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8 | B U L L E T I N D E C E M B E R 2 0 1 2
H y p e r - g M i c r o s c o p y
L I V E C E L L I M A G I N G U N D E R H Y P E R - G R A V I T Y
C O N D I T I O N S
YOUSSEF CHEBLI1, JACK J.W.A. VAN LOON2,3 AND ANJA GEITMANN1
INTRODUCTION
Long-term space missions and implementation
of permanent bases on the Moon and Mars
will require the detailed understanding of the
functioning of biological processes under altered
gravity conditions. Investigations at cellular level
generally rely on or comprise microscopic studies
- be it to investigate cellular structures, transport
processes, expression of proteins or localization of
cellular components. To study the effects of altered
gravity conditions at cellular level, researchers
have resorted to installing microscopes at the
International Space Station (to operate under
micro-gravity conditions) or to tilt microscopes
to allow for imaging of objects such as plant
roots positioned parallel to Earth's gravity vector
[1-5]. Both set-ups are associated with certain
technical challenges, but their long-term use does
hardly pose a mechanical risk to the microscope
during its operation. The third experimental
condition that is traditionally used for gravity
research, hyper-gravity, is more challenging in
this regard. At least two technical solutions can be
envisaged to perform microscopic imaging under
hyper-gravity conditions: either a microscope is
built specifically to spin around itself exposing
the specimen to centrifugal force, or an adapted
conventional microscope is placed into a large
centrifuge that is rotated to apply centrifugal
acceleration on the entire device. The former, a
light microscope unit mounted on a centrifuge
plate has been built in the 1990s for experiments
ranging between 1 and 5g [6]. However, this
particular microscope (the "Nizemi" or "slow
rotating centrifuge microscope") is only able
to acquire images in bright/dark field, phase
contrast, and differential interference contrast. It
was not conceived for fluorescence imaging. To
enable fluorescence imaging, we opted to place
a conventional epi-fluorescence microscope in
the Large Diameter Centrifuge (LDC) at the
facilities of the European Space Research and
Technology Centre (ESTEC) of the European
Space Agency (ESA) located in The Netherlands
[7]. In the following we describe the experimental
set-up and challenges as well as the biological
system and the motivation for this project. To
our knowledge this is the first time that live
cell imaging using fluorescence has been used at
hyper-gravity conditions.
MICROSCOPE SET-UP IN THE LARGE
DIAMETER CENTRIFUGE
The LDC has four arms, each of which can
support up to two gondolas with a maximum
payload of 80 kg per gondola (Figs. 1A,B). The
total diameter of the device is 8 m and g-forces of
up to 20g can be achieved. An optical microscope
(inverted Zeiss Axiovert 10) equipped for epi-
fluorescence was fixed in one of the payload
gondolas. During rotation the gondolas swing out
so that the vertical axis of any object placed inside
remains positioned parallel to the acceleration
vector resulting from the centrifugal force and
1 - Institut de recherche en biologie végétale, Département de sciences biologiques, Université de Montréal, Montréal, Québec, Canada
2 - Department of Oral and Maxillofacial Surgery & Oral Cell Biology, Academisch Centrum Tandheelkunde Amsterdam (ACTA), University of
Amsterdam and Vrije Universiteit Amsterdam, Research Institute MOVE, Amsterdam, The Netherlands
3 - Life & Physical Sciences Instrumentation and Life Support Section (TEC-MMG), European Space Agency (ESA), Noordwijk, The Netherlands
Corresponding author: [email protected]
B U L L E T I N D E C E M B E R 2 0 1 2 | 9
H y p e r - g M i c r o s c o p y
Figure 1. Large Diameter Centrifuge and experimental set-up(A) The LDC is located at the European Space Research and Technology Centre of the European Space Agency in Noordwijk,
The Netherlands. It is composed of four arms supporting a total of up to 6 gondolas. (B) An inverted Zeiss Axiovert 10
microscope equipped with a mercury lamp was fixed inside one of the gondolas allowing live observations of growing pollen
tubes in Ibidi® cells (C).
10 | B U L L E T I N D E C E M B E R 2 0 1 2
H y p e r - g M i c r o s c o p y
Earth's gravity. The microscope was equipped
with a digital camera (Leica DFC 300 FX) for
brightfield and fluorescence imaging. Since the
weight of the whole system increases linearly with
the applied level of centrifugal acceleration, and
since the microscope used was a conventional
model, we had to ensure that the system resists
hyper-g conditions. Several major challenges had
to be met when preparing the microscope for
operation during centrifugation runs.
1. Increased centrifugal acceleration caused
a displacement of the condenser requiring
refocusing of the samples during time-lapse
imaging. To be able to perform this adjustment,
the x-y position of the stage and the focus of
the microscope could be remote controlled
from an outside control room.
2. Due to the centrifugal acceleration force, the
optical and electronic equipment had to be
firmly attached and reinforced. The objectives
used here had spring-loaded retractable ends,
which had the tendency to retract away from
the sample at hyper-g. Fixation measures were
applied to tightly fix the spring retractable
end to the objective barrel and prevent any
displacement.
3. A conventional microscope slide/cover
glass sample mounting would have risked
dehydrating the sample. To ensure that the
sample would not dehydrate or squeeze out
during centrifugation, it was placed in tightly
sealed wells of a 0.4 µm Ibidi® cell (µ-Slide VI
0.4, IbiTreat) (Fig. 1C).
4. Centrifuge operation always causes slight
vibrations and made imaging difficult at high
g-levels and high magnifications. To decrease the
transfer of the vibrations from the gondola to the
microscope, we suspended the whole microscope
within the gondola using bungee cords (Fig. 1B).
This set-up eliminated perceptible vibrations at
acceleration levels of up 13g.
THE BIOLOGICAL SYSTEM:
POLLEN TUBE GROWTH
The availability of ambient air, sustainable
food supply and treatment of human waste are
crucial for long-term space mission. All these
requirements can be fulfilled through cultivation
of plants on board the spacecraft or in the
permanent bases on e.g. Moon or Mars. Because
of their multiple roles, plants will play a primordial
role in future space missions and understanding
the plant metabolic and morphogenetic responses
to altered gravity conditions is indispensable for
the development of space craft ecosystems or
long-term planetary colonization [8]. Cultivation
of plants on orbital platforms affects growth of
organs and individual cells as was shown in many
plant species [9].
Plants perceive the magnitude and orientation
of gravity using at least two different principles:
statolith based perception is based on the
sedimentation of small starch filled bodies inside
the cytoplasm that are of higher density than
the surrounding cytosol - the statoliths. This
mechanism is known to operate in specialized
tissues such as the root cap. Hormone signaling is
used to transmit the signal from the root cap to the
rest of the plant and trigger a growth response in
the growing portions of the organs. Interestingly,
the majority of plant cells are not equipped with
statoliths but are nonetheless known to respond
to a change in the magnitude or direction of
gravity using a different mechanism: statolith-
less perception of gravity occurs in most plant
cells, but the mechanical principle is not well
understood. It is assumed that the protoplast is
compressed under its own weight and/or that the
cytoskeletal arrays are deformed [8,10].
To assess the effect of altered g-force on plant cell
growth and metabolism in a statolith-free system,
we chose the model system pollen tube. The pollen
tube is a protuberance formed by the pollen grain
B U L L E T I N D E C E M B E R 2 0 1 2 | 11
H y p e r - g M i c r o s c o p y
upon contact with a receptive stigma. It carries
the male gametes, the sperm cells, from the
pollen grain to the female gametophyte nestled
deep within the pistillar tissues of the flower.
Since speed is a direct selection factor, pollen tube
growth is the fastest cellular growth process in the
plant kingdom. Because of its unique features,
its simple geometry, and its extremely active
metabolism and growth behavior, this cell is
ideal for short-term experiments. At the growing
apex of the cell, both exocytosis and endocytosis
occur at high rates and we used this system to
investigate the effect of hyper-gravity on plant
cell growth and metabolism.
Like all plant cells, pollen tubes are surrounded
by a cell wall, an outer envelope rich in
carbohydrates and polymers that provides the cell
with protection from pathogen infections, acts as a
filtering mechanism, plays a primordial structural
role and confers to the cell its characteristic
mechanical properties. In pollen tubes the cell
wall is formed by two major mechanisms, the
first is based on a very active vesicle exocytosis
of cell wall components like pectins and cell
wall synthesis enzymes like cellulose and callose
synthases. Intracellular trafficking is therefore
very important in this context as vesicles filled
with cell wall precursors have to be delivered to
their appropriate destination at the apex before
fusing and delivering their content. Any mis-
localization of the exocytotic events can lead to
the alteration of the self-similar tubular growth of
B
Meridional distance from the pole ( m)
Cellulose
5g – Crystalline Cellulose
E
1g
D
C
F
Rela
tive f
luore
scence inte
nsity
Membranes labeled with FM1-43
A Figure 2. Imaging of growing Camellia pollen tubes(A) Scanning electron micrograph of pollen tubes of
Camellia japonica. Bar = 100 µm. (B-E) The spatial
distribution of cellulose was affected by the hyper-gravity
conditions. The apex of the cell which is normally devoid
of cellulose (B,D) displays intense label for cellulose at
5g (B,E). (F) Intracellular trafficking was assessed using
FM1-43, a styryl dye taken up by endocytosis. The surface
area of orthographic projection of the apical vesicle cone
and its fluorescence intensity varied with gravity conditions
indicating altered vesicle trafficking. Bar in C (applies to
C-F) = 10 μm.
12 | B U L L E T I N D E C E M B E R 2 0 1 2
H y p e r - g M i c r o s c o p y
the pollen tube. The second mechanism is based
on the synthesis of cell wall components directly
at the plasma membrane by specific enzymes [11-
13]. Our objective was to determine the effect of
hyper-gravity on both the trafficking and delivery
of cell wall material as well as the resulting spatial
distribution of cell wall components.
EXPERIMENTAL DESIGN
Two types of experiments were conducted:
1. To assess the effect of hyper-gravity on the
spatial distribution of cell wall components,
pollen tubes were chemically fixed with a
37% formaldehyde solution after 3 hours of
growth at defined g levels and subsequently
labelled with specific cell wall components
antibodies. Pectins were labelled with JIM5
and JIM7 antibodies recognizing non esterified
and highly esterified pectins, respectively,
crystalline cellulose was labelled with Cellulose
Binding Module 3a (CBM3a) [14] and callose
was labelled with anti-callose as indicated
in [15]. We used a quantification method
developed previously [16] to determine the
spatial profiles of these cell wall components
on fluorescence micrographs.
2. To understand the effect of hyper-gravity on
the intracellular traffic, styryl dye FM1-43
was added to the pollen tubes. This dye is
rapidly internalized by the cells and then labels
intracellular membrane structures as well as the
plasma membrane. It allows for observation of
organelle and vesicle motion during live cell
imaging. Videos of growing pollen tubes were
acquired at different g levels as described in [15].
RESULTS
Our findings published this month in PloS One [15]
show that, while pectin localization does not seem
to be altered in pollen tubes grown in hyper-gravity
conditions, cellulose and callose are relocalized
closer to the pollen tube apex (Fig.2). This suggests
a role of these two components in reinforcing the
cell wall against the pressure (internal and external)
generated by hyper-g conditions.
Moreover we found that hyper-g alters the
intracellular transport and delivery of cell wall
material at the growing apex of the tube (Fig.
2F), suggesting that molecular factors implicated
in the regulation of intracellular transport are
dependent on and/or affected by the magnitude
of gravitational acceleration. These findings have
implications beyond the field of plant science.
Intracellular transport processes are crucial in
all eukaryotic cells and failures in their proper
regulation causes diseases such as Alzheimer
and Huntington [17]. Assessing how hyper-
and microgravity affect these processes will be
essential for future space missions.
ACKNOWLEDGMENTS
This project was financed by the European Space
Agency Spin Your Thesis! educational program.
We would like to acknowledge the support of Mr.
Alan Dowson from ESA-ESTEC-TEC-MMG.
Research in the Geitmann lab is supported by
grants from the Natural Sciences and Engineering
Research Council of Canada (NSERC) and the
Fonds Québécois de la Recherche sur la Nature
et les Technologies (FQRNT). Youssef Chebli
is funded by the Ann Oaks doctoral scholarship
of The Canadian Society of Plant Biologists
/ La Société canadienne de biologie végétale.
Van Loon is supported Microgravity Research
Program of NWO-Netherlands Space Office
(NSO), grant ALW-GO-MG-057.
B U L L E T I N D E C E M B E R 2 0 1 2 | 13
H y p e r - g M i c r o s c o p y
REFERENCES
1. Braun M, Buchen B, Sievers A (1999) Ultrastructure and cytoskeleton of Chara
rhizoids in microgravity. Advances in Space Research 24: 707-711.
2. Braun M (1997) Gravitropism in tip-growing cells. Planta 203: S11-S19.
3. Hejnowicz Z, Sondag C, Alt W, Sievers A (1998) Temporal course of graviperception in
intermittently stimulated cress roots. Plant, Cell & Environment 21: 1293-1300.
4. Kuznetsov O, Brown C, Levine H, Piastuch W, Sanwo-Lewandowski M, et al. (2001) Composition and physical
properties of starch in microgravity-grown plants. Advances in Space Research 28: 651-658.
5. Baluška F, Hasenstein KH (1997) Root cytoskeleton: its role in perception
of and response to gravity. Planta 203: S69-S78.
6. Friedrich ULD, Joop O, Pütz C, Willich G (1996) The slow rotating centrifuge microscope
NIZEMI — A versatile instrument for terrestrial hypergravity and space microgravity research
in biology and materials science. Journal of Biotechnology 47: 225-238.
7. van Loon JJWA, Krause J, Cunha H, Goncalves J, Almeida H, et al. (2008) The Large Diameter
Centrifuge, LDC, for life and physical sciences and technology. Proceedings of the 'Life in
Space for Life on Earth Symposium'. Angers, France: ESA SP-663. pp. 22–27.
8. Chebli Y, Geitmann A (2011) Gravity research on plants: use of single cell
experimental models. Frontiers in Plant Science 2: 1-10.
9. Morita MT (2010) Directional gravity sensing in gravitropism. Annual Review of Plant Biology 61: 705-720.
10. Morita MT, Tasaka M (2004) Gravity sensing and signaling. Current Opinion in Plant Biology 7: 712-718.
11. Chebli Y, Geitmann A (2007) Mechanical principles governing pollen tube growth.
Functional Plant Science and Biotechnology 1: 232-245.
12. Geitmann A, Steer MW (2006) The architecture and properties of the pollen tube cell
wall. In: Malhó R, editor. The pollen tube: a cellular and molecular perspective, Plant
Cell Monographs. Berlin Heidelberg: Springer Verlag. pp. 177-200.
13. Kroeger JH, Bou Daher F, Grant M, Geitmann A (2009) Microfilament orientation constrains vesicle
flow and spatial distribution in growing pollen tubes. Biophysical Journal 97: 1822-1831.
14. Blake AW, McCartney L, Flint JE, Bolam DN, Boraston AB, et al. (2006) Understanding the
biological rationale for the diversity of cellulose-directed carbohydrate-binding modules
in prokaryotic enzymes. Journal of Biological Chemistry 281: 29321-29329.
15. Chebli Y, Pujol L, Shojaeifard A, Brouwer I, Van Loon JJWA, et al. (2013) Cell wall assembly and intracellular trafficking
in plant cells are directly affected by changes in the magnitude of the gravitational force PloS one In Press.
16. Chebli Y, Kaneda M, Zerzour R, Geitmann A (2012) The cell wall of the Arabidopsis thaliana pollen tube - spatial
distribution, recycling and network formation of polysaccharides. Plant Physiology 160: 1940-1955.
17. Aridor M, Hannan LA (2000) Traffic jam: A compendium of human diseases
that affect intracellular transport processes. Traffic 1: 836-851.