Download An immunocytochemical voyage throug the endomembrane system

An immunocytochemical voyage throug the endomembrane
system of plant cells: from electrons to photons
B. Satiat-Jeunemaitre”,
C. Hawes+ and Spencer Brown*.
* Institutdes
Sciences V6g&ales, C.N.R.S., 91198 Gif-sur-Yvette Cedex, France.
http://www.cnrs-gif.fr/ISV/
’ Research School of Biological and Molecular Sciences, Oxford Brookes University, Oxford, U.K.
http://www.brookes.ac.uk/schools/bms/reseEirch/molcell/hawes/
Eukaryotic cells are characterised by the compartmentalisation
of their cytoplasm.
Biological studies of cell endomembranes
have elucidated their specialised functions such as
assembling, sorting, and transporting newly synthesized proteins, glycoproteins, and polysaccharides
to their final destination for action, storage, deposition
or degradation. The movement
of
macromolecules in membrane-bounded
vesicles or even tubules appears to be a rapid and efficient
mechanism for transfer between the various compartments in the secretory pathway.
Some features
of membrane biology are however specific to plants (1) and are currently being investigated in our
laboratory.
Both light and electron microscopy have been combined with immunocytochemistry
to
visualise and identify the components of endomembrane system and some of its associated regulatory
proteins in various cell types. Immuno-“visualisation”
of the plant endoplasmic reticulum and the
Golgi apparatus is currently performed by immunofluorescence
and confocal microscopy, in order to
clarify the 3-D organisation of these compartments. Data confirm the earliest electron microscope
studies suggesting that the architecture of the plant Golgi apparatus (GA) and its structural (and
functional) relations with the endoplasmic reticulum (ER) may vary with the cell type observed (2).
Moreover, such versatility may also appear as a response to exogenous stress or physiological
factors. We will illustrate this latter point by presenting immunocytochemistry
describing
(i)
spontaneous variations of the 3-D organisation of the endomembrane system during cell proliferation,
and (ii) experimental modifications of the ER/Golgi compartment by brefeldin A (BFA), a drug
known to inhibit the exocytotic pathway (3). After comparison with observations in animal or yeast
membrane biology, our results outline some plant specificities regarding the dynamics of the
endomembrane system and the organisation of trafficking pathways.
1. Evolution of ER and Golgi Membranes
in Proliferating Cells
The 3D organisation of the ER and GA in proliferating
monitored by confocal laser scanning microscopy.
maize or tobacco cells has been
In interphase cells, the ER ramifies throughout the cytoplasm and circumscribes the
nucleus. In prophase, the ER surrounding the nucleus ,breaks down. During metaphase, there is a
strong rearrangement of ER membranes as they concentrate in the polar area of the mitotic spindle,
following a similar pattern usually described for microtubules. In anaphase, the ER seems to envelop
the moving set of chromosomes,
and concentrate at the future cell plate. In telophase, the
phragmoplast is strongly marked by the ER.
During interphase, the Golgi stacks are dispersed throughout the cytoplasm.
At
prophase, metaphase and anaphase, they are pushed away from the dividing nuclear zone. At late
anaphase Golgi stacks migrate to the equatorial plane. At early telophase the phragmoplast
is
strongly mark by Go&derived
membranes, while late telophase is characterised by an accumulation
of Golgi units at the end of the growing cell plate.
It is well known that ER and Golgi apparatus are the initial compartments involved in
the secretory pathway. The clear dissociation of this arrangement during mitosis questions the
functioning of the secretory pathway during this phase of the cell cycle.
2. BFA-induced
Modifications
Under BFA
Multiparametric analyses of
cytometry. It shows that the
change in the total quantity
proliferating cells.
3. Comparison
treatment,
plant Golgi stacks tend to gather and vesiculate (4).
BFA-treated cells stained with a Golgi marker have been made by flow
reorganisation of the GA observed at the cellular level is not linked to a
of GA membranes. BFA-induced Golgi clusters are also described in
with Animal Cells
Structural differences between the plant GA and its animal counterpart are evident,
notably distinct architecture throughout the cell and a different behavior during cytokinesis (5).
However, data on the evolution of the endomembrane system along the cell cycle in tobacco cells,
compared with literature data and our own experiments on HeLa cells, point out interesting
similarities in the behaviour of Golgi membranes in the two systems despite the currently described
differences: (i) The first step of disassembly of the GA in mitotic HeLa cells ressembles the GA in
interphase plant cells; (ii) The homotypic fusion of GA-derived vesicles during the end of mitosis
described in animal cells are occurring in plant cells too. However, in plant cells they do not lead to
the formation of de novo Golgi stacks as in animal cells; instead they contribute to the building of a
new cell structure, the cell plate, taking place between two daughter cells. (iii) Immunocytochemistry
combined with flow cytometry shows that the major increment in Golgi membrane occurs in Gl
phase, both in animal and plant cells.
References
1. Gunning B. and Steer M., Plant Cell Biology. Jones and Barlett F’ublishers.(l996)
2. Hawes B. and Satiat-Jeunemaitre B. , Trends in Plant Sciences, 1, 395-401 (1996)
3. Satiat-Jeunemaitre B. et al., J. Microsc., 181, 162-167 (1996)
4. Satiat-Jeunemaitre B. and Hawes C., J. Cell Ski., 103, 1153-1166 (1992)
5. Warren G. and Wickner W. Cell 84, 395-400. (1996)