We show that Schwann cell-derived Desert hedgehog (Dhh) signals the formation of the connective tissue sheath around peripheral nerves. mRNAs for dhh and its receptor patched (ptc) are expressed in Schwann cells and perineural mesenchyme, respectively. In dhh-/- mice, epineurial collagen is reduced, while the perineurium is thin and disorganized, has patchy basal lamina, and fails to express connexin 43. Perineurial tight junctions are abnormal and allow the passage of proteins and neutrophils. In nerve fibroblasts, Dhh upregulates ptc and hedgehog-interacting protein (hip). These experiments reveal a novel developmental signaling pathway between glia and mesenchymal connective tissue and demonstrate its molecular identity in peripheral nerve. They also show that Schwann cell-derived signals can act as important regulators of nerve development.

Abstract. We have purified the transformation and shape change sensitive isoform of an actin associated polypeptide doublet previously described by us (Shapland, C., P. Lowings, and D. Lawson. 1988. J. Cell Biol. 107:153-161) and have shown that it is evolutionarily conserved as far back as yeast. The purified protein: (a) binds directly to actin filaments at a ratio of 1:6 actin monomers, with a binding constant (Ka) of •7.5 x 105 M-~; and (b) causes actin filament gelation within 2 min. Although these activities are controlled by ionic strength (and may be mediated by positively charged amino acid residues) the molecule remains as a monomer irrespective of ionic conditions. EM reveals that the addition of this protein to actin filaments converts them from a loose, random distribution into a tangled, cross-linked meshwork within 1 min, and discrete tightly aggregated foci after 10 min. By use of an "add-back" cell permeabilization system we can rebind this molecule specifically to actin filaments in cells from which it has previously been removed. Since the protein is transformation sensitive and gels actin, we have named it transgelin. TIN is crucial for a variety of cellular events such as motility, division and cell surface receptor movement (40,43,55,56). Actin organization can be controlled by a large number ('~70 are so far known) (3, 37) of actin-associated proteins which act by bundling, cross-linking, severing, gelating, sequestering monomers, or preventing actin polymerization (3,9,37,40,43,44,49). These molecules, acting either individually or in concert, regulate (a) the physical status of actin (that is the ratio of globular to filamentous actin) (43, 56), (b) actin geometry (5,19,20,27,44), and (c) provide both the fine control and the driving force required for the cellular events mentioned above.Disruption of the actin network is known to accompany events such as neoplasia (21, and see reference 4D and, in this instance, can involve alterations to both the actin microfilament network itself (21) and the expression of selected actin-associated proteins (45). However, only seven of the 70 or so proteins associated with actin are thus far known to be affected by transformation; namely, the higher molecular weight tropomyosins (11), nonmuscle caldesmon (14), smooth muscle myosin light chain 2 (16), gelsolin and actin-binding protein (17), protein C4 (41), and gelsolin (50). Since some actin-associated proteins probably act synergistically to control and organize actin, e.g., tropomyosin, gelsolin, and caldesmon (50), it seems very likely that the major changes to the actin microfiament network that occur following transformation may reflect the coordinated downregulation of several of these important molecules rather than alterations to the ratio of globular: filamentous actin itself.We have previously identified a transformation-sensitive polypeptide doublet (protein C4h'9 present in all cells and tissues apart from skeletal muscle, red blood ceils and neurons, and have shown that the higher rela...

We have used thin section and freeze-fracture electron microscopy to study membrane changes occurring during exocytosis in rat peritoneal mast cells. By labeling degranulating mast cells with ferritin-conjugated lectins and anti-immunoglobulin antibodies, we demonstrate that these ligands do not bind to areas of plasma membrane or granule membrane which have fused with, or are interacting with, granule membrane. Moreover, intramembrane particles are also largely absent from both protoplasmic and external fracture faces of plasma and granule membranes in regions where these membranes appear to be interacting. Both the externally applied ligands and intramembrane particles are sometimes concentrated at the edges of fusion sites. These results indicate that membrane proteins are displaced laterally into adjacent membrane regions before the fusion process and that fusion occurs between protein-depleted lipid bilayers. The finding of protein-depleted blebs in regions of plasma and granule membrane interaction raises the interesting possibility that blebbing may be a process for exposing the granule contents to the extracellular space and for the elimination of excess lipid while conserving membrane proteins.In most secretory cells, the triggered release of specific materials stored in secretory granules occurs by exocytosis (2,18,37,43). This process involves the fusion of granule membrane with plasma membrane, and results in the exposure of the granule contents to the extracellular space (2,18,37,43). The molecular events in exocytosis, and in membrane fusion in general, are unknown. We have studied the behavior of membrane lipids and proteins during and after the fusion events occurring in the process of degranulation of rat peritoneal mast cells. The extensive membrane interactions occurring all around the circumference and inside degranulating mast cells make these cells particularly attractive for studying membrane fusion.Mast cells can easily be obtained in homogeneous cell suspension (9). Having high affinity surface receptors for the Fc region of immunoglobulin E (IgE) (so-called Fc receptors) (5), mast cells obtained from appropriately immunized rats have cytophilic IgE antibodies on their surface which serve as receptors for the specific antigen (6). Such sensitized cells can be stimulated to secrete histamine (and other substances) by exposure to the specific antigen (4, 6), or to anti-Ig antibody (6,

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Abstract. The association and interaction of plectin (Mr 300,000) with intermediate filaments and filament subunit proteins were studied. Immunoelectron microscopy of whole mount cytoskeletons from various cultured cell lines (rat glioma C6, mouse BALB/c 3T3, and Chinese hamster ovary) and quick-frozen, deepetched replicas of Triton X-100-extracted rat embryo fibroblast cells revealed that plectin was primarily located at junction sites and branching points of intermediate filaments. These results were corroborated by in vitro recombination studies using vimentin and plectin purified from C6 cells. Filaments assembled from mixtures of both proteins were extensively crosslinked by oligomeric plectin structures, as demonstrated by electron microscopy of negatively stained and rotary-shadowed specimens as well as by immunoelectron microscopy; the binding of plectin structures on the surface of filaments and cross-link formation occurred without apparent periodicity. Plectin's crosslinking of reconstituted filaments was also shown by ultracentrifugation experiments. As revealed by the rotary-shadowing technique, filament-bound plectin structures were oligomeric and predominantly consisted of a central globular core region of 30-50 nm with extending filaments or filamentous loops. Solidphase binding to proteolytically degraded vimentin fragments suggested that plectin interacts with the helical rod domain of vimentin, a highly conserved structural element of all intermediate filament proteins. Accordingly, plectin was found to bind to the glial fibrillar acidic protein, the three neurofilament polypeptides, and skin keratins. These results suggest that plectin is a cross-linker of vimentin filaments and possibly also of other intermediate filament types.