishimoto et al.,
1977). Oocytes in the maturation hormone 1-methyladenine (1 µM)
were used as soon as a majority had undergone nuclear envelope breakdown
(20 min); they were layered on 23% 70 kDa ficoll (Sigma Chemical Co., St.
Louis, MO) in sea water (50 µl oocytes on 50 µl ficoll in a 1.5-ml
Eppendorf tube) and centrifuged at 14,000 x g for 15 min at
4°C.
Squid (Loligo pealeii) were obtained through the Marine Resources
Center at the Marine Biological Laboratory (Woods Hole, MA). The hindmost
stellar nerve or giant axon (380–450-µm diameter) and the adjoining
fin nerve bundle were dissected under running seawater and maintained in
artificial seawater (Galbraith et
al., 1999). For wounding experiments both the squid giant
axon and the fin nerve bundle were mounted between a pair of number 0
coverslips with parallel lines of silicone grease acting as spacers. The
chamber was periodically perfused with artificial seawater. In experiments
that fluorescently labeled the mitochondria the axon was incubated in 10
µg/ml Rhodamine 123 for 30 min before imaging.
Zebrafish embryos (Brachydanio rerio) were obtained from the
zebrafish facility at the Marine Biological Laboratory. Embryos were injected
at the one-cell stage with a transfection vector containing a HuC promoter
driving expression of GFP (Park et
al., 2000); this results in mosaic expression in embryonic
neurons. At 1.5 d, embryos were embedded in 1.5% agarose at 45°C with MS
222 (0.15 mg/ml) to inhibit movement and phenylthiocarbamide (25 µg/ml) to
block development of pigment cells. Embryos were held within a well cut in a
culture dish with a coverslip bottom and were kept at room temperature
(22°C). Wounded embryos were imaged using the Bio-Rad confocal
microscope with a Zeiss 40x numerical aperture (NA) 0.75 water immersion
objective lens.
Multiphoton Microscopy
Experiments were done with a Zeiss LSM 510 multiphoton microscope using a
Coherent Mira 900 Ti:Sapphire laser pumped with a either 5 W or 10 W Verdi
solid state laser (Coherent Inc., Santa Clara, CA) using software that allows
for line scan or spot mode and a Meta attachment for spectrum analysis.
Emission spectra were corrected for background autofluorescence. The laser was
coupled to a Zeiss Axiovert 200 (Carl Zeiss Inc.) inverted microscope. A Zeiss
25x multi-immersion lens (NA 0.8) was used for most experiments. We were
also able produce multiphoton damage with Bio-Rad and Leica multiphoton
microscope systems.
Most experiments were performed using 800-nm light; for the Ti:Sapphire
laser pumped with the 5 W Verdi laser, the maximum laser output was 600
mW as measured with a Coherent Fieldmaster energy level meter. After passing
through the intervening optics between the laser and the back aperture plane
of the objective lens this energy level was reduced to 100 mW. With the
25x multi-immersion lens, the energy at the specimen was 60 mW; to
make this measurement, a pair of back to back matched objectives were used to
eliminate errors associated with internal reflection
(Misawa et al., 1991;
Liang et al., 1996).
The Ti:Sapphire laser pulse frequency was 76 MHz, so that at full laser power,
each pulse was 0.8 nJ at the specimen; the pulse duration was 100
femtoseconds. We found that the best way to cause controlled damage was to use
the line scan mode of the 510 LSM microscope with the laser at full power
(lowest attenuation setting) and slow scan speed (72 µs per pixel, or
5000 pulses per pixel). In many cases a single line scan was sufficient
to cause damage. A single scan of 100 pixels in length provided an exposure of
7.2 ms and 430 µJ.
It is interesting to compare the exposure settings used for wounding to
those used for live cell imaging. A typical setting for obtaining images in a
long-term time-lapse sequence is 10% laser power and 1-µs dwell
time. To create deliberate damage, each pulse has 10x as many photons
(100% laser power), and each pixel is exposed to 70x as many pulses
(72-µs pixel dwell time), so that there is 700x difference in
exposures between live cell imaging and creating this type of damage.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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;
Cole et al., 1995;
Khodjakov et al.,
1997). We set out to make damage at greater depths using the
Ti:Sapphire laser in a multiphoton microscope.
We used echinoderm eggs as a test specimen and 800 nm light from the
Ti:Sapphire laser. We also used a water immersion lens because these are less
subject to spherical aberration at large distances from the coverslip than oil
immersion lenses (e.g., Keller,
1995). Laser power levels that would be used for long-term live
cell observations caused no detectable damage. When the laser power and the
pixel dwell time were increased to the maximum settings on the microscope (see
MATERIALS AND METHODS), a distinct, condensed "scar" was produced
that is visible by both transmitted light and fluorescence microscopy
(Figure 1A). The same exposure
created a scar in A6 cells (a frog epithelial cell line) and human cheek
epithelial cells. In these cells, as well as all other specimens we used, the
transmitted light scar and fluorescent scar appeared together; we saw no
instances of a transmitted light scar without fluorescence, or a fluorescent
scar without a transmitted light scar.
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Scars could be created with wavelengths between 720 nm and 850
nm, but it was more difficult to produce damage with longer wavelengths. This
wavelength dependence is similar to that of multiphoton autofluorescence,
which also decreases at longer wavelengths. In starfish oocytes, cytoplasm is
more autofluorescent at 800 nm than the nuclear interior and also was easier
to damage (Figure 1A);
similarly, in the squid fin nerve, variations in autofluorescence corresponded
with the ease of making wounds. However, autofluorescence itself is not
sufficient, because the autofluorescent oil cap of centrifuged oocytes (see
Figure 5) was not easier to
damage. Also, oocytes injected with fluorescent dextran were not easier to
damage than uninjected oocytes, so that the damage is not simply due to
fluorescence excitation.
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The bright fluorescence from the scar is visible by multiphoton excitation or by conventional fluorescence with 488- or 568-nm excitation. The emission spectrum (Figure 1B) has a broad peak between 475 and 525 nm. The fluorescence develops immediately and can be used to monitor the degree of damage during the scanning process. The scar persisted in the specimens that we tried and was useful for identifying the damage site during long-term experiments.
The Ti:Sapphire laser can provide photons either continuously (continuous wave mode) or as brief, intense, repetitive pulses (mode lock). For these two cases, the same amount of photons (or equivalently, energy) is delivered when averaged over time. The normal operating condition is mode lock, because the pulses provide the high intensities required for multiphoton excitation of fluorescence. Likewise, damage occurred with the laser in mode lock, but not in continuous wave mode, where the same total amount of energy is delivered to the specimen at a steady, low rate.
We examined the dependence of wounding on pulse energy. Within the
multiphoton microscope, an acousto-optic device attenuates pulse energy,
whereas the number of pulses delivered to the specimen is controlled by the
scan speed of the galvanometer mirror through the pixel dwell time setting.
For each pixel dwell time setting, we determined the minimum energy required
to create a fluorescent scar (Figure
1C). The data was fit by E 
(1/T)2.5 where E = energy, T = pixel
dwell time. This means, for instance, that pulses of twice the energy require
1/6, instead of 1/2, the number of pulses to cause the same damage. The
nonlinear dependence indicates that the damage is due to a multiphoton
process.
We investigated the size of the damage caused by multiphoton wounding. For
high exposures to light energy, there must be some spreading of damage from
the region that is exposed to light; this is evident from
Figure 1C, where all of the
wounds were created by a line scan that delivers light to a thin strip of
cytoplasm. In wounds larger than 25 µm, the shape of the wound was
often very irregular, and a small air bubble often appeared at the damage
site, possibly because of plasma formation from decomposition of water
(Noack and Vogel, 1999). We
found that we could make much smaller wounds by carefully controlling the
exposure levels. We used 0.8 and 1.2 NA water immersion lenses (25x and
63x magnification, respectively) to make wounds in the squid fin nerve
(Figure 2, A–F). The
x, y, and z full width half-maximal values for the smallest wound
produced by the 25x lens were 2.69, 1.88, and 4.14 µm (middle wound;
Figure 2, A–C) and for
the 63x lens were 0.79, 0.61, and 1.0 µm (bottom wound;
Figure 2, D–F). This
shows that it is possible to create small, precise wounds by using appropriate
energy levels and optical conditions.
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It is possible that the region of structural damage extends beyond the fluorescent scar or that diffusible toxins can damage regions beyond the fluorescent scar. To investigate this, a wound was made in a squid axon with fluorescently stained mitochondria. In time lapse sequences, mitochondria were observed to move through regions just beyond the edges of the fluorescent scar (Figure 2G), which had x and y dimensions of 2.3 µm and a depth of 4.7 µm. This suggests that the damage is restricted to the region of the fluorescent scar.
Biological Applications: Damage of Axons
Individual axons from the fin nerve bundle of the squid >50 µm
beneath the surface were damaged, demonstrating the ability of multiphoton
illumination to produce a small volume wound deep within a tissue. The damage
shown in Figure 3A was
generated using a line scan across a single axon and a 25x 0.8 NA lens
water immersion lens. These axons were typically 10 µm in diameter, and
Z-series images confirmed that disruption was limited to one axon. In
addition, transmitted light images showed an accumulation of organelles on the
proximal side of the scar, indicating that transport was blocked at that
location. This transport block was observed only in the damaged axon and not
in any of the surrounding axons.
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A more localized interruption of axonal transport was also accomplished by
disrupting the axoplasm of the giant axon again with the 25x lens and a
line scan 25 µm from the axon surface
(Figure 3B). Fluorescently
labeled mitochondria were observed by timelapse confocal microscopy to move
linearly in a saltatory manner through the axoplasm before damage. However,
after damage, mitochondria moving along a line that intersected the scar
tended to stop and accumulate at the scar, whereas mitochondria moving along
lines that did not intersect the scar were not affected
(Figure 3B). Like other large
scars (>25 µm) some variation is seen in the fluorescent intensity along
the length, but transport was blocked at all points along the band.
Severing of a Dendrite
We cut single neuronal processes within living zebrafish embryos. A
transfection vector with a neuronal promoter driving GFP expression was
injected into one cell stage zebrafish embryos, resulting in mosaic expression
of GFP in a small fraction of embryonic neurons. GFP-labeled Rohon-Beard cells
were used as targets. These cells are mechanosensory neurons that are easily
identifiable in 2-d-old embryos. The cell bodies, which are located in the
dorsal spinal cord, send out dendritic processes that innervate the skin
(Figure 4).
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GFP-labeled Rohon-Beard cells were located by conventional confocal
microscopy. The dendritic branches are much thinner (2–3 µm) than the
squid axons described in the previous section, so that multiphoton line scan
wounds resulted in severing rather than creation of damage within the
dendrite. The presence of GFP did not seem to reduce the amount of energy
required for wounding. Over the next 6 h Z-series images of the neuron were
obtained by conventional confocal microscopy at 30–45-min
intervals. The severed dendritic branch remained intact for 5 h and then
began to vesiculate and eventually disappeared
(Figure 4). Multiphoton
excitation was also effective at severing axons within the spinal cord
(unpublished data).
Ablation of a Mitotic Pole
We used sea urchin embryos, a classic model for mitosis. At about the time
of anaphase onset, microtubules grow from the two mitotic poles away from the
spindle and are easily seen in bright field microscopy as prominent star-like
patterns (mitotic asters). The microtubules of the aster are involved in
setting up the position of the cleavage furrow, and the cell becomes committed
to divide sometime before the cell begins to constrict
(Rapoport, 1981). Multiphoton
damage was made to the mitotic pole at the center of the aster during the
3-min period between the first appearance of the asters and the beginning
of constriction.
Two cell stage embryos were used. As control, a wound was made deliberately away from the mitotic pole (Figure 5A). The blastomere that inherited the fluorescent scar divided normally and in synchrony with the undamaged blastomere (2/2 embryos). In embryos where the damage was made at the center of a mitotic aster, the damaged cell completed the first division normally. However, of the four resulting daughter cells, the cell that inherited the fluorescent scar failed to divide in the next cycle (Figure 5B; 3/3 embryos). In the case of the first division, the cell was probably able to divide because the commitment to form a cleavage furrow had already occurred at the time of the wounding. The failure of the second division indicates that sea urchin blastomeres cannot continue normal development without a centrosome.
Plasma Membrane Wounding
We used the starfish oocyte for wounding of the plasma membrane and the
nuclear envelope (next section). Repair of plasma membrane wounds requires
fusion of intracellular organelles at the injury site
(Steinhardt et al.,
1994; McNeil and Steinhardt,
1997; Reddy et al.,
2001). In echinoderm eggs, the primary organelle involved in
repair of large wounds appears to be the yolk platelets
(Terasaki et al.,
1997; McNeil et al.,
2000).
When echinoderm eggs are centrifuged, the yolk platelets are displaced to
the centrifugal end, leaving the other half optically clear. Heilbrunn
(1958) noted that surface
wounds become repaired on the yolk containing end but not on the clear end;
not believing in the existence of the plasma membrane, he advocated protein
coagulation for repair. We confirmed Heilbrunn's observation using microneedle
wounds, then used multiphoton excitation to make wounds. No cytoplasmic
changes occurred when a line scan wound was made at the yolk containing end
(3/3 eggs), whereas a wound at the clear end resulted in degeneration of the
clear cytoplasm and the establishment of an apparent new cellular boundary
between the clear and yolk containing cytoplasm (6/6 eggs)
(Figure 6). Multiphoton damage
within the clear zone cytoplasm did not cause any changes in the oocyte (6/6
eggs; Figure 6). The simplest
interpretation of these results is that the plasma membrane of the clear end
does not become sealed because of the lack of yolk platelets and that the
raised intracellular Ca in the clear end causes fusion of the bordering yolk
platelets to create a new boundary (McNeil
et al., 2000).
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Nuclear Envelope Wounding
The starfish oocyte has a 60–70-µm diameter nucleus (germinal
vesicle) positioned close to the cell surface at the animal pole. The oocyte
is arrested in prophase of meiosis I, which is the stage just before nuclear
envelope breakdown. Meiotic maturation is initiated by the hormone
1-methyladenine, whereupon nuclear envelope breakdown occurs at
20–30 min, followed by polar body extrusion and formation of the
female pronucleus by 3 h.
The nuclear envelope is a double membrane boundary associated with an
underlying lamina. As in other cells, the starfish nucleus can be injected,
indicating that a rupture in the nuclear envelope can be sealed. However, the
nuclear envelope does not reseal multiphoton excitation wounds. A line scan
wound at the nuclear envelope resulted in slow leakage of cytoplasmic
molecules into the nucleus; fluorescent 70 kDa dextran came to equilibrium by
40–50 min (Figure 7, A and
B).
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The outline of wounded nuclei remained unchanged until 45–90 min
when the nuclear envelope outline collapsed
(Figure 6C). Small remnants of
the nuclear envelope remained visible, and the collapse differed from the
breakdown of the nucleus that normally occurs during meiotic maturation
because polar bodies and the female pronuclei never formed. The oocyte was
stable for 12 h then underwent blebbing and deterioration. Oocytes that
were wounded in the cytoplasm instead of at the nuclear envelope survived and
were as healthy as unwounded oocytes for at least 2 d.
During meiotic and mitotic cell cycles, nuclear envelope breakdown is
preceded by the translocation of several cell cycle regulatory proteins
between nucleus and cytoplasm (Pines,
1999; further details in DISCUSSION). By wounding the starfish
nuclear envelope, the normal nucleocytoplasmic compartmentalization is
disrupted so that the normal interactions and translocations that involve
compartmentalization will not be able to occur. We tested whether this
disruption would impair the normal meiotic cell cycle by adding maturation
hormone to oocytes 2 h after the nuclear envelope had been wounded.
Surprisingly, the oocytes completed the meiotic divisions on a normal time
schedule as evidenced by formation of polar bodies and the female pronucleus
(14/15 oocytes from three animals; Figure
7D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Two recent studies have investigated damage in multiphoton microscopes,
primarily with the purpose of minimizing photodamage during imaging of living
cells, rather than the deliberate creation of damage. Damage was assessed by
nonphysiological rises in intracellular calcium in neurons and chromaffin
cells or degranulation in the chromaffin cells
(Koester et al.,
1999; Hopt and Neher,
2001). Both studies found a 2.5 power dependence on energy for
damage, indicating a multiphoton process (a one photon process is expected to
produce a linear power dependence). Hopt and Neher speculated that damage
involves a significant amount of three photon absorption. This seems to
correspond with our observation that multiphoton damage is easiest with
wavelengths < 850 nm, because 800/3 = 266 nm is a highly absorbing
region of the spectrum for the aromatic amino acids and nucleotides, while
there is less absorption at 900/3 = 300 nm. We found a similar 2.5 power
dependence for deliberately induced multiphoton damage as assayed by
fluorescent scar formation. This indicates that the wounds should be
restricted to the focal plane region (Denk
et al., 1995) and is consistent with our experimental
observations that wounds with dimensions of 1 µm or less can be made under
appropriate conditions.
As with other forms of laser induced damage, the physical/chemical
mechanisms in multiphoton damage are not well understood and are likely to be
complex. There may be interesting differences, because the Ti:Sapphire laser
makes fluorescent scars and has no effect on glass at the power levels used
for wounding, whereas the Nd-YAG and dye lasers make nonfluorescent scars and
damage glass (Bargmann and Avery,
1995; Khodjakov et
al., 1997).
A related issue is the spatial extent of multiphoton damage. Because high
numerical aperture objective lenses can focus light to a volume comparable to
the wavelength of light, it may be feasible to restrict damage to a similar
volume. With a Nd-YAG laser focused to a stationary spot by an oil immersion
lens (NA 1.4), it is possible to make very precise wounds that are 0.4
µm in diameter at a depth of a few micrometers into a tissue culture cell
(Khodjakov et al.,
1997). Using a water immersion lens (NA 1.2), we were able to make
and detect a fluorescent scar as small as 1 µm at a depth of 20 µm
in the squid fin nerve. It is possible that nonfluorescent structural damage
extends beyond the observed fluorescent scar or that diffusible toxins may be
generated. However, in squid axons, we observed that mitochondria are able to
move in the region just adjacent to the fluorescent scar; this is evidence
that damage is restricted to the region of the fluorescent scar.
Two features of multiphoton wounding are advantageous for the practical use of this technique. The fluorescent scar is very useful for monitoring the damage as it is occurring and also for locating the damage site later in long-term observations. It is also very convenient that the Ti:Sapphire laser is integrated within a scanning microscope. The location and intensity of the laser illumination are controlled precisely with high time resolution, resulting in much spatial and temporal flexibility in making damage. The normal imaging mode of the multiphoton microscope is also convenient for characterizing the damage or wound response afterward.
We used multiphoton wounding on four different biological systems. In the first, axons within the squid fin nerve as well as the giant axon were selectively damaged. This shows that it is possible to make wounds deep within tissues or cytoplasm without damaging surrounding regions, offering the possibility of laser microsurgery with the multiphoton microscope. In the second system, a dendritic branch of Rohon-Beard neurons within zebrafish embryos was selectively cut; because of the thinness of the dendrites, multiphoton wounding caused severing rather than damage within the dendrite. This ability to sever neuronal processes at locations that have previously been difficult to reach provides a better opportunity to investigate mechanisms of axonal degeneration, regeneration, and pathfinding.
In the third system, a mitotic pole in sea urchin embryos was damaged. In
previous studies where cultured mammalian cell centrosomes were ablated with a
Nd-YAG laser, the centrosomes could be ablated early in the cell cycle because
they were labeled by GFP-gamma tubulin; it was found that the cell can
assemble a mitotic spindle and divide, but the daughter cell that inherits a
damaged centrosome becomes arrested in G1
(Khodjakov and Rieder, 2001).
Multiphoton damage should provide the opportunity to pursue these findings in
other systems such as embryonic cells or cells within tissues.
Starfish oocytes were the fourth system used. Previous work had indicated
that yolk platelets are the primary intracellular organelle involved in repair
of large wounds in echinoderm eggs
(Terasaki et al.,
1997; McNeil et al.,
2000). We tested this by examining the ability of the plasma
membrane to repair wounds in stratified oocytes, and the results were
consistent this idea. Other work shows that fluorescently labeled plasma
membranes can be specifically damaged by multiphoton excitation
(McNeil et al.,
2001), whereas our studies involve unstained membranes.
Multiphoton wounds in the starfish oocyte nuclear envelope were not healed,
as shown by the slow leak of large cytoplasmic fluorescent dextrans into the
nucleus. After 45–90 min, the nucleus underwent collapse, indicating
that this disruption somehow makes the nuclear structure unstable. Dissipation
of the nuclear Ran GTP gradient (Kalab
et al., 2002) would disrupt active nucleocytoplasmic
transport and might lead to destabilization of the normal nuclear structure.
The collapse of nucleus was not immediately detrimental to the oocyte, which
survived for 12 h and then degenerated.
We used multiphoton disruption of the nucleus to investigate requirements
for nuclear compartmentation and translocation during meiosis. The presence of
the nucleus is not always required for cell cycle–related events. For
instance, in enucleated fertilized eggs, a system in which the rapid embryonic
division cycle has already been initiated, centrosome duplication and surface
contraction cycles continue (Hara et
al., 1980; Sluder et
al., 1986). The presence of the nucleus is required though,
during meiotic maturation in starfish oocytes. The full level of maturation
promoting factor appears in the cytoplasm only after nuclear envelope
breakdown, and removal of the nucleus prevents full development of maturation
promoting factor activity (reviewed in
Kishimoto, 1999); these
effects are not well understood.
There are also complex movements across the nucleus before breakdown occurs
(e.g., Pines, 1999;
Takizawa and Morgan, 2000).
Nuclear envelope breakdown is preceded by the entry into the nucleus of
cdc2 kinase (Pines and Hunter,
1991; Ookata et al.,
1992). Depending on the species and cell type, the regulators of
cdc2 are compartmentalized differently and undergo translocations
between nucleus and cytoplasm. In starfish, the inhibitory myt1 and
the activating phosphatase cdc25 are both cytoplasmic, with
cdc25 translocating into the nucleus before nuclear envelope
breakdown (Okano-Uchida et al.,
1998; Kishimoto,
1999), whereas in mammalian cells, wee1 is nuclear and
cdc25C is cytoplasmic and undergoes translocation to the nucleus
(Heald et al., 1993).
In addition, cyclin B apparently must be phosphorylated for cdc2 to
enter the nucleus (Toyoshima-Morimoto
et al., 2001). The reasons for the localizations and
movements in the activation and function of cdc2 are not well
understood.
The collapse of the starfish oocyte nucleus caused by multiphoton wounding was not followed by meiotic divisions. This shows that disruption of the nucleocytoplasmic compartmentalization by itself is not sufficient to start meiosis; in these oocytes, for instance, cdc2 and cdc25 presumably can mix with the former nuclear contents. We tested whether the loss of nucleo-cytoplasmic compartmentalization would affect the ability of the oocyte to undergo meiotic maturation. In the oocytes with a collapsed nucleus, many of the normal interactions or translocations that involve compartmentalization will not be able to occur. Surprisingly, these oocytes were still able to complete meiotic divisions when exposed to maturation hormone. This seems to show that the localization of the cdc2 and its regulatory molecules and their translocations are not required for initiation and completion of starfish meiotic maturation. It is possible that the compartmentalizations and translocations are only involved in nuclear envelope breakdown and are not required for initiating any of the following events in meiosis. Another possibility is that they are involved in monitoring whether nuclear envelope has occurred, and if inactivated, will simply allow meiotic divisions to occur.
In conclusion, we have used multiphoton excitation to make controlled damage in several biological systems and have shown that it is possible to make precise wounds within thick specimens. This significantly expands the capabilities for controlled damage within cells and tissues. We foresee that controlled damage by multiphoton excitation will become a useful tool for experimental cell biology.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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Online version of this article contains video materials. Online version is
available at
www.molbiolcell.org. 
Corresponding author. E-mail address:
terasaki{at}neuron.uchc.edu.
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REFERENCES |
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