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Cytokeratin patterns during the first cell cycle of hamster embryos
Cell Biology International
W65
Reports, Vol. 14, Abstracts Supplement
PAT
Id mLmMENT AND METAiiloRPHosIs ~~.~,. _Barry Gumbinns. 1) Physiopath.du Giovanni Levit, Jean-Paul‘Iblerv’. Develop., F!coleNonnale gupaietue. 4MNecrUlm,Paris,Prwce2)Dep. of piuamacology,univ. of calif. SfatPrancii. CA 94143-0450USA.
E-cad&h is a cell adhesionmokxle dinxtly implicatedia the conuol of Ca+Uep&ent intemctioosbetweenepithelial cells. We have investigated by immanohistofluore~~~~ the patternsof eqreaskm of EcadMio during Xemqwslaevis early developmeatandmetamqhosis. The fast expressioo of E-cad&& takes place in the preaamptiveectodexmof early gastmlas; regionsof the external cell mantledestinedto invaginatedating gasmdation do not express the molecule. Dming late gasimlation and neamlation the eaodem overlying and the neural plate is E-cad&in-negative while both layers of the non-neural ectodermare strongly positive. Diierently from mostother species,&odemul cells do not expressesE-cadhexioup to stage 20NF. In the gut, endodermaJcells are E-cadherin-positive only when a welldiffereatiated epithelium is detectable.No mesodamal structure is stainedduring early development.In the placodes,at variance with other species,E-cadkio d@paars very rapidly after placodethickening.During further embryonic developmeatE-cadhezinis presentin the SkiR the gut epitltelium. the pancreas,many monosuatitkd epithelia and most glands. The liver primordiam and the pronephmsare stainedvery weakly. In the mesonephmsthe Wolffian duct aodcollecting ductsarepositive. During amphibii metamoqhosis a completerestmctaring of the body plan occursundexthe strict control of thyroid hormones. All newly formed epithelia are strongly positive for E-cadhezin.Satprisingly, degenemtiag epithelia of both skin and intestln maintain high levels of the protein even after sming to disorganize. In the adult, staining is strongia the skin, the glands,the luogs, the.gut epitheliamand the panueas,andweak in the liver. In all casesE-cadherin is preseN in the cell membraneio regions of cell-to-ceil cantact.% resultsare discus& in view of thepossiblemle of E-cadherinin theformation anddiierentiation of epithelia.
ISOLATION OF JNTEGRIN a.SUBUNlT BINDING CYTOPLASMIC PROTEINS Shot&at Dedhar, Mumtaz Rojiani. Advanced Therapeutics Department, Cancer Control Agency of B.C., Vancouver, B.C., VSZ 4B6, Canada. We have endeavored to identify proteins that interact with the cytoplasmic domain of the a subunits of integrins the cell surface receptors involved in interactions with the Affinity chromatography, in the extracellular matrix. presence of divalent cations, on sepharose coupled to a synthetic peptide, KLGFFKR, which corresponds to a common motif on the cytoplasmic domain of all a subunits resulted in the isolation of several proteins. Along with actin, a-actinin. talin, tmpomyosin and vinculin as determined by western blot analysis, we have isolated a 59K protein following elution with BDTA-containing buffer. Similar proteins were isolated from extracts of human osteosatcoma as well as neuroblastoma cells. The N-terminal amino acid sequence of the 59K protein was found to be identical to Ro/SS-A, a 59K antigen to which antibodies am found in a number of autoimmune diseases. This protein is highly charged and therefore exhibits an aberrant migration pattern on SDS-PAGE, is retained on the inner surface of the plasma membrane and is a calcium binding protein. We am presently carrying out in vitro binding studies to determine if this protein interacts directly with integrins. and are also carrying out experiments to determine which of the isolated protein binds directly to the KLGF’FKR sequence.
W67
59
1990
Carlos
E Plan&a'.
Inst.
Histol.
-1..
Call
Biol.,
Llepsrt.
Rria
cycle
of
ikmunofluorescence
and
unfertilized
oocyte
distribution,
confined
to
pattern.
the
disassembled persisting
responsible
cocytes
to for
cycle, ware
patches cytokeratin
into
Z-
also
canpared.
fibrils.
with
region
nust
a scholarship
INDUCES
LRnININ-BINDING
INVOLVED IN FLLS. , G. Tarone@& , M. _Abbadini
patchy-fibrillar fibrils
are
reappears,
In
contrast
and
fertilized
to
the
now surround
zygotic
factors
the
first
and
parthenogenetic
indicate transfiguration
fertilization-dapondmependent
to the cortical
NSF
overall
of
becones
neshwrks
during
in
Our results the
a
matamal
reorganization patterns
meshworks
granules.
between
reproduce
tight
stage.
The
cytokeratin
distribution
filenant
the both
methods.
cytokeratin
of interchranatin-like
cytokeratin
using
cytokeratin
cell
cytokaratin
distinguish
'Supported
W66
early the
during
studied
exhibiting
spotty
cytokeratin
parthenogenotes
region.
a homogeneous
clusters
order
to
and
Portugal.
patterns
spotty
fertilization.
Co&x
Oeiras.
ware
corresponding
the
the
David-Ferroira.
microscopy
mitosis,
oocyte,
cytoplaseic
cell
and
a&ryos
a hoeogeneous
After
first
until
unfertilized
In
spot
cortical
During
of Science,
imunoelectron
filaents.
F
1699 Lisboa
and distribution
hamster
presents
each
intermediate
School.
Institute
organization
nitotic
Jos.6
Mica1
Gulbankian
The cytokaratin first
Caw-Fonsaca.
Lisbon
that
antnyonic
although of
factors
the
cytokeratin
that
restrict
EXPRESSlON
OF
be considered. fram "INIC"
INCRECISED INTESRIN
NEUFtITE
A
i160/135kDal
WTSROUTH
IN
PC12
I. Gavazzi’, R. Tirnpl*, P. Flossing 3. , , F. Giancctti, L. Silengo Aumailley di Genetlca Biologia e ‘Dipart. P.C. MarcQisloT. Chimica, Dipar t. dl Sci$n:e Biamedlche4 IIIIIV. di fiir Max-Planck-Institttt Italy Tortno, and Biochemie, Martinsried, FRG. Rat pheochromocr,toma PC12 cells exposed to NGF differentiate as sympathetic neurons and extend new i tes on laminirl and to a much lesser extent on Neurite outgrowth occur mainly on the fibronectin. laminin fragment Pl corresponding to the center of long the cross, and only poorly on fragment EE, a arm structure, that is active with other neuronal cells. Integrin antibodies prevented adhesion and neur i t e sprouting of these cells on larninin, fragment PI. By affinity chromatography on laminin we isolated an integrin consisting of two subunits of 1EOkDa and 135kDa. The latter is recognized by ah antieerllm to integrin 131 l ubuhit. The bound 1 amihin receptor could be displaced by EDTA, but In,t by RGD OlAffinity YIGSR peptides. chromatography 01) laminin fragments showed that the 180!135kDa receptor binds to fragment Pl. The 1GOkDa o subunit of the laminin receptor was increased tenfold after NGF treatment. The effect of NGF is specific since the amount of a 15OkDa fibronectin-bindicig lntegrin a subunit remained unchanged. Noreover , the i "creased expression of the 180 135kDa +eceptor at the cell surface corresponded to a selective increa5e in cell adhesion to laminin and to fragment Pl. The lEO/lXkDa complex is, thus, an integrin receptor fril larniullll wl~oee expression and blhdihg specificity correlates witit the capacity of NGF-induced PC12 cells to extend heurltes on laminin.
W65
Reports, Vol. 14, Abstracts Supplement
PAT
Id mLmMENT AND METAiiloRPHosIs ~~.~,. _Barry Gumbinns. 1) Physiopath.du Giovanni Levit, Jean-Paul‘Iblerv’. Develop., F!coleNonnale gupaietue. 4MNecrUlm,Paris,Prwce2)Dep. of piuamacology,univ. of calif. SfatPrancii. CA 94143-0450USA.
E-cad&h is a cell adhesionmokxle dinxtly implicatedia the conuol of Ca+Uep&ent intemctioosbetweenepithelial cells. We have investigated by immanohistofluore~~~~ the patternsof eqreaskm of EcadMio during Xemqwslaevis early developmeatandmetamqhosis. The fast expressioo of E-cad&& takes place in the preaamptiveectodexmof early gastmlas; regionsof the external cell mantledestinedto invaginatedating gasmdation do not express the molecule. Dming late gasimlation and neamlation the eaodem overlying and the neural plate is E-cad&in-negative while both layers of the non-neural ectodermare strongly positive. Diierently from mostother species,&odemul cells do not expressesE-cadhexioup to stage 20NF. In the gut, endodermaJcells are E-cadherin-positive only when a welldiffereatiated epithelium is detectable.No mesodamal structure is stainedduring early development.In the placodes,at variance with other species,E-cadkio d@paars very rapidly after placodethickening.During further embryonic developmeatE-cadhezinis presentin the SkiR the gut epitltelium. the pancreas,many monosuatitkd epithelia and most glands. The liver primordiam and the pronephmsare stainedvery weakly. In the mesonephmsthe Wolffian duct aodcollecting ductsarepositive. During amphibii metamoqhosis a completerestmctaring of the body plan occursundexthe strict control of thyroid hormones. All newly formed epithelia are strongly positive for E-cadhezin.Satprisingly, degenemtiag epithelia of both skin and intestln maintain high levels of the protein even after sming to disorganize. In the adult, staining is strongia the skin, the glands,the luogs, the.gut epitheliamand the panueas,andweak in the liver. In all casesE-cadherin is preseN in the cell membraneio regions of cell-to-ceil cantact.% resultsare discus& in view of thepossiblemle of E-cadherinin theformation anddiierentiation of epithelia.
ISOLATION OF JNTEGRIN a.SUBUNlT BINDING CYTOPLASMIC PROTEINS Shot&at Dedhar, Mumtaz Rojiani. Advanced Therapeutics Department, Cancer Control Agency of B.C., Vancouver, B.C., VSZ 4B6, Canada. We have endeavored to identify proteins that interact with the cytoplasmic domain of the a subunits of integrins the cell surface receptors involved in interactions with the Affinity chromatography, in the extracellular matrix. presence of divalent cations, on sepharose coupled to a synthetic peptide, KLGFFKR, which corresponds to a common motif on the cytoplasmic domain of all a subunits resulted in the isolation of several proteins. Along with actin, a-actinin. talin, tmpomyosin and vinculin as determined by western blot analysis, we have isolated a 59K protein following elution with BDTA-containing buffer. Similar proteins were isolated from extracts of human osteosatcoma as well as neuroblastoma cells. The N-terminal amino acid sequence of the 59K protein was found to be identical to Ro/SS-A, a 59K antigen to which antibodies am found in a number of autoimmune diseases. This protein is highly charged and therefore exhibits an aberrant migration pattern on SDS-PAGE, is retained on the inner surface of the plasma membrane and is a calcium binding protein. We am presently carrying out in vitro binding studies to determine if this protein interacts directly with integrins. and are also carrying out experiments to determine which of the isolated protein binds directly to the KLGF’FKR sequence.
W67
59
1990
Carlos
E Plan&a'.
Inst.
Histol.
-1..
Call
Biol.,
Llepsrt.
Rria
cycle
of
ikmunofluorescence
and
unfertilized
oocyte
distribution,
confined
to
pattern.
the
disassembled persisting
responsible
cocytes
to for
cycle, ware
patches cytokeratin
into
Z-
also
canpared.
fibrils.
with
region
nust
a scholarship
INDUCES
LRnININ-BINDING
INVOLVED IN FLLS. , G. Tarone@& , M. _Abbadini
patchy-fibrillar fibrils
are
reappears,
In
contrast
and
fertilized
to
the
now surround
zygotic
factors
the
first
and
parthenogenetic
indicate transfiguration
fertilization-dapondmependent
to the cortical
NSF
overall
of
becones
neshwrks
during
in
Our results the
a
matamal
reorganization patterns
meshworks
granules.
between
reproduce
tight
stage.
The
cytokeratin
distribution
filenant
the both
methods.
cytokeratin
of interchranatin-like
cytokeratin
using
cytokeratin
cell
cytokaratin
distinguish
'Supported
W66
early the
during
studied
exhibiting
spotty
cytokeratin
parthenogenotes
region.
a homogeneous
clusters
order
to
and
Portugal.
patterns
spotty
fertilization.
Co&x
Oeiras.
ware
corresponding
the
the
David-Ferroira.
microscopy
mitosis,
oocyte,
cytoplaseic
cell
and
a&ryos
a hoeogeneous
After
first
until
unfertilized
In
spot
cortical
During
of Science,
imunoelectron
filaents.
F
1699 Lisboa
and distribution
hamster
presents
each
intermediate
School.
Institute
organization
nitotic
Jos.6
Mica1
Gulbankian
The cytokaratin first
Caw-Fonsaca.
Lisbon
that
antnyonic
although of
factors
the
cytokeratin
that
restrict
EXPRESSlON
OF
be considered. fram "INIC"
INCRECISED INTESRIN
NEUFtITE
A
i160/135kDal
WTSROUTH
IN
PC12
I. Gavazzi’, R. Tirnpl*, P. Flossing 3. , , F. Giancctti, L. Silengo Aumailley di Genetlca Biologia e ‘Dipart. P.C. MarcQisloT. Chimica, Dipar t. dl Sci$n:e Biamedlche4 IIIIIV. di fiir Max-Planck-Institttt Italy Tortno, and Biochemie, Martinsried, FRG. Rat pheochromocr,toma PC12 cells exposed to NGF differentiate as sympathetic neurons and extend new i tes on laminirl and to a much lesser extent on Neurite outgrowth occur mainly on the fibronectin. laminin fragment Pl corresponding to the center of long the cross, and only poorly on fragment EE, a arm structure, that is active with other neuronal cells. Integrin antibodies prevented adhesion and neur i t e sprouting of these cells on larninin, fragment PI. By affinity chromatography on laminin we isolated an integrin consisting of two subunits of 1EOkDa and 135kDa. The latter is recognized by ah antieerllm to integrin 131 l ubuhit. The bound 1 amihin receptor could be displaced by EDTA, but In,t by RGD OlAffinity YIGSR peptides. chromatography 01) laminin fragments showed that the 180!135kDa receptor binds to fragment Pl. The 1GOkDa o subunit of the laminin receptor was increased tenfold after NGF treatment. The effect of NGF is specific since the amount of a 15OkDa fibronectin-bindicig lntegrin a subunit remained unchanged. Noreover , the i "creased expression of the 180 135kDa +eceptor at the cell surface corresponded to a selective increa5e in cell adhesion to laminin and to fragment Pl. The lEO/lXkDa complex is, thus, an integrin receptor fril larniullll wl~oee expression and blhdihg specificity correlates witit the capacity of NGF-induced PC12 cells to extend heurltes on laminin.