Efficient targeting to storage granules of human proinsulins with altered propeptide domain.

In neuronal and endocrine cells, peptide hormones are selectively segregated into storage granules, while other proteins are exported continuously without storage. Sorting of hormones by cellular machinery involves the recognition of specific structural domains on prohormone molecules. Since the propeptide of insulin is known to play an important role in its three-dimensional structure, it is reasonable to speculate that targeting of proinsulin to storage granules would require a functional connecting peptide. To test this hypothesis, we constructed two mutations in human proinsulin with different predicted structures. In one mutation, Ins delta C, the entire C peptide was deleted, resulting in an altered insulin in which the B and the A chains are joined contiguously. In the other mutation, Ins/IGF, the C peptide of proinsulin was replaced with the unrelated 12-amino acid connecting peptide of human insulin-like growth factor-I; this substitution should permit correct folding of the B and A chains to form a tertiary structure similar to that of proinsulin. By several biochemical and morphological criteria, we found that Ins/IGF is efficiently targeted to storage granules, suggesting that the C peptide of proinsulin does not contain necessary sorting information. Unexpectedly, Ins delta C, which presumably cannot fold properly, is also targeted to granules at a high efficiency. These results imply that either the targeting machinery can tolerate changes in the tertiary structure of transported proteins, or that the B and A chains of insulin can form a relatively intact three-dimensional structure even in the absence of C peptide

Isolation of EpH4 mammary epithelial cell subpopulations which differ in their morphogenetic properties

Summary: EpH4 is a nontumorigenic cell line derived from spontaneously immortalized mouse mammary gland epithelial cells (Fialka et al., 1996). When grown in collagen gels, EpH4 cells give rise to different types of structures, e.g., solid cords or branching tubes. By removing and subsequently dissociating single three-dimensional colonies of defined morphology, we have isolated six clonal subpopulations of EpH4 cells which display distinct morphogenetic properties in collagen gel cultures. Thus, cells from the H1B clone form branching cords devoid of a central lumen, K3A3 cells from cords enclosing small multifocal lumina, and J3B1 cells form large cavitary structures containing a wide lumen. I3G2 cells form either cords or tubes, depending on the type of serum added to the culture medium. Finally, when grown in serum-free medium, Be1a cells form spherical cysts, whereas Be4a cells form long, extensively branched tubes. In additional assays of morphogenesis, i.e., cell sandwiching between two collagen gels or culture on a thick layer of Matrigel (a laminin-rich extracellular matrix), all clones form epithelial-cell-lined cavitary structures, except H1B cells which are unable to generate lumina under these conditions. The EpH4 sublines we have isolated provide an in vitro system for studying the mechanisms responsible for lumen formation and branching morphogenesis, as well as for identifying the factors which subvert these developmental processes during mammary carcinogenesi

Architecture of coatomer: Molecular characterization of delta-COP and protein interactions within the complex

Copyright © 2011 by The Rockefeller University Press.Coatomer is a cytosolic protein complex that forms the coat of COP I-coated transport vesicles. In our attempt to analyze the physical and functional interactions between its seven subunits (coat proteins, [COPs] alpha-zeta), we engaged in a program to clone and characterize the individual coatomer subunits. We have now cloned, sequenced, and overexpressed bovine alpha-COP, the 135-kD subunit of coatomer as well as delta-COP, the 57-kD subunit and have identified a yeast homolog of delta-COP by cDNA sequence comparison and by NH2-terminal peptide sequencing. delta-COP shows homologies to subunits of the clathrin adaptor complexes AP1 and AP2. We show that in Golgi-enriched membrane fractions, the protein is predominantly found in COP I-coated transport vesicles and in the budding regions of the Golgi membranes. A knock-out of the delta-COP gene in yeast is lethal. Immunoprecipitation, as well as analysis exploiting the two-hybrid system in a complete COP screen, showed physical interactions between alpha- and epsilon-COPs and between beta- and delta-COPs. Moreover, the two-hybrid system indicates interactions between gamma- and zeta-COPs as well as between alpha- and beta' COPs. We propose that these interactions reflect in vivo associations of those subunits and thus play a functional role in the assembly of coatomer and/or serve to maintain the molecular architecture of the complex.This work was supported by The Deutsche Forschungsgemeinschaft (SFB 352), the Human Frontier Science Program, and the Swiss National Science Foundation No. 31-43366.95

Endothelial diaphragmed fenestrae: in vitro modulation by phorbol myristate acetate

Cultured microvascular endothelial cells isolated from fenestrated capillaries have been shown to express many properties of their in vivo differentiated phenotype, yet they contain very few diaphragmed fenestrae. We show here that treatment of capillary endothelial cells with the tumor promoter, 4 beta-phorbol 12-myristate 13-acetate, induces more than a fivefold increase in the frequency of fenestrae per micron 2 of cell surface, as determined from a quantitative evaluation on freeze-fracture replicas. In quick-frozen, deep-etched preparations, the endothelial fenestrae appeared to be bridged by a diaphragm composed of radial fibers interweaving in a central mesh, as previously observed in vivo. These results indicate that diaphragmed fenestrae are inducible structures, and provide an opportunity to study them in vitro