Use of green fluorescent proteins linked to cytoskeletal proteins to analyze myofibrillogenesis in living cells.

Once the appropriate site has been selected for the attachment of GFP to the sarcomeric protein, it is quite remarkable that the large size of the GFP molecule does not appear to interfere with the localization of the fluorescent sarcomeric proteins into the sarcomeric regions of the myofibrils. A similar approach using truncated parts of sarcomeric proteins linked to GFP should allow studies of the targeting properties of other sarcomeric domains for localization and assembly studies.

Myofibrillogenesis visualized in living embryonic cardiomyocytes.

Myofibril formation was visualized in cultured live cardiomyocytes that were transfected with plasmids expressing green fluorescent protein (GFP) linked to the Z-band protein, alpha-actinin. The expression of this fluorescent protein provided an in vivo label for structures containing alpha-actinin. The GFP-alpha-actinin fusion protein was incorporated into Z-bands, intercalated discs, and attachment plaques, as well as into the punctate aggregates, or Z-bodies, that are thought to be the precursors of Z-bands. Observations of live cells over several days in culture permitted us to test aspects of several theories of myofibril assembly that had been proposed previously based on the study of fixed cells. Fine fibrils, called premyofibrils, that formed de novo at the spreading edges of cardiomyocytes, contained punctate concentrations of alpha-actinin, termed Z-bodies. The punctate Z-bodies grew and aligned with Z-bodies in adjacent fibrils. With increasing time, adjacent fibrils and Z-bodies appeared to fuse and form mature myofibrils and Z-bands in cytoplasmic regions where the linear arrays of Z-bodies had been. These new myofibrils became aligned with existing myofibrils at their Z-bands to form myofibrils that spanned the length of the spread cell. These results are consistent with a model that postulates that the fibrils that form de novo near the cell membrane are premyofibrils-i.e., the precursors of mature myofibrils.

An N-terminal fragment of titin coupled to green fluorescent protein localizes to the Z-bands in living muscle cells: overexpression leads to myofibril disassembly.

Cultures of nonmuscle cells, skeletal myotubes, and cardiomyocytes were transfected with a fusion construct (Z1.1GFP) consisting of a 1.1-kb cDNA (Z1.1) fragment from the Z-band region of titin linked to the cDNA for green fluorescent protein (GFP). The Z1.1 cDNA encodes only 362 amino acids of the approximately 2000 amino acids that make up the Z-band region of titin; nevertheless, the Z1.1GFP fusion protein targets the alpha-actinin-rich Z-bands of contracting myofibrils in vivo. This fluorescent fusion protein also localizes in the nascent and premyofibrils at the edges of spreading cardiomyocytes. Similarly, in transfected nonmuscle cells, the Z1.1GFP fusion protein localizes to the alpha-actinin-containing dense bodies of the stress fibers in vivo. A dominant negative phenotype was also observed in living cells expressing high levels of this Z1.1GFP fusion protein, with myofibril disassembly occurring as titin-GFP fragments accumulated. These data indicate that the Z-band region of titin plays an important role in maintaining and organizing the structure of the myofibril. The Z1.1 cDNA was derived from a chicken cardiac lambda gt11 expression library, screened with a zeugmatin antibody. Recent work has suggested that zeugmatin is actually part of the N-terminal region of the 81-kb titin cDNA. A reverse transcriptase polymerase chain reaction using a primer from the distal end (5' end) of the Z1.1 zeugmatin cDNA and a primer from the nearest known proximal (3' end) chicken titin (also called connectin) cDNA resulted in a predicted 0.3-kb polymerase chain reaction product linking the two known chicken titin cDNAs to each other. The linking region had a 79% identity at the amino acid level to human cardiac titin. This result and a Southern blot analysis of chicken genomic DNA hybridized with Z1.1 add further support to our original suggestion that zeugmatin is a proteolytic fragment from the N-terminal region of titin.

Zeugmatin is part of the Z-band targeting region of titin.

Originally, zeugmatin was identified as a 600-800 kD muscle specific protein in Z-bands of cardiac and skeletal muscles by Maher et al. (1985). In this presentation we review our work on myofibrillogenesis and present evidence that zeugmatin is actually part of the Z-Band region of titin and that this region of titin plays an important role in the assembly of the Z-bands and myofibrils. Rhee et al. (1994) reported that during myofibrillogenesis, zeugmatin antibody localization is detected in fully formed Z-bands in the mature myofibrils, in the Z-bodies of the nascent myofibrils, but not in the Z-bodies of the premyofibrils. These observations lead to the suggestion that zeugmatin might be responsible for the fusion of the Z-bodies to form the solid Z-bands of the mature myofibrils (Rhee et al. 1994). As part of a study to test aspects of this model of myofibrillogenesis, we isolated a 1.8 kb cDNA from a chicken cardiac expression library using an anti-zeugmatin antibody (Turnacioglu et al., 1996). We found this chicken cDNA to be 60% identical at the amino acid level to a segment of the Z-band region of human cardiac titin (connectin) sequenced by Labeit and Kolmerer (1995). This homology along with Western blot analysis with purified titin, suggested that zeugmatin is in fact part of the N-terminal region of chicken titin. When expressed in non-muscle cells, Z1.1 product colocalized with the alpha-actinin in stress fiber dense bodies and focal adhesions. Cultures of non-muscle cells, skeletal myotubes and cardiomyocytes were also transfected with a fusion construct (Z1.1GFP) consisting of the Z1.1 kb cDNA linked to the cDNA for green fluorescent protein (GFP). The Z1.1 kb cDNA encodes only 362 of the approximately 2,000 amino acids which comprise the Z-band region of titin; nevertheless, the Z1.1GFP fusion protein targets in vivo to the alpha-actinin rich Z-bands of contracting myofibrils. A dominant negative phenotype was observed in living cells expressing high levels of this Z1.1GFP fusion protein with inhibition of myofibrillogenesis as well as the disassembly of preexisting myofibrils in these cells. These data indicate that the Z-band region of titin (connectin) plays an important role in organizing and maintaining the structure of the myofibril.

Editing of the GLuR-B ion channel RNA in vitro by recombinant double-stranded RNA adenosine deaminase.

Double-stranded RNA (dsRNA)-specific adenosine deaminase (DRADA) has been implicated as an enzyme responsible for the editing of RNA transcripts encoding glutamate-gated ion channel subunits (GLuR) in brain. In one case, the editing alters the gene-encoded glutamine (Q) to an arginine (R) located within the channel-forming domain of the alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptor subunit GLuR-B. The result of editing at this site, called the 'Q/R' site, is a profound alteration of the Ca2+ permeability of the GLuR channel. Using recombinantly expressed DRADA proteins, we now demonstrate in vitro that DRADA is indeed involved in editing of the GLuR-B RNA. In addition to the formation of an RNA duplex structure involving exon and intron sequences, Q/R site-selective editing by DRADA also requires a cofactor protein(s) commonly present even in non-neuronal cells. The accuracy and efficiency of this RNA editing system appear to be determined by the quantitative balance between DRADA, cofactor and substrate GLuR-B RNA.

Molecular cloning of human macrophage capping protein cDNA. A unique member of the gelsolin/villin family expressed primarily in macrophages.

Macrophage capping protein (MCP) is a Ca(2+)-sensitive protein which reversibly blocks the barbed ends of actin filaments but does not sever preformed actin filaments. The human cDNA for MCP has been cloned and sequenced. The derived amino acid sequence predicts a polypeptide of 38.4 kDa. Human MCP expressed in Escherichia coli using a pET12a vector was functionally identical to the native protein purified from rabbit alveolar macrophages with respect to Ca2+ sensitivity and ability to block monomer exchange at the barbed end of actin filaments. Sequence comparison with other actin-binding protein sequences indicates that MCP is a member of the gelsolin/villin family of barbed end blocking proteins. Unlike gelsolin, this protein has a limited tissue distribution being detected primarily in macrophages where it was abundant, representing 0.9-1% of the total cytoplasmic protein. Northern blot analysis of U937 and HL60 cells differentiated to macrophage-like cells demonstrated that MCP message increases to 2.6 and greater than 7 times initial levels, respectively. Human MCP displays a 93% amino acid sequence identity with two recently described mouse proteins, gCap39 and Mbh1. Its abundance in macrophages and the corresponding increases in mRNA levels upon promyelocyte and monocyte development into macrophages indicate that MCP may play an important role in macrophage function.

Listeria monocytogenes moves rapidly through the host-cell cytoplasm by inducing directional actin assembly.

Listeria monocytogenes is an intracellular parasite that can readily infect the macrophage-like cell line J774 and the kidney epithelial cell PtK2. After being ingested, the organism escapes from the phagolysosome into the host-cell cytoplasm. N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)-phallacidin, a specific stain for actin filaments (F-actin), demonstrates that within 1 hr of initiation of infection, the bacteria become surrounded by host-cell cytoplasmic actin filaments. By 3 hr, long projections of F-actin begin to form at one end of the bacteria. These actin structures colocalize with the actin-bundling protein alpha-actinin as well as with tropomyosin. Microinjection of fluorescently labeled alpha-actinin in living cells demonstrates that the formation of these F-actin projections is associated with bacterial movement, actin filaments rapidly assembling behind the bacteria as they migrate through the cytoplasm. These F-actin tails attain lengths up to 40 microns. The movement of the bacteria through the cytoplasm is rapid, 0.12-1.46 microns/sec. Within 2 min of cytochalasin D (0.5 micrograms/ml) treatment, all bacterial intracellular movement stops, and additional bacteria-associated actin assembly is blocked. A nonmotile Listeria mutant induces comparable actin assembly and moves at speeds similar to the wild type, indicating that the forces required for intracellular bacterial movement are generated by the host cell. L. monocytogenes can dramatically stimulate host-cell actin assembly in a directional manner, which serves to rapidly propel the bacteria through the cytoplasm, allowing the organisms to move to peripheral membranes and spread to uninfected cells.

Polymorphonuclear leukocyte adherence induces actin polymerization by a transduction pathway which differs from that used by chemoattractants.

Nitrobenzoxadiazole-phallacidin in combination with quantitative fluorescent microscopy have been used to measure F-actin concentrations in human polymorphonuclear leukocytes (PMN) as they adhere to a plastic surface. Like stimulation with chemoattractants, adherence is associated with a twofold rise in F-actin content. However unlike the rapid rise in F-actin induced by chemoattractants which peaks within 30 s, actin assembly induced by adherence is slower, maximum F-actin values not being observed until 10 min. Furthermore the rise in F-actin induced by adherence is persistent, remaining constant over 60 min while F-actin returns to near basal levels after 20 min exposure to chemoattractant. The combination of adherence (5 min) followed by chemoattractant (FMLP 5 x 10(-8) M for 40 s) resulted in an additive rise in F-actin content to greater than threefold over unstimulated values. Unlike chemoattractant induced actin assembly, adherence-associated PMN actin polymerization was not inhibited by pertussis toxin, but was markedly reduced by lowering extracellular Ca2+. Fluorescent micrographs of adherent PMN stained with nitrobenzoxadiazole-phallacidin revealed F-actin in the lamellipodia and in small foci on the adherent surface. These findings suggest that the transduction mechanisms by which adherence induces PMN actin polymerization differ from those used by chemoattractant receptors.

Neutrophil actin dysfunction is a genetic disorder associated with partial impairment of neutrophil actin assembly in three family members.

A male infant with a severe neutrophil motility disorder and poorly polymerizable actin in PMN extracts was reported over a decade ago to have neutrophil actin dysfunction (NAD) (1974. N. Engl. J. Med. 291:1093-1099). Polymerized actin (F-actin) content of fixed and permeabilized intact neutrophils from the father, mother, and sister of the NAD index case have been measured using nitrobenzoxadiazole-phallacidin, a fluorescent compound which binds specifically to actin filaments. F-actin content of unstimulated PMN from all three family members was significantly lower than unstimulated control PMN (mean 23.6 +/- 0.4 SEM fluorescent units vs. 32.6 +/- 0.6 for controls). After stimulation with the chemotactic peptide FMLP, maximal F-actin content of NAD family member PMN was below that of controls (52.7 +/- 1.3 vs. 72.6 +/- 1.8). F-actin content of detergent insoluble cytoskeletons after stimulation with FMLP was also significantly lower in PMN from NAD family members as compared with controls (21 +/- 6% vs. 73 +/- 8%). PMN extracts from the father and mother, when treated with 0.6 M KCl, polymerized half as much actin as controls. Whereas diisopropylfluorophosphate treatment of normal PMN decreased actin polymerizability in cell extracts, this treatment increased the assembly of actin in parental PMN extract. Addition of purified actin to NAD extracts failed to reveal an abnormal actin polymerization inhibitory activity, and no obvious structural defect in actin purified from the father's PMNs was noted by HPLC and two dimensional thin layer chromatography of tryptic digests. The present studies of actin assembly in intact PMNs confirm that NAD is associated with a true defect in PMN actin assembly and is a genetic disorder that is recessively inherited.

Platelet-activating factor both stimulates and "primes" human polymorphonuclear leukocyte actin filament assembly.

The phospholipid inflammatory mediator, platelet-activating factor (PAF), can stimulate polymorphonuclear leukocyte (PMN) chemotaxis. Conversion of cytoplasmic actin from monomers to filaments is associated with PMN motile functions. Using the fluorescent actin filament stain nitrobenzodiaxole phallicidin, we have investigated PAF's effects on human PMN actin polymerization. Concentrations of PAF between 1 x 10(-11) to 1 x 10(-6) mol/L induced actin filament (F-actin) assembly. An optimal concentration of PAF (1-5 x 10(-8) mol/L) induced a significantly lower rise in relative F-actin content (1.72 +/- 0.07 SEM) than an optimal concentration (5 x 10(-7) mol/L) of the chemotactic peptide FMLP (2.21 +/- 0.06). Unlike FMLP (F-actin content: 1.25 +/- 0.04 at five seconds), PAF stimulation was associated with a delay of more than five seconds (1.04 +/- 0.01 at five seconds) before an increase in F-actin could be detected. F-actin concentration reached maximum levels by 30 to 60 seconds. Prolonged stimulation (20 minutes) with PAF was associated with two phases of polymerization and depolymerization. Like FMLP, the initiation of actin filament assembly by PAF required receptor occupancy, this reaction being totally blocked by the PAF receptor inhibitor, SKI 63-441. As evidenced by the lack of inhibition by nordihydroguaiaretic acid (5 to 20 mumol/L), the production of leukotriene B4 was not required for the PAF-induced changes in F-actin. Like FMLP, PAF's ability to stimulate PMN actin polymerization was inhibited by pertussis toxin (.05 to 2.5 micrograms/mL) but not impaired by the addition of EGTA and/or the calcium ionophore A23187. Preincubation with 1 x 10(-11) to 1 x 10(-8) mol/L PAF for 2 to 60 minutes enhanced the rise in F-actin content induced by low concentrations of FMLP (5 x 10(-12) to 1 x 10(-10) mol/L) indicating that this phospholipid was capable of "priming" the PMN actin polymerization response.