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Nuclear structures involved in transcription and splicing of pre-mRNA
Colloque SBCF-ATIPEXNRS MORPHOLOGICAL
CIIARACTERISATION OF RAT ISOLATED IIEPATOCYTE MULTIPLETS
TRAN Dien, COMBETTES Laurent, TORDIMANN Brigitte et CLARET Michel INSERM
Biologie du D&eloppement,
u2 74. UPS: orsoy,
Thieny, BERTHON
Frmcr
The stimulation of rat hepatocytes by Ca2+ mobilising agonists induces cytosolic free Ca2+ oscillations. We have recently reported that, in contrast to single cells, multiplets (doublet or triplet) of associated hepatocytes whose bile canaliculus is maintained, showed coordinated oscillations and that a Ca2+ wave propagated from one cell to another. In the present work, we have studied the cell polarity and junctional complexes of these hepatocyte multiplets using different antibodies specifically recognising protein markers of particular plasma membrane domains and cell junctions. Multiplets (30% of the cell preparation) were obtained by moderate collagenase digestion of rat hepatocytes.. After one hour adherence on collagen coated coverslips, only multiplets having bile canaliculus were studied. The lateral domain of the plasma membrane was visualized by a monoclonal antibody B 1 and the canalicular (apical) domain was studied by two different approaches: I- utilisation of an anti-DPP4 polyclonal antibody directed against dipeptidyl peptidase. an enzyme predominant in the apical domain of epithelial cells. 2- specific labelling of F-actin by phalloidin (Bodipy 581/591) revealing a circle of F-a&n around the bile canaliculus. Two types of cell junction were also visualized: the tight junction (zonula occludens) decorated by an antibody directed against ZO-1, a high molecular weight polypeptide associated with tight junction, and the gap junction revealed by an anti-connexin 32 antibody These results obtained by different specific antibodies showed that, in contrast to single cells where molecules specific for cell polarity were distributed all over the cell surface, hepatocyte multiplets maintained their cell polarity and junctional complexes, The preservation of these structures are likely lo be essential to the Ca2+ wave propagation observed in these multiplets under hormonal stimulation.
PROTEINS DINOPLAGELLATES.
RELATED
Dinoflagellate
TO A BIOCHEMICAL
Protists
display
characteristicsare
permanent
persists
all along
the cell cycle
stretched
out between
spindle
We
recently
presence
of
(PERRET
r/n/,
cells.
kDa
cohnii, t 1~5).
was also analysed whole
chromosome
the IWO centrosomal
%I.. in
in confocal
pus).
laser scanning
One of the mnin
interests
microtubules
do not depolymerize
cells (PERRY
er 01. (1991). RwSystcms.
With
mono-
and
actin,
nuclear
both in Prnrwmtrm~
actin
especially
actin
(44kDa).
from
purified
nuclei
late
S-phase
during presence
of
Ctypthecodinium
revealed
the
micunr
cohnii.
chain The
of
cells during
to most
cell SCI.,
104.639-0513.
zones
and Crypthrcodinium
the
presence
of of
cells.
of proteins
on cryosections.
kDa)
eukatyotic
and the presence
cohnii
and immunoblotting
We in the
mitosis
is that the cortex
we demonstrated
(200
dinoflagellate
on permeabilized
system
~1 nl.. submtlled). myosin
the
in the contrary
as by immunofiuorescence
(SOYER-GOBILLARD heavy
mitosis
the
microscopy
in the centrosome
by electrophoresis
as well
fluorescence
antibodies,
located
in
to human
of the p-tubulin
25, 53.65: (1s43). J.
polyclonal
cytoplasmic
protein
of this microtubular
during
which
immunocytochemistry
from dinoflagellate
also
NUCLEAR STRUCTURES INVOLVED IN TRANSCRIPTION AND SPLICING OF PRE-mRNA. PUVION-DUTlLLEUL Francinc, BESSE Sylvie and PUVlON Edmond. Laboratoire BP& 94801
: Oiganisation fonctionnelle - VILLULJIF - Cedex.
du Noyau
- UPR 272 - IFC,
Compared with fluorescence microscopy, combinations at the electron microscopic level of high resolution autoradiography, cytochemisay, immunomicroscopy and in situ hybridization are methods of choice for identifying the structures involved in transcription and splicing of pre-mRNA. We established that the perichromatin fibrils extending in the interchromatin space correspond to newly synthesized pre-mRNP and represent the morphological substrate of splicing. The clusters of interchromatin granules and coiled bodies which also accumulate components of spliceosomes would rather be involved in pre and/or post-splicing events such as the assembly of spliceosomes, snRNP recycling, intron degradation, sorting of RNA molecules (Visa N.. PuvionDutilleul F., Harper F., Bachellerie J.P. and Puvion E. (1993) Exp. Cell Res.. 208, 1934). In addition, we recently identified a new compartment always associated with clusters of interchromatin granules and therefore termed : interchromatin granule-associated zone. This structure contains Ul but not U2 snRNA, this suggests that it might be a site of storage and/or final maturation of the Ul snRNP particle (Visa N.. Puvion-Dutitleul F.. Bachellerie J.P.and F’uvionE. (1993) Europ.J.cell Bioi., 60.308-321). We also found that the interchromatin granule associated zones accumulate the protein p80 coilin which until now was considered as being specific of coiled bodies. In chromatin-depleted nuclei, the snRNA content of the coiled bodies, clusters of interchromatin granules and their associated zones, all structures which were easily recognizable within the residual nuclear RNP network was unmodified. The data indicate, therefore, that all spliceosomes accumulation sites including coilin-containing interchromatin granule-associated zones are integral components of the nUCkar rntitt’ix (Puvion-Dutilteut I. Cell Sci. in press).
F. Besse S.. Ghan E.. Tan E.. and Puvion
E. (1995)
Christophe KLEIN, Nathalie GILBERT, Laurent LUCAS and Dominique PLOTON? Unit6 INSERh4 3 14, HGpital Ma&n Blanche, 45 rue Cogacq Jay, 5 1092 Relms Cedex. France.
microtubular
zones.
and
The distribution
main
envelope
extranuclear
polarized
chaperonin
is conserved
whose
into the nuclear
103
31, LOCALISATION AND KENDERING 01; TIlE RNA POLYMERASE I TRANSCRII’TION FACTOR UBI” DURING INTERPHASE AND MITOSIS OBSERVED IN CONFOCAI, MICROSCOPY
UNICELLULAR
(dinomitosis)
of a labile
by biochemistry
centrosome
J CCII
mitosis
compaction
and the presence
which
IN STUDY.
an original
demonstrated
a 72
Ctythrcodinirrm
MITOSIS
mars 1995
Nuclear extracted
is maximum demonstrate
whote
proteins
functionof thesediverseproteinsis discussed.
the of
The study of the topological organisation of genetic material during interphasis and mitosis has considerably developped these last years. This improvement was helped by new technics especially confocal microscopy. It is with such a microscope that we studied the three-dimensional (3D) organisation of ribosomic genes brought out by immunofluorescent labbeling of US!, a rDNA bound RNA polymerase1 transcription factor. UBF was colocahsed with chromomycine A3 specifically stained DNA.The series of optical slices obtained were reconstructed and visualised thanks to a new software based on ray tracing method that we developped. This enabled us to obtain with a very high resolution and in the whole nucleus volume, the structure, the number,, the position and the 3D organisation of UBF containing sites during Interphase and mitosis. The examination of interphasic nuclei showed that UBF containing sites are organ&d as close beads forming a necklace like svucture in the nucleolus. The 3D localisation of UBF containing chromosomic sites enable us to state that these are very close together during metaphase and very symmetrically organised in each of the chromosome sets dividing during anaphase.
CIIARACTERISATION OF RAT ISOLATED IIEPATOCYTE MULTIPLETS
TRAN Dien, COMBETTES Laurent, TORDIMANN Brigitte et CLARET Michel INSERM
Biologie du D&eloppement,
u2 74. UPS: orsoy,
Thieny, BERTHON
Frmcr
The stimulation of rat hepatocytes by Ca2+ mobilising agonists induces cytosolic free Ca2+ oscillations. We have recently reported that, in contrast to single cells, multiplets (doublet or triplet) of associated hepatocytes whose bile canaliculus is maintained, showed coordinated oscillations and that a Ca2+ wave propagated from one cell to another. In the present work, we have studied the cell polarity and junctional complexes of these hepatocyte multiplets using different antibodies specifically recognising protein markers of particular plasma membrane domains and cell junctions. Multiplets (30% of the cell preparation) were obtained by moderate collagenase digestion of rat hepatocytes.. After one hour adherence on collagen coated coverslips, only multiplets having bile canaliculus were studied. The lateral domain of the plasma membrane was visualized by a monoclonal antibody B 1 and the canalicular (apical) domain was studied by two different approaches: I- utilisation of an anti-DPP4 polyclonal antibody directed against dipeptidyl peptidase. an enzyme predominant in the apical domain of epithelial cells. 2- specific labelling of F-actin by phalloidin (Bodipy 581/591) revealing a circle of F-a&n around the bile canaliculus. Two types of cell junction were also visualized: the tight junction (zonula occludens) decorated by an antibody directed against ZO-1, a high molecular weight polypeptide associated with tight junction, and the gap junction revealed by an anti-connexin 32 antibody These results obtained by different specific antibodies showed that, in contrast to single cells where molecules specific for cell polarity were distributed all over the cell surface, hepatocyte multiplets maintained their cell polarity and junctional complexes, The preservation of these structures are likely lo be essential to the Ca2+ wave propagation observed in these multiplets under hormonal stimulation.
PROTEINS DINOPLAGELLATES.
RELATED
Dinoflagellate
TO A BIOCHEMICAL
Protists
display
characteristicsare
permanent
persists
all along
the cell cycle
stretched
out between
spindle
We
recently
presence
of
(PERRET
r/n/,
cells.
kDa
cohnii, t 1~5).
was also analysed whole
chromosome
the IWO centrosomal
%I.. in
in confocal
pus).
laser scanning
One of the mnin
interests
microtubules
do not depolymerize
cells (PERRY
er 01. (1991). RwSystcms.
With
mono-
and
actin,
nuclear
both in Prnrwmtrm~
actin
especially
actin
(44kDa).
from
purified
nuclei
late
S-phase
during presence
of
Ctypthecodinium
revealed
the
micunr
cohnii.
chain The
of
cells during
to most
cell SCI.,
104.639-0513.
zones
and Crypthrcodinium
the
presence
of of
cells.
of proteins
on cryosections.
kDa)
eukatyotic
and the presence
cohnii
and immunoblotting
We in the
mitosis
is that the cortex
we demonstrated
(200
dinoflagellate
on permeabilized
system
~1 nl.. submtlled). myosin
the
in the contrary
as by immunofiuorescence
(SOYER-GOBILLARD heavy
mitosis
the
microscopy
in the centrosome
by electrophoresis
as well
fluorescence
antibodies,
located
in
to human
of the p-tubulin
25, 53.65: (1s43). J.
polyclonal
cytoplasmic
protein
of this microtubular
during
which
immunocytochemistry
from dinoflagellate
also
NUCLEAR STRUCTURES INVOLVED IN TRANSCRIPTION AND SPLICING OF PRE-mRNA. PUVION-DUTlLLEUL Francinc, BESSE Sylvie and PUVlON Edmond. Laboratoire BP& 94801
: Oiganisation fonctionnelle - VILLULJIF - Cedex.
du Noyau
- UPR 272 - IFC,
Compared with fluorescence microscopy, combinations at the electron microscopic level of high resolution autoradiography, cytochemisay, immunomicroscopy and in situ hybridization are methods of choice for identifying the structures involved in transcription and splicing of pre-mRNA. We established that the perichromatin fibrils extending in the interchromatin space correspond to newly synthesized pre-mRNP and represent the morphological substrate of splicing. The clusters of interchromatin granules and coiled bodies which also accumulate components of spliceosomes would rather be involved in pre and/or post-splicing events such as the assembly of spliceosomes, snRNP recycling, intron degradation, sorting of RNA molecules (Visa N.. PuvionDutilleul F., Harper F., Bachellerie J.P. and Puvion E. (1993) Exp. Cell Res.. 208, 1934). In addition, we recently identified a new compartment always associated with clusters of interchromatin granules and therefore termed : interchromatin granule-associated zone. This structure contains Ul but not U2 snRNA, this suggests that it might be a site of storage and/or final maturation of the Ul snRNP particle (Visa N.. Puvion-Dutitleul F.. Bachellerie J.P.and F’uvionE. (1993) Europ.J.cell Bioi., 60.308-321). We also found that the interchromatin granule associated zones accumulate the protein p80 coilin which until now was considered as being specific of coiled bodies. In chromatin-depleted nuclei, the snRNA content of the coiled bodies, clusters of interchromatin granules and their associated zones, all structures which were easily recognizable within the residual nuclear RNP network was unmodified. The data indicate, therefore, that all spliceosomes accumulation sites including coilin-containing interchromatin granule-associated zones are integral components of the nUCkar rntitt’ix (Puvion-Dutilteut I. Cell Sci. in press).
F. Besse S.. Ghan E.. Tan E.. and Puvion
E. (1995)
Christophe KLEIN, Nathalie GILBERT, Laurent LUCAS and Dominique PLOTON? Unit6 INSERh4 3 14, HGpital Ma&n Blanche, 45 rue Cogacq Jay, 5 1092 Relms Cedex. France.
microtubular
zones.
and
The distribution
main
envelope
extranuclear
polarized
chaperonin
is conserved
whose
into the nuclear
103
31, LOCALISATION AND KENDERING 01; TIlE RNA POLYMERASE I TRANSCRII’TION FACTOR UBI” DURING INTERPHASE AND MITOSIS OBSERVED IN CONFOCAI, MICROSCOPY
UNICELLULAR
(dinomitosis)
of a labile
by biochemistry
centrosome
J CCII
mitosis
compaction
and the presence
which
IN STUDY.
an original
demonstrated
a 72
Ctythrcodinirrm
MITOSIS
mars 1995
Nuclear extracted
is maximum demonstrate
whote
proteins
functionof thesediverseproteinsis discussed.
the of
The study of the topological organisation of genetic material during interphasis and mitosis has considerably developped these last years. This improvement was helped by new technics especially confocal microscopy. It is with such a microscope that we studied the three-dimensional (3D) organisation of ribosomic genes brought out by immunofluorescent labbeling of US!, a rDNA bound RNA polymerase1 transcription factor. UBF was colocahsed with chromomycine A3 specifically stained DNA.The series of optical slices obtained were reconstructed and visualised thanks to a new software based on ray tracing method that we developped. This enabled us to obtain with a very high resolution and in the whole nucleus volume, the structure, the number,, the position and the 3D organisation of UBF containing sites during Interphase and mitosis. The examination of interphasic nuclei showed that UBF containing sites are organ&d as close beads forming a necklace like svucture in the nucleolus. The 3D localisation of UBF containing chromosomic sites enable us to state that these are very close together during metaphase and very symmetrically organised in each of the chromosome sets dividing during anaphase.