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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: anja.geitmann@umontreal.ca

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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).

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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

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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.

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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.

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