Provide Attachment Sites Research Articles

Characterization of membrane glycoproteins involved in attachment of microfilaments to the microvillar membrane.

We have characterized a novel integral membrane glycoprotein, from intestinal microvilli, with a relative molecular mass (Mr) of 140 000 as measured by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). This glycoprotein has been purified to homogeneity, and specific antibodies have been prepared to localize it immunocytochemically and to determine its topological organization with respect to the membrane bilayer. This protein's major features are: (1) in contrast to other major glycoproteins of the microvillus membrane it cannot be quantitatively extracted by detergents; (2) treatment of the core residue (an insoluble fraction which remains after Triton X-100 treatment of purified microvilli) with low-ionic-strength buffer in the presence of chelating agents promotes partial solubilization of the amphipathic glycoprotein; (3) controlled proteolysis with papain, using right-side-out sealed vesicles derived from microvilli, results in solubilization of the protein. Once solubilized by papain, the protein co-migrates (on SDS-PAGE) with the protein obtained by dialysis but, unlike the low-ionic-strength form, it does not bind detergents or exhibit hydrophilic properties. These observations are consistent with the protein having a small hydrophobic domain that anchors it to the microvillar membrane. Most of these features were reported some years ago for aminopeptidase, but this newly described protein has a distinct behaviour with respect to its association with microfilaments. We have demonstrated that the 110K protein, a major cytoskeletal protein of the lateral bridges, will bind to the glycoprotein in vitro. These observations suggest that this 140K polypeptide is a transmembrane protein and may provide attachment sites in vivo for cytoskeletal proteins.

Platelet interaction with synthetic copolypeptide films.

Kinetic and equilibrium studies of blood platelet binding to copolypeptide films show that attachment and serotonin release are not dependent upon the composition of the copolypeptide. Data may be explained by postulating that platelets frequently collide elastically with the surface but leave behind material that modifies subsequent behavior. Similarly, material released from platelets adsorbs at the interface and the extent of attachment and serotonin release are modified and controlled by these adsorbed species. Basically, if the platelet is exposed to a clean surface, its collision with the surface leads to activation and release. In the presence of inert protein, the collision is cushioned by the protein and platelets do not attach or release to any extent. Finally, if protein (or other entities) released from the platelet provide attachment sites, then attachment occurs without release. It is postulated that the behavior of platelets at surfaces is controlled by these interrelated processes.

Development of Vertebrate Skeletal Muscle

An investigation of developing skeletal muscle necessitates the study of three categories; the derivation of muscle cells or fibers, myofilament synthesis and interactions, assembly of myofilaments into functional sarcomeres of striated myofibrils. With few exceptions, skeletal muscle cells are of mesodermal origin, and consist of rounded mononucleated cells which elongate and fuse with one another to become myotubes. Within the sarcoplasm, myofibrillar proteins are synthesized and grouped into interacting thick and thin filaments. Crude, non-striated myofibrils result from linear arrangements of thick and thin filaments which are horizontally aligned by the invaginating sarcotubular system. After Z-lines form, providing attachment sites for thin filaments, a typical banding pattern follows. The newly formed Z-lines pull apart, followed by the attached thin filaments, and repeating “relaxed” sarcomeres are the resulting striated myofibrillar pattern.

Dopa and Cysteine Incorporation into Premelanosomes: Effects of Cycloheximide and Gene Substitution

Electron microscopic autoradiography was used to investigate the relationship between melanin formation and thiol incorporation in premelanosomes of genetically defined chick embryos. 3 H-dopa was used as the indicator of melanogenic capacity, 35 S-cysteine as a representative thiol compound, and 3 H-leucine for control purposes since the latter is not specifically incorporated into melanoprotein. 3 H-dopa was selectively incorporated into wild type (e + / e + ) melanocytes and was localized in melanogenic organelles as determined by comparison with 3 H-leucine incorporation, 35 S-cysteine also showed specificity for premelanosomes when compared to 3 H-leucine, but its incorporation was not as great as that of 3 H-dopa. Cycloheximide reduced the mean number of 35 S-cysteine grains per μ 2 of premelanosome although specificity was maintained for premelanosomes, probably due to non-peptide bond formation between cysteine molecules and melanin intermediates. Genetic substitution showed that both 3 H-dopa and 35 S-cysteine were incorporated in conjunction with premelanosome production. When premelanosome synthesis was high ( E/E tissue), both dopa and cysteine incorporation were high. When premelanosome synthesis was low (e y /e y tissue), both dopa and cysteine incorporation were low. Of the cysteine molecules incorporated into premelanosomes, some reacted with melanin intermediates as free cysteines, while some became incorporated into the premelanosomal proteins. These autoradiographic results support several previous reports which suggest that thiol-bearing premelanosomal polypeptides provide attachment sites for melanin intermediates through thioether bonds. These thiol polypeptides may be the mechanism by which melanin is securely linked to premelanosomes.