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L I Fessler

Publications and source records attributed to L I Fessler.

At least 37 records · Page 2Linked to original sources
PubMed1990

A role for integrin in the formation of sarcomeric cytoarchitecture.

We propose that integrins help to coordinate the differentiation of the internal, sarcomeric cytoarchitecture of a muscle fiber with its immediate environment and are essential for correct integration of muscle cells into tissue. We found that integrin alpha PS2 beta PS accumulated at contact regions of Drosophila embryo cells cultured in D-22 medium on Drosophila laminin. Myotubes formed, but subsequent addition of serum or fibronectin was needed for sarcomere formation: integrin and actin became concentrated at Z-bands; myosin and actin occurred between the Z-bands. This change failed to occur in the multinucleate myotubes derived from integrin beta PS null myospheroid mutants. In normal embryos/early larvae, integrin was located at Z-bands and at muscle insertions. Myogenesis and Z-bands were defective in myospheroid embryos. Attachment, spreading, and growth of myoblasts and neurons on the laminin substrate utilized different binding proteins and were independent of integrin.

Animals↗
PubMed1990

Glutactin, a novel Drosophila basement membrane-related glycoprotein with sequence similarity to serine esterases.

Glutactin, a new acidic sulfated glycoprotein, was isolated from Drosophila Kc cell culture media. Immunofluorescence microscopy located it to embryonic basement membranes, particularly to the sequentially invaginated envelope of the central nervous system, muscle apodemes and dorsal median cell processes. Its chromosome locus is 29D. The nucleic acid sequence coding for the 1023 residue long polypeptide contains one intron and was confirmed by partial amino acid sequencing. Glutactin has a signal peptide and an amino domain of greater than 500 residues that strongly resembles acetylcholine esterases and other serine esterases, but lacks the catalytically critical serine residue. The amino and carboxyl domains of glutactin are separated by 13 contiguous threonine residues. Glutamine and glutamic acid make up 44% of glutactin's very acidic carboxyl domain. Glutactin preferentially binds Ca2+ in the presence of excess Mg2+ and four of its tyrosines are O-sulfated. Several similarities with mammalian entactin caused our previous, preliminary mention of glutactin as a putative Drosophila entactin, but sequence comparison now shows them to be different proteins.

Amino Acid Sequence↗
PubMed1989

The homeotic gene Sex Combs Reduced of Drosophila: gene structure and embryonic expression.

The homeotic gene Sex Combs Reduced (Scr) of Drosophila is required during embryogenesis for labial and first thoracic segment development. We define the Scr gene structure, showing that the major embryonic transcript is proximal to the fushi tarazu gene, and report the sequence of the transcript, which encodes a 413-amino acid, homeodomain-containing protein. We describe Scr protein distribution throughout embryogenesis. Expression begins at gastrulation and is eventually apparent in three tissues, epidermis, nervous system and visceral mesoderm, though there are clear contrasts in the domains of expression in these three tissues.

Amino Acid Sequence↗
PubMed1988

Drosophila basement membrane procollagen IV. I. Protein characterization and distribution.

A collagen was isolated from Drosophila E85, Schneider line 2L and Kc cell cultures. The purified protein was characterized and antibodies were raised against it. Immunofluorescence microscopy locates this material to the regions of basement membranes of Drosophila embryos, larvae, and adults. The molecules are mostly, or entirely, homotrimers of one polypeptide chain linked by interchain disulfide bonds. The partial amino acid sequences of a cyanogen bromide cleavage product of this chain are identical with a part of the virtual translation product of the Drosophila pro alpha 1(IV) nucleotide sequence that is reported in the accompanying paper. This gene is at Drosophila chromosome location 25C and was identified by the high homology of one part of it with the noncollagenous carboxyl terminus (NC1) of vertebrate type IV basement membrane collagens (Blumberg, B., MacKrell, A. J., Olson, P. F., Kurkinen, M., Monson, J. M., Natzle, J. E., and Fessler, J. H. (1987) J. Biol. Chem. 262, 5947-5950). In the electron microscope each molecule appears as a thread with a knob at one end, which contains the carboxyl peptide domains. The variation of flexibility of the thread was mapped along its length. Pulse-chase labeling of cell cultures showed that these molecules associate into disulfide-linked dimers and higher oligomers that can be partly separated by velocity sedimentation and are resolved by sodium dodecyl sulfate-agarose gel electrophoresis. Dimers and higher oligomers formed by overlap of the amino ends of molecules were found. Mild pepsin digestion of Drosophila embryos and larvae solubilized the corresponding disulfide-linked collagen molecules, and Staphylococcus aureus V8 protease peptide maps showed the identity of the collagen derived from animals and from cell cultures. Individual, native molecules have a sedimentation coefficient s20,w = 4.1 S, the dichroic spectrum and amino acid composition of a collagen, and a Tm = 31 degrees C. Positive in situ hybridization with a specific probe for this collagen began 6-8 h after egg laying and showed message in the locations of embryos and larvae which reacted with the antibodies. This included some prominent individual cells in the hemolymph.

Amino Acids↗
PubMed1987

Papilin: a Drosophila proteoglycan-like sulfated glycoprotein from basement membranes.

A sulfated glycoprotein was isolated from the culture media of Drosophila Kc cells and named papilin. Affinity purified antibodies against this protein localized it primarily to the basement membranes of embryos. The antibodies cross-reacted with another material which was not sulfated and appeared to be the core protein of papilin, which is proteoglycan-like. After reduction, papilin electrophoresed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a broad band of about 900,000 apparent molecular weight and the core protein as a narrow band of approximately 400,000. The core protein was formed by some cell lines and by other cells on incubation with 1 mM 4-methylumbelliferyl xyloside, which inhibited formation of the proteoglycan-like form. The buoyant density of papilin in CsCl/4 M guanidine hydrochloride is 1.4 g/ml, that of the core protein is much less. Papilin forms oligomers linked by disulfide bridges, as shown by sodium dodecyl sulfate-agarose gel electrophoresis and electron microscopy. The protomer is a 225 +/- 15-nm thread which is disulfide-linked into a loop with fine, protruding thread ends. Oligomers form clover-leaf-like structures. The protein contains 22% combined serine and threonine residues and 25% combined aspartic and glutamic residues. 10 g of polypeptide has attached 6.4 g of glucosamine, 3.1 g of galactosamine, 6.1 g of uronic acid, and 2.7 g of neutral sugars. There are about 80 O-linked carbohydrate chains/core protein molecule. Sulfate is attached to these chains. The O-linkage is through an unidentified neutral sugar. Papilin is largely resistant to common glycosidases and several proteases. The degree of sulfation varies with the sulfate concentration of the incubation medium. This proteoglycan-like glycoprotein differs substantially from corresponding proteoglycans found in vertebrate basement membranes, in contrast to Drosophila basement membrane laminin and collagen IV which have been conserved evolutionarily.

Amino Acids↗
PubMed1987

Drosophila laminin: characterization and localization.

Drosophila laminin was isolated from the medium of Drosophila Kc cell cultures. It was purified by velocity sedimentation, gel filtration, and chromatography. Drosophila laminin is a disulfide-linked molecule consisting of three chains with apparent molecular masses of 400, 215, and 185 kD. In electron micrographs, it has the cross-shaped appearance with globular domains characteristic of vertebrate laminin with closely similar dimensions. The amino acid composition and lectin-binding properties of Drosophila laminin are given. Polyclonal antibodies to Drosophila laminin were prepared and their specificity was established. In developing embryos immunofluorescence staining was detected between 6 and 8 h of development; and in sections of 8-9-h and older embryos immunostaining was seen at sites where basement membranes are present surrounding internal organs, muscles, underlying the hypodermal epithelium, and in the nervous system. Basement membrane staining was also seen in larva and adults. Cells from Drosophila embryos dissociated at the cellular blastoderm stage were grown in culture and some specific, differentiated cells synthesized laminin after several hours of culture as shown by immunofluorescence. The significance of the evolutionary conservation of the structure of this basement membrane component is discussed.

Amino Acids↗
PubMed1986

Localization of the Antennapedia protein in Drosophila embryos and imaginal discs.

Antibodies have been raised against a fusion protein containing the 3' region of the coding sequence of the Antennapedia (Antp) gene fused to beta-galactosidase. The distribution of the protein on whole mount embryos and imaginal discs of third instar larvae was examined by immunofluorescence. In young embryos, expression of the Antp protein was limited to the thoracic segments in the epidermis, whereas it was found in all neuromeres of head, thorax and abdomen. At the end of embryogenesis, the Antp protein mainly accumulated in the ventral nervous system in certain parts of the thoracic neuromeres, from posterior T1 to anterior T3, with a gap in posterior T2. Comparison of Antp protein distribution in nervous systems from wild-type and Df P9 embryos, lacking the genes of the Bithorax-complex (BX-C), revealed a pattern of expression which indicated that the BX-C represses Antp in the posterior segments with the exception of the last abdominal neuromeres (A8-9) which are regulated independently. The protein pattern in nervous systems from Sex combs reduced(Scr) mutant embryos was indistinguishable from that found in wild-type embryos; thus, neurogenic expression of Antp in T1 and the more anterior segments does not appear to be under the control of Scr. All imaginal discs derived from the three thoracic segments express Antp protein. The distribution was distinct in each disc; strongest expression was observed in the proximal parts of the discs. In the leg discs the protein distribution seemed to be compartmentally restricted, whereas in the wing disc this was not the case. Antp protein was not detected in the eye-antennal disc. In embryos, as well as in imaginal discs, the protein is localized in the nucleus.

Journal Article↗
PubMed1986

Intracellular transport and tyrosine sulfation of procollagens V.

Several tyrosine residues of the extracellular p-collagens V and collagens V are sulfated [Fessler, L. I., Brosh, S., Chapin, S. and Fessler, J. H. (1986) J. Biol. Chem. 261, 5034-5040]. Here, the sulfation of their intracellular precursors, the procollagens V, was studied. A Golgi-enriched subcellular fraction of chick embryo tendon catalyzed the sulfation of tyrosine residues in both endogenous and added, unsulfated procollagens V with the sulfate donor 3'-phosphoadenosine 5'-[35S]phosphosulfate. Intracellular tyrosine sulfation of procollagen V occurred at a point distal to the cis Golgi compartment as judged by change of the N-linked carbohydrate of procollagen V from being endoglycosidase-H-sensitive to being resistant. The time course of the intracellular modifications of procollagen V was determined by incubating tendons with 3H-labeled amino acids and with [35S]sulfate. The pro alpha(V) chains were synthesised in about 10 min and then assembled into unsulfated procollagen V molecules. Tyrosine sulfation occurred 50 min after completion of polypeptide synthesis and the molecules were successively sulfated in the order in which they had been synthesized. The antimicrotubular drug Nocodazole, which disrupts the spatial organization of the Golgi, decreased the time interval between synthesis of procollagens V and sulfation. The sulfated procollagens V were soon secreted and cut to sulfated p-collagens V. Sulfated pro alpha 1(V) chains were cleaved faster than sulfated pro alpha 1'(V) chains. The relationship of sequential protein modification to spatial cellular organization is discussed.

Animals↗
PubMed1986

Tyrosine sulfation in precursors of collagen V.

Radioactive labeling of p-collagens V, collagens V, and, to a small extent, of procollagen V occurred when [35S]sulfate was incubated with tendons or primary tendon cell cultures, or blood vessels and crops of 17- to 19-day-old chick embryos, or with lung slices from neonatal rats. Most or all of this label is in the form of 1 or more sulfated tyrosine residues/chain of p alpha 1(V), alpha 1(V), p alpha 1'(V), alpha 1'(V), p alpha 2(V), and alpha 2(V), and it remains attached through purification by dialysis, ammonium sulfate precipitation, CsCl-GdnCl2 equilibrium buoyant density and velocity sedimentations, ion-exchange chromatography, and sodium dodecyl sulfate gel electrophoresis. Radioactive tyrosine sulfate was identified in alkaline hydrolysates of these collagen V chains, after labeling the tissues with either [35S]sulfate or [3H]tyrosine, by electrophoretic and chromatographic comigration with a tyrosine sulfate standard. Tunicamycin A1, which inhibits the attachment of N-linked complex carbohydrate, did not interfere with the sulfation process. The tyrosine sulfate is located in a noncollagenous domain, which is probably adjacent to the amino end of the collagen helix, and is retained throughout the physiological proteolytic processing of procollagens V. After digestion with Staphylococcus aureus V8 protease, 35S-labeled p alpha 1(V) and alpha 1(V) chains gave the same map of labeled peptides, and this differed from the map given by p alpha 1'(V) and alpha 1'(V) chains. Little sulfation of p alpha 2(V) and alpha 2(V) chains occurs. The implications of these observations for the structure and properties of procollagens V and their derivatives are considered.

Animals↗
PubMed1986

Biosynthesis and supramolecular assembly of procollagen IV in neonatal lung.

The rate of biosynthesis of procollagen IV, the principal collagen of basement membranes, and the concentration of specific RNAs coding for procollagen IV were measured in neonatal rat lungs. Both decreased sharply at birth and then recovered again a few days later. The supramolecular assembly of procollagen IV was followed in neonatal rat, mouse, and chick lungs, which actively elaborate endothelial and alveolar basement membranes, and in chick embryo gizzard which is rich in smooth muscle. The tetramer of four procollagen IV molecules linked covalently through their amino ends was isolated as an assembly intermediate from all these tissues. While noncovalent association of the carboxyl ends of two procollagen IV molecules occurred readily, the subsequent establishment of covalent cross-links was substantially slower in the junctional complexes of the carboxyl ends than of the amino ends. Both disulfide bonds and other, unidentified covalent links formed. The six component carboxyl peptides of a junctional complex became progressively covalently linked into two kinds of carboxyl peptide pairs. We conclude that both amino-linked tetramers and carboxyl-linked dimers of procollagen IV molecules are intermediates in the biological assembly of the collagen networks of these basement membranes.

Age Factors↗
PubMed1985

Isolation of a new procollagen V chain from chick embryo tendon.

Whole tendons of chick embryos synthesize procollagens V which consist of three pro-alpha chains: pro-alpha 1'(V), pro-alpha 1(V) and pro-alpha 2(V). This report shows that while the pro-alpha 1'(V) chain is similar to the pro-alpha 1(V) chain in many respects, such as similar but not identical peptide maps, it also distinctly differs from it in size and in other ways. The new chain is denoted as pro-alpha 1' to indicate the relationship. We have failed to see conversion of one chain into the other and they are regarded as variants, although we do not know whether they are different transcripts of one gene or products of two closely related genes. The pro-alpha(V) chains are assembled into the disulfide-linked homotrimer (pro-alpha 1'(V))3 and the heterotrimer [(pro-alpha 1'(V)S-S-pro-alpha 2(V))pro-alpha 1(V)] and a smaller amount of [(pro-alpha 1(V)2pro-alpha 2(V)]. The pro-alpha 1'(V) chains are processed similarly to the pro-alpha 1(V) by the initial removal of the presumed carboxyl propeptide yielding p-alpha 1'(V) and then by reduction in the size of the noncollagenous, presumed amino propeptide to yield alpha 1'(V). A size difference between the alpha 1'(V) and alpha 1(V) series of molecules is demonstrated by velocity sedimentation and by electrophoretic mobility of the reduced molecules. This difference is ascribed to a difference in the size of the propeptides because after pepsin digestion the products of both series of molecules are the same size and electrophorese like alpha 1(V)(pepsin). The carboxyl propeptides of pro-alpha 1(V) and pro-alpha 1'(V) are the same size, but the amino propeptide of pro-alpha 1'(V) is smaller than that of pro-alpha 1(V). The amino propeptide of pro-alpha 1'(V) and p-alpha 1'(V) also lacks asparagine-linked complex carbohydrate, which is linked to propeptides of the p-alpha 1(V) and p-alpha 2(V) chains. Differences between the alpha 1(V) and alpha 1'(V) series of molecules remain in material synthesized in the presence of tunicamycin. Primary cultures of tendon cells synthesize procollagen V consisting of the above three chains, but the procollagen V made by cultured tendon sheath synovial cells predominantly contains [(pro-alpha 1(V))2pro-alpha 2(V)].

Animals↗
PubMed1985

Biosynthesis of collagen.

During the biosynthesis and assembly of collagen structures, disulfide links can serve several functions. During biosynthesis they successively stabilize intrapeptide folding and associations of three chains into one molecule. Studies on the refolding and reassociation of reduced and denatured carboxyl propeptides of procollagen I showed that successive interactions of folding and assembly are successively weaker. Disulfide bridges were reestablished within correctly refolded carboxyl propeptides. Rearrangements of disulfide bridges may occur during the processing of type V procollagen molecules as these collagens become incorporated into extracellular matrix. The basement membrane procollagen IV molecules become disulfide linked at each end into networks, and there are indications that further rearrangements of disulfide links may allow additional modulation.

Animals↗
PubMed1984

Characterization of the procollagen IV cleavage products produced by a specific tumor collagenase.

The specific mammalian collagenase isolated from cultures of metastatic mouse PMT sarcoma cells cleaves murine procollagen IV into two segments, of approximate mass ratio 3:1. These fragments were separated by velocity sedimentation, visualized by electron microscopy, and analyzed. The longer COOH-terminal procollagen segment has a 270-nm collagenous portion with a knob at one end. This knob consists of the three previously identified, noncollagenous carboxyl propeptides, of approximately 30,000 daltons each. These carboxyl propeptides are chain-specific, and the three chains of each segment have the same amino to carboxyl orientation. The collagenase cuts through all three chains at one site, and the three-component chains of both the longer COOH-terminal procollagen segment and the shorter NH2-terminal procollagen segment are linked by interchain disulfide bridges. The enzyme cuts off the same COOH-terminal procollagen segment from procollagen IV monomers and tetramers, and the flexibility of this segment is similar to that of interstitial collagen helices. The amino ends of the NH2-terminal procollagen segments derived from tetramers remain joined as the 32-nm long "7 S collagen" junctional complex, and the remaining 89-nm long projecting threads are significantly more flexible than the COOH-terminal procollagen segment. The electrophoretic mobilities of the enzyme cleavage products are consistent with a heterotrimeric composition of this procollagen IV.

Amino Acid Sequence↗
PubMed1983

Procollagen IV. Association to tetramers.

Procollagen IV was isolated from culture media of the mouse endodermal cell line PF-HR9. Some of the triple helical procollagen IV molecules were associated at their NH2 ends to tetramers which were identified by electron microscopy, velocity sedimentation, and electrophoresis. The formation of these tetramers in cell cultures and from isolated procollagen IV molecules was investigated. After an initial noncovalent association, which is reversible, disulfide bonds form between molecules. Even alkylated molecules form disulfide-linked tetramers when exposed to a mixture of reduced and oxidized glutathione. This reaction requires an adequate concentration of procollagen and is not facilitated by added laminin, Ca2+, or Mg2+ ions. Cystine, as a normal constituent of cell culture media, interferes in tetramer assembly, presumably by forming mixed disulfides. Tetramers formed normally and under the influence of glutathione are similar, but probably not identical, and resemble those isolated from fragmented basement membranes. We conclude that the NH2 ends of procollagen IV molecules can associate into tetramers without the help of other molecules and that disulfide bridges subsequently stabilize the association in various ways.

Animals↗
PubMed1982

Structural implications from an electronmicroscopic comparison of procollagen V with procollagen I, pC-collagen I, procollagen IV, and a Drosophila procollagen.

Chick embryo procollagen V, procollagen I, and pC-collagen I were sprayed, rotary shadowed, and compared electronmicroscopically with mouse procollagen IV and Drosophila procollagen produced by cell cultures and prepared in the same way. All the molecules appeared as threads and had a prominent knob protrusion at one end. For procollagen I and pC-collagen I this must correspond to the carboxyl propeptides, and most likely corresponds to the similarly sized carboxyl propeptides of procollagen IV. Procollagen V appeared as a thread of nearly the same length as procollagen I but with substantial knobs at both ends, corresponding to its known propeptides. The length of procollagen V and the known, successive processing of its propeptides make procollagen V more similar to the interstitial collagens than to the basement membrane procollagen IV, which is 1.4 times as long and has so far been found not to be processed. Drosophila procollagen resembles procollagen IV in length and appearance.

Animals↗
PubMed1982

Identification of the carboxyl peptides of mouse procollagen IV and its implications for the assembly and structure of basement membrane procollagen.

Clusters of mouse PF-HR9 endoderm cells derived from teratocarcinoma PCC4-F cells were incubated with [3H]proline and [35S]methionine. The synthesis of pro alpha 1 IV and pro alpha 2 IV chains and their association into triple helically folded disulfide-linked molecules were followed. Short incubations and incubations with pactamycin showed that approximately 30,000 molecular weight collagenase-resistant peptides, which are destroyed by pepsin, form the carboxyl end of the pro alpha IV chains. While disulfide links bridge parts of individual peptides, the carboxyl peptides of the three chains of a molecule are not disulfide linked to each other. We propose that these peptides form the knob protrusion seen in electron micrographs of rotary shadowed procollagen IV molecules. The implications of these findings, especially for the relatively slow assembly of procollagen IV, are discussed.

Animals↗