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Villin is an evolutionarily well conserved, Ca2+ regulated actin‐binding protein, and a major structural component of the brush border of specialized absorptive cells. Using paraffin sections and an affinity purified polyclonal anti‐villin antibody, we have investigated the early expression of villin during mouse embryogenesis. Villin is first detectable at the early post‐implantation stage in visceral endodermal cells at the periphery of the egg cylinder. In this extra embryonic layer, the expression of villin increases and then persists until full term gestation. In the embryo, villin first appears in gut anlage during the axial rotation. Using the same methodology, villin expression is also demonstrated in differentiating embryoid bodies from a teratocarcinoma. Both in extra embryonic and embryonic extracts, villin expression is confirmed by immunoblot and Northern blot analysis which reveal, respectively, a single polypeptide of 93 kd and an mRNA of 3.4 kb in length, two well defined parameters for adult mouse villin gene expression. The results presented here show that paraffin sections allow very sensitive and highly resolutive detection of antigens in early embryogenesis. They provide a detailed developmental profile of villin expression and demonstrate the usefulness of villin as a marker for epithelial cells involved in absorptive processes.

A monoclonal antibody (53FC3) has been produced against a Golgi membrane protein with a mol. wt. of 135 000 which was originally identified using a polyclonal antiserum. Treatment of isolated, intact Golgi vesicles with protease caused a decrease in mol. wt. of 5000‐10 000, whereas in the presence of Triton X‐100, the protein was completely degraded. This shows that the protein spans the bilayer and that most of its mass is on the luminal side of Golgi membranes. Using two immunoelectron microscopic techniques, the protein was found in one or two cisternae on one side of the Golgi stack which, in normal rat kidney cells, had 4‐6 cisternae. As an illustration of the use to which this monoclonal antibody can be put we present a light microscopic study of the disassembly and reassembly of the Golgi complex during mitosis.

Villin, a Ca2+‐regulated actin‐binding protein is a major component of microvilli of intestinal epithelial cells and kidney proximal tubule cells. Villin expression during assembly of the brush border can be investigated using a human colon adenocarcinoma cell line HT29‐18. This cell line is able to differentiate under nutritional control and develops an enterocyte‐like phenotype. A cDNA library from a subclone HT29‐18‐C1 was constructed in an expression vector and a cDNA specific for human villin was isolated. This cDNA codes for the 110 carboxy‐terminal residues of villin. Within that region, the 76 carboxy‐terminal residues present 65% homology with the chicken villin ‘head piece’. We show that two mRNA species 4.0 kb and 3.2 kb long hybridize with this cDNA probe in humans, whereas in rat and chicken only one mRNA species can be detected. The two villin mRNA species are co‐expressed in normal human small and large intestinal mucosa and tumoral HT29‐18 cells as well as in normal kidney. No villin mRNAs were detected in other normal or malignant epithelial cell types. Finally, we observed an accumulation of the two mRNA species coding for villin when HT29‐18 cells become differentiated, suggesting that control of villin expression during terminal differentiation can occur at the transcription level or by RNA stabilization.

An integral membrane glycoprotein of pig intestinal microvilli which exists in two polypeptide forms [mol. wt. 140 K and 200 K as measured by SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE)] was purified to homogeneity and characterized. The 200‐K form is probably a precursor of the 140‐K species. We have localized the glycoprotein by electron microscope immunochemistry using specific antibodies and determined its topological organization with respect to the membrane bilayer. Triton X‐100 treatments which solubilize most other microvillar membrane glycoproteins from purified, closed, right‐side out vesicles do not efficiently extract this protein. The protein can be partially solubilized from the detergent‐insoluble residue, either by treatment with proteases (trypsin or papain) or by exposure to low ionic strength buffer in the presence of chelating agents and detergents. Once solubilized by papain or trypsin, the protein co‐migrates on SDS‐PAGE with the protein obtained by low ionic strength extraction. However, the form of the protein released by papain does not bind detergents and exhibits hydrophilic properties. Our observations are consistent with the 140‐K protein having a small hydrophobic domain that anchors it to the microvillar membrane. The 140‐K glycoprotein binds in vitro to a 110‐K protein of the core cytoskeleton residue. These observations suggest that the 140‐K glycoprotein may be a transmembrane protein which may in vivo provide attachment sites for direct or indirect association with polypeptides of the microvillus cytoskeleton.

Monoclonal antibodies have been raised to pig brain triskelions and one clone, DC41, was found to recognize the clathrin heavy chain by immunoblotting. However, both by immunofluorescence and immunoelectron microscopy, and in complete contrast to polyclonal anti‐clathrin antibodies, monoclonal DC41 did not label either coated pits or coated vesicles anywhere in the cell. Instead it appeared to label the cell cytoplasm. These data suggest that DC41 recognizes a cytoplasmic form of clathrin, perhaps that form produced by uncoating of coated vesicles which is then ready to re‐build another coated pit.

The actin binding sites of actobindin and thymosin beta 4, two small polypeptides that inhibit actin polymerization by interacting with monomeric actin, have been localized using peptide mimetics. Both sites are functionally similar and extend over 20 residues and are located in the NH2‐terminus of the polypeptides. They can be dissected into two functional entities: a conserved hexapeptide motif (LKHAET or LKKTET), which forms the major contact site through electrostatic interactions with actin, and a non‐conserved NH2‐terminal segment preceding the motif, which exerts the inhibitory activity on actin polymerization probably by steric hindrance. The introduction of a glutamic acid at the third position in the motif, creating LKEAET or LKETET sequences, which are similar to those found in some F‐actin binding proteins, converts the peptide's inhibitory phenotype into an F‐actin stimulatory property. These results allow the proposal of a simple model for G‐ to F‐actin modulation.

This paper describes the properties of rabbit polyclonal antibodies directed against purified human insulin receptor which strongly stimulate the intrinsic tyrosine kinase activity. The stimulatory effect of the antibodies on the kinase activity was obtained on the insulin receptor autophosphorylation as well as on the kinase activity towards a synthetic substrate. This stimulation is additive to that induced by insulin. Moreover, rabbit antibodies do not impair insulin binding. These data strongly suggest that antibodies and insulin act through separate pathways. This conclusion is reinforced by the differences observed on the phosphopeptide maps of the receptor's beta subunit whose phosphorylation was performed either in the presence of insulin or rabbit antibodies. Interestingly, these polyclonal antibodies can also induce an activation of the receptor autophosphorylation by interacting only with extracellular determinants. The anti‐insulin receptor antibodies mimic insulin in their stimulatory effect on amino acid (AIB) uptake, but they have a different effect to that found on the kinase activity; the simultaneous addition of the antiserum and insulin failed to stimulate this amino acid transport over the level induced by a saturating concentration of hormone.