Marilyn A. Levy

Elastic fiber formation: A dynamic view of extracellular matrix assembly using timer reporters

To study the dynamics of elastic fiber assembly, mammalian cells were transfected with a cDNA construct encoding bovine tropoelastin in frame with the Timer reporter. Timer is a derivative of the DsRed fluorescent protein that changes from green to red over time and, hence, can be used to distinguish new from old elastin. Using dynamic imaging microscopy, we found that the first step in elastic fiber formation is the appearance of small cell surface‐associated elastin globules that increased in size with time (microassembly). The elastin globules are eventually transferred to pre‐existing elastic fibers in the extracellular matrix where they coalesce into larger structures (macroassembly). Mechanical forces associated with cell movement help shape the forming, extracellular elastic fiber network. Time‐lapse imaging combined with the use of Timer constructs provides unique tools for studying the temporal and spatial aspects of extracellular matrix formation by live cells. J. Cell. Physiol. 207: 87–96, 2006.

Site-directed mutations in the Sindbis virus 6K protein reveal sites for fatty acylation and the underacylated protein affects virus release and virion structure

A small hydrophobic polypeptide of 55 amino acids, noted as the 6K protein, is formed during processing of the polyprotein translated from the Sindbis virus subgenomic 26 S mRNA. In the accompanying paper we show that this 6K protein can be found in purified preparations of virions and that it is palmitoylated via thioester bonds with about four covalently bound fatty acids per molecule. To determine acylation sites on 6K and define a role for these fatty acids, we used site-directed mutagenesis to alter cysteine codons in the 6K gene of Sindbis virus cDNA. One of these mutants had a single cysteine replaced with a serine and the second had two adjacent cysteines replaced with an alanine-serine sequence. Transfection of the transcribed RNA from these two cDNAs produced infectious virus which contained 6K proteins that had decreased amounts of fatty acids. Intracellular formation and maturation of virus glycoproteins appeared to be unaffected by the mutations but the release of virus particles from mutant-infected cells was decreased about 70 to 90% from that observed with wild-type virus. Electron microscopy of virus-infected cells and of isolated virions showed that the 6K mutations led to large numbers of aberrant enveloped particles containing multiple nucleocapsids. These results indicate that the 6K protein and its state of acylation are important factors in Sindbis virus assembly and budding. Additional phenotypic changes are reported for virions released from cells infected with the mutationally altered viruses.

Reduced Vessel Elasticity Alters Cardiovascular Structure and Function in Newborn Mice

Elastic blood vessels provide capacitance and pulse-wave dampening, which are critically important in a pulsatile circulatory system. By studying newborn mice with reduced (Eln+/−) or no (Eln−/−) elastin, we determined the effects of altered vessel elasticity on cardiovascular development and function. Eln−/− mice die within 72 hours of birth but are viable throughout fetal development when dramatic cardiovascular structural and hemodynamic changes occur. Thus, newborn Eln−/− mice provide unique insight into how a closed circulatory system develops when the arteries cannot provide the elastic recoil required for normal heart function. Compared with wild type, the Eln−/− aorta has a smaller unloaded diameter and thicker wall because of smooth muscle cell overproliferation and has greatly reduced compliance. Arteries in Eln−/− mice are also tortuous with stenoses and dilations. Left ventricular pressure is 2-fold higher than wild type, and heart function is impaired. Newborn Eln+/− mice, in contrast, have normal heart function despite left ventricular pressures 25% higher than wild type. The major vessels have smaller unloaded diameters and longer lengths. The Eln+/− aorta has additional smooth muscle cell layers that appear in the adventitia at or just before birth. These results show that the major adaptive changes in cardiovascular hemodynamics and in vessel wall structure seen in the adult Eln+/− mouse are defined in late fetal development. Together, these results show that reduced elastin in mice leads to adaptive remodeling, whereas the complete lack of elastin leads to pathological remodeling and death.

The importance of elastin to aortic development in mice.

Elastin is an essential component of vertebrate arteries that provides elasticity and stores energy during the cardiac cycle. Elastin production in the arterial wall begins midgestation but increases rapidly during the last third of human and mouse development, just as blood pressure and cardiac output increase sharply. The aim of this study is to characterize the structure, hemodynamics, and mechanics of developing arteries with reduced elastin levels and determine the critical time period where elastin is required in the vertebrate cardiovascular system. Mice that lack elastin (Eln(-/-)) or have approximately one-half the normal level (Eln(+/-)) show relatively normal cardiovascular development up to embryonic day (E) 18 as assessed by arterial morphology, left ventricular blood pressure, and cardiac function. Previous work showed that just a few days later, at birth, Eln(-/-) mice die with high blood pressure and tortuous, stenotic arteries. During this period from E18 to birth, Eln(+/-) mice add extra layers of smooth muscle cells to the vessel wall and have a mean blood pressure 25% higher than wild-type animals. These findings demonstrate that elastin is only necessary for normal cardiovascular structure and function in mice starting in the last few days of fetal development. The large increases in blood pressure during this period may push hemodynamic forces over a critical threshold where elastin becomes required for cardiovascular function. Understanding the interplay between elastin amounts and hemodynamic forces in developing vessels will help design treatments for human elastinopathies and optimize protocols for tissue engineering.

Immunolocalization of Endosomal Cathepsin D in Rabbit Alveolar Macrophages

Intravacuolar proteolysis appears to be an important component of antigen presentation, the activation of peptide hormones, and the conversion of biologically important mediators from inactive precursors. Cathepsin D has been identified in the endosomes of rabbit alveolar macrophages by biochemical analyses [Diment and Stahl, J. Biol. Chem. 260,15311, 1985; Diment et al., J. Biol. Chem. 263,6901, 1988]. Using affinity‐purified goat antirabbit cathepsin D IgG, we have localized cathepsin D to the endosomes of rabbit alveolar macrophages. Immunofluorescent staining of frozen sections showed labeling in lysosomes and small vesicles in the periphery of the cell. Label was not seen on the plasma membrane. With immunoperoxidase labeling at the electron microscopic level on cells containing endocytosed mannose‐BSA gold, we saw labeling in endosomes and classical lysosomes. When the results were quantrtated using immunogold labeling of thin cryosections, we found that the majority of cathepsin D (62.2%) was present in lysosomes, 4.0% in large clear vacuoles, a surprisingly high percentage (29.3%) in small vesicles, 4.9% in endosomes, and none on the plasma membrane. We conclude from this study that, in addition to being present in lysosomes, cathepsin D is present in endosomes and in small peripheral vesicles.

CONTACT FORMATION AND TRANSFER OF MANNOSE BSA GOLD FROM MACROPHAGES TO COCULTURED FIBROBLASTS

When macrophages were cocultured with fibroblasts many of the cells formed firm contacts. In some of these contacts both cell types were closely apposed and in others they were more clearly separated with numerous pseudopodia extending from macrophages toward the fibroblasts. Many small vesicles similar in structure to caveoli were observed immediately beneath the plasma membrane of some fibroblasts in regions immediately adjacent to areas of contact with macrophages. The membrane integrity of both cell types was always maintained and no connecting cytoplasmic strands were observed between contacting cells. Junctions were freely permeable to ruthenium red and less permeable to the larger cationized ferritin. Gold conjugated to mannose BSA was taken up readily by macrophages but not by fibroblasts. When fibroblasts were cocultured with macrophages that had been labeled with endocytosed gold, increasing amounts were transferred to them. Gold was observed within gaps formed between cocultured cells and within recipient fibroblasts in vesicles anatomically similar to lysosomes. These points of contact thus appear to provide a series of specialized protected clefts into which directed exocytosis of ligands from donor cells can take place and from which endocytosis into recipient cells is facilitated.

Clathrin-coated vesicles from human placenta contain GTP-binding proteins

Biochemical and morphological techniques were used to investigate the GTP-binding proteins of clathrin-coated vesicles. Binding of [3H]GTP to clathrin coats was demonstrated by electron microscopic autoradiography. Purified coated vesicles bound 5.2 pmol [35S]GTP gamma S/mg protein. Addition of GTP or GTP gamma S, but not ATP nor GMP, inhibited binding of [35S]GTP gamma S to intact coated vesicles and Triton X-100-extracted coats. A series of 23-24 kDa GTP-binding proteins with isoelectric points between pH 5-8 were detected in coated vesicles. We suggest that the low molecular weight ras-like GTP-binding protein(s) play a role in regulating vesicle-mediated protein transport or signal transduction within intracellular organelles.

Developmental Cardiovascular Remodeling Associated With Reduced Elastin Levels in Mice Occurs After Embryonic Day 18

Elastin provides reversible extensibility and stores energy during the cardiac cycle in large blood vessels. It is only present in vertebrate animals with a closed circulatory system and high, pulsatile blood pressures. Elastin is crucial for proper cardiac function in vertebrates, as mice lacking elastin (eln-/-) die soon after birth with cardiovascular abnormalities including long, tortuous, stenotic, stiff arteries and high left ventricular (LV) pressure with low cardiac output [1, 2].© 2009 ASME