- Email: [email protected]
Developmental changes in keratin phosphorylation during stratified squamous epithelial differentiation
184
185
CELL SURFACE PROTEOGLYCAN ANCHORS THE EXTRACELLULAR MATRIX TO THE CYTOSKELETON A_~. R a v r a e g e r a n d M__j_.B e r n f i e l d . Dept. of Pediatrics, Stanford University School of Medicine, Stanford, CA., 9 4 3 0 5 The c e l l s u r f a c e h e p a r a n s u l f a t e - r i c h proteoglycan (PG) o f NMuMG mammary e p i thelia has two domains, a membrane d o m a i n a n d an e c t o d o m a i n which binds matrix molecules. Cross-linkin$ ligands promote association of the membrane domain with the cytoskeleton because (i) PG c r o s s - l i n k e d by antibodies forms clusters which are removed from the cell surface, a process blocked by azide or cytochalasin D, a n d ( i i ) the clusters h a v e an i n c r e a s e d resistance to Triton extraction. Analogously, PG c r o s s - l i n k e d b y an i n s o l u b l e matrix may estabish a stable association with the cytoskeleton. In the presence of type I collagen, a physiological ligand, clusters distribute along collagen fibrils but r e m a i n at t h e c e l l surface. We p r o p o s e that the cell s u r f a c e PG i s a t r a n s m e m brane matrix receptor which transmits matrix organization into cytoskeletal stability and, ultimately, cell behavior. (Supported b y NIH g r a n t s HD17146 and CA28735 and ACS g r a n t BC409.)
TRANSMEMBRANE INTERACTIONS DURING MOUSE PREIMPLANTATION DEVELOPMENT. M.F. Maylie-Pfenninger, Dept. Anatomy and Cell Biology, Columbia University, P&S, New York. The i n t e r a c t i o n of d e v e l o p m e n t a l l y regulated surface g]ycoproteins with the c y t o p ] a s m i c m a t r i x of b l a s t o m e r e s is monitored by incubation of the embryos with fluorescent (F]) ]ectins followed by treatment of the embryos with the nonionic detergent Lubro] PX. The detergent treatment yields intact inso]uble ghosts consisting of filamentous material, paracrystalline structures and nuclear remnants. These ghosts are labeled by all the ]ectins tested. However, if embryos are detergent-treated prior to F]-]ectin ]abeling, no fluorescence is visible on the surface of the ghosts. These transmembrane interactions are disrupted by incubation of the ghosts with 2% Emplgen BB and 0.1% SDS. The i d e n t i t y of the g]ycoproteins as well as of the matrix proteins involved in these interactions is being determined. Our results indicate that developmentally regulated surface g]ycoproteins can interact with the cytoplasmic matrix and that this interaction is not of covalent nature.
186 DEVELOPMENTAL CHANGES IN KERATIN PHOSPHORYLATION DURING STRATIFIED SQUAMOUS EPITHELIAL DIFFERENTATION C. F. Shuler and S. A. Schwartz University Of Chicago, Chicago, lllinois USA 60637. Rat palatal epithelium synthesizes an identical set of keratin proteins at all stages of fetal and p o s t n a t a l differentiation. A distinct change in immunereactivity is temporally related to the initial stratification of the epithelium. We examined keratin phosphorylation with respect to epithelial differentiation and keratin immunoreactlvlty. Palatal mucosa from known gestational stages was incubated in [00uCi/ml 32-P orthophosphate in Medium 199 for 3 hours at 37°C. The keratins were extracted, resolved by SDSPAGE, silver stained and autoradiographed. until 15 days in utero only a 52kd keratin was labelled. All 17-day in utero keratins were phosphorylated and at ~egrees differing from their relative concentration. The 67kd keratin was prominently labelled at 17 days but not at later stages of development. The changes in keratin phosphorylation were temporally correlated with both the changes in k e r a t i n imunoreactivity and initial epithelial stratification and may represent a post-translational modification related to a differentiated function of the kerati~s i n the epithelial cell cytoskeleton.
187 EVIDENCE FOR A DUAL MECHANISM OF THE ASSES~BLY OF THE PLASMA MEMBRANE DURING CELL DIVISION IN THE DROSOPHILA MELANOGASTER EMBRYO. J.Bluemink, S.Biliriski &W.Hage; Hubrecht Laboratory, Utrecht, the Netherlands. We have used tannic acid as a m o r d a n t t o preserve phospholipids in ultrathin sections, as well as freeze fracture electron microscopy, to analyse how membrane is assembled in the cleavage furrows during blastoderm formation. Evidence will be presented for a dual mechanism of the assembly of the plasma membrane involving coated vesicles derived from the Golgi system that package integral membrane proteins, and multilamellar bodies (mlb's) derived from endoplasmic reticulum that package bulk membrane phospholipids. Both elements probably are transported by microtubules, which are numerous. Coated vesicles insert into the furrow and appear as coated pits, which probably give rise to specialised membrane domains such as focal desmosomes. Mlb's fuse by focal defects in the lipid bilayer which in replicas appeaY as lipidic particles. It is probably the nLlb's that make the major contribution to the surface area needed to form some 5000 cells within 90 rain.
74S
185
CELL SURFACE PROTEOGLYCAN ANCHORS THE EXTRACELLULAR MATRIX TO THE CYTOSKELETON A_~. R a v r a e g e r a n d M__j_.B e r n f i e l d . Dept. of Pediatrics, Stanford University School of Medicine, Stanford, CA., 9 4 3 0 5 The c e l l s u r f a c e h e p a r a n s u l f a t e - r i c h proteoglycan (PG) o f NMuMG mammary e p i thelia has two domains, a membrane d o m a i n a n d an e c t o d o m a i n which binds matrix molecules. Cross-linkin$ ligands promote association of the membrane domain with the cytoskeleton because (i) PG c r o s s - l i n k e d by antibodies forms clusters which are removed from the cell surface, a process blocked by azide or cytochalasin D, a n d ( i i ) the clusters h a v e an i n c r e a s e d resistance to Triton extraction. Analogously, PG c r o s s - l i n k e d b y an i n s o l u b l e matrix may estabish a stable association with the cytoskeleton. In the presence of type I collagen, a physiological ligand, clusters distribute along collagen fibrils but r e m a i n at t h e c e l l surface. We p r o p o s e that the cell s u r f a c e PG i s a t r a n s m e m brane matrix receptor which transmits matrix organization into cytoskeletal stability and, ultimately, cell behavior. (Supported b y NIH g r a n t s HD17146 and CA28735 and ACS g r a n t BC409.)
TRANSMEMBRANE INTERACTIONS DURING MOUSE PREIMPLANTATION DEVELOPMENT. M.F. Maylie-Pfenninger, Dept. Anatomy and Cell Biology, Columbia University, P&S, New York. The i n t e r a c t i o n of d e v e l o p m e n t a l l y regulated surface g]ycoproteins with the c y t o p ] a s m i c m a t r i x of b l a s t o m e r e s is monitored by incubation of the embryos with fluorescent (F]) ]ectins followed by treatment of the embryos with the nonionic detergent Lubro] PX. The detergent treatment yields intact inso]uble ghosts consisting of filamentous material, paracrystalline structures and nuclear remnants. These ghosts are labeled by all the ]ectins tested. However, if embryos are detergent-treated prior to F]-]ectin ]abeling, no fluorescence is visible on the surface of the ghosts. These transmembrane interactions are disrupted by incubation of the ghosts with 2% Emplgen BB and 0.1% SDS. The i d e n t i t y of the g]ycoproteins as well as of the matrix proteins involved in these interactions is being determined. Our results indicate that developmentally regulated surface g]ycoproteins can interact with the cytoplasmic matrix and that this interaction is not of covalent nature.
186 DEVELOPMENTAL CHANGES IN KERATIN PHOSPHORYLATION DURING STRATIFIED SQUAMOUS EPITHELIAL DIFFERENTATION C. F. Shuler and S. A. Schwartz University Of Chicago, Chicago, lllinois USA 60637. Rat palatal epithelium synthesizes an identical set of keratin proteins at all stages of fetal and p o s t n a t a l differentiation. A distinct change in immunereactivity is temporally related to the initial stratification of the epithelium. We examined keratin phosphorylation with respect to epithelial differentiation and keratin immunoreactlvlty. Palatal mucosa from known gestational stages was incubated in [00uCi/ml 32-P orthophosphate in Medium 199 for 3 hours at 37°C. The keratins were extracted, resolved by SDSPAGE, silver stained and autoradiographed. until 15 days in utero only a 52kd keratin was labelled. All 17-day in utero keratins were phosphorylated and at ~egrees differing from their relative concentration. The 67kd keratin was prominently labelled at 17 days but not at later stages of development. The changes in keratin phosphorylation were temporally correlated with both the changes in k e r a t i n imunoreactivity and initial epithelial stratification and may represent a post-translational modification related to a differentiated function of the kerati~s i n the epithelial cell cytoskeleton.
187 EVIDENCE FOR A DUAL MECHANISM OF THE ASSES~BLY OF THE PLASMA MEMBRANE DURING CELL DIVISION IN THE DROSOPHILA MELANOGASTER EMBRYO. J.Bluemink, S.Biliriski &W.Hage; Hubrecht Laboratory, Utrecht, the Netherlands. We have used tannic acid as a m o r d a n t t o preserve phospholipids in ultrathin sections, as well as freeze fracture electron microscopy, to analyse how membrane is assembled in the cleavage furrows during blastoderm formation. Evidence will be presented for a dual mechanism of the assembly of the plasma membrane involving coated vesicles derived from the Golgi system that package integral membrane proteins, and multilamellar bodies (mlb's) derived from endoplasmic reticulum that package bulk membrane phospholipids. Both elements probably are transported by microtubules, which are numerous. Coated vesicles insert into the furrow and appear as coated pits, which probably give rise to specialised membrane domains such as focal desmosomes. Mlb's fuse by focal defects in the lipid bilayer which in replicas appeaY as lipidic particles. It is probably the nLlb's that make the major contribution to the surface area needed to form some 5000 cells within 90 rain.
74S