Green Fluorescent Protein Sequence Research Articles

Adenoviral Expression of Calmodulin Antisense Reduces Hypertrophy in Cultured Cardiomyocytes

Sustained myocardial hypertrophy is associated with an increased risk of sudden death and progression to heart failure. Multiple signal pathways are involved in cardiac hypertrophy and understanding their interaction may point to new therapeutic targets. In this work, we tested the hypothesis that adenovirus-mediated calmodulin (CaM) antisense expression will reduce the intracellular availability of CaM and inhibit the hypertrophic response. Three recombinant adenoviruses were constructed: AdASCaM, containing the AntiSense sequence of CaM and the enhanced green fluorescent protein (GFP) coding sequence; AdCaM, containing the coding sequence of CaM and the GFP sequence; and the AdGFP, containing the GFP coding sequence. Neonatal rat ventricular cardiomyocytes were infected with AdASCaM, AdCaM, or AdGFP and stimulated with phenylephrine (PE, 50 microM) or angiotensin II (AngII, 10 microM) for 48 h and cell surface area measured with planimetry. After PE treatment, the surface areas of cardiomyocytes infected with AdASCaM or AdGFP were 411 +/- 174.3 micro(2) and 832.6 +/- 372.3 micro(2), respectively (P < 0.01). After AngII treatment, the surface areas of cardiomyocytes infected with AdASCaM or AdGFP were 441.5 +/- 149.2 micro(2) and 726 +/- 328.3 micro(2), respectively (P < 0.01). Adenoviral expression of the CaM antisense (AdASCaM) significantly inhibited PE or AngII-induced cardiomyocyte hypertrophy. Cardiomyocytes infected with the AdCaM showed increased area when compared with those infected with the AdGFP. These results suggest that adenovirus-mediated changes in CaM expression may alter hypertrophy in cardiac myocytes.

Expression of Vascular Endothelial Growth Factor Receptor 1 in Bone Marrow-derived Mesenchymal Cells Is Dependent on Hypoxia-inducible Factor 1

Bone marrow-derived cells are recruited to sites of ischemia, where they promote tissue vascularization. This response is dependent upon the expression of vascular endothelial growth factor (VEGF) receptor 1 (VEGFR1), which mediates cell migration in response to VEGF or placental growth factor (PLGF). In this study, we found that exposure of cultured mouse bone marrow-derived mesenchymal stromal cells (MSC) to hypoxia or an adenovirus encoding a constitutively active form of hypoxia-inducible factor 1 (HIF-1) induced VEGFR1 mRNA and protein expression and promoted ex vivo migration in response to VEGF or PLGF. MSC in which HIF-1 activity was inhibited by a dominant negative or RNA interference approach expressed markedly reduced levels of VEGFR1 and failed to migrate or activate AKT in response to VEGF or PLGF. Thus, loss-of-function and gain-of-function approaches demonstrated that HIF-1 activity is necessary and sufficient for basal and hypoxia-induced VEGFR1 expression in bone marrow-derived MSC.

Conversion of Green Fluorescent Protein into a Toxic, Aggregation-prone Protein by C-terminal Addition of a Short Peptide

A non-natural 16-residue "degron" peptide has been reported to convey proteasome-dependent degradation when fused to proteins expressed in yeast (Gilon, T., Chomsky, O., and Kulka, R. (2000) Mol. Cell. Biol. 20, 7214-7219) or when fused to green fluorescent protein (GFP) and expressed in mammalian cells (Bence, N. F., Sampat, R. M., and Kopito, R. R. (2001) Science 292, 1552-1555). We find that expression of the GFP::degron in Caenorhabditis elegans muscle or neurons results in the formation of stable perinuclear deposits. Similar perinuclear deposition of GFP::degron was also observed upon transfection of primary rat hippocampal neurons or mouse Neuro2A cells. The generality of this observation was supported by transfection of HEK 293 cells with both GFP::degron and DsRed(monomer)::degron constructs. GFP::degron expressed in C. elegans is less soluble than unmodified GFP and induces the small chaperone protein HSP-16, which co-localizes and co-immunoprecipitates with GFP::degron deposits. Induction of GFP::degron in C. elegans muscle leads to rapid paralysis, demonstrating the in vivo toxicity of this aggregating variant. This paralysis is suppressed by co-expression of HSP-16, which dramatically alters the subcellular distribution of GFP::degron. Our results suggest that in C. elegans, and perhaps in mammalian cells, the degron peptide is not a specific proteasome-targeting signal but acts instead by altering GFP secondary or tertiary structure, resulting in an aggregation-prone form recognized by the chaperone system. This altered form of GFP can form toxic aggregates if its expression level exceeds the capacity of chaperone-based degradation pathways. GFP::degron may serve as an instructive "generic" aggregating control protein for studies of disease-associated aggregating proteins, such as huntingtin, alpha-synuclein, and the beta-amyloid peptide.

Evidence for Direct Roles of Two Additional Factors, SECp43 and Soluble Liver Antigen, in the Selenoprotein Synthesis Machinery

Selenocysteine (Sec) is inserted into selenoproteins co-translationally with the help of various cis- and trans-acting factors. The specific mechanisms of Sec biosynthesis and insertion into protein in eukaryotic cells, however, are not known. Two proteins, SECp43 and the soluble liver antigen (SLA), were previously reported to interact with tRNA([Ser]Sec), but their functions remained elusive. Herein, we report that knockdown of SECp43 in NIH3T3 or TCMK-1 cells using RNA interference technology resulted in a reduction in the level of methylation at the 2'-hydroxylribosyl moiety in the wobble position (Um34) of Sec tRNA([Ser]Sec), and consequently reduced glutathione peroxidase 1 expression. Double knockdown of SECp43 and SLA resulted in decreased selenoprotein expression. SECp43 formed a complex with Sec tRNA([Ser]Sec) and SLA, and the targeted removal of one of these proteins affected the binding of the other to Sec tRNA([Ser]Sec). SECp43 was located primarily in the nucleus, whereas SLA was found in the cytoplasm. Co-transfection of both proteins resulted in the nuclear translocation of SLA suggesting that SECp43 may also promote shuttling of SLA and Sec tRNA([Ser]Sec) between different cellular compartments. Taken together, these data establish the role of SECp43 and SLA in selenoprotein biosynthesis through interaction with tRNA([Ser]Sec) in a multiprotein complex. The data also reveal a role of SECp43 in regulation of selenoprotein expression by affecting the synthesis of Um34 on tRNA([Ser]Sec) and the intracellular location of SLA.

Induction of Replication in Human Corneal Endothelial Cells by E2F2 Transcription Factor cDNA Transfer

Corneal endothelial cells in humans do not replicate to any meaningful extent. Diminishing density of the cell monolayer with age and in the disease states is a major cause of loss of corneal transparency. This study was conducted to test the hypothesis that overexpression of the transcription factor E2F2 results in replication in nonproliferating human corneal endothelial cells. Whole human corneas were incubated for 2 hours in a solution of recombinant E1(-)/E3(-) adenovirus incorporating cDNA encoding E2F2 and green fluorescent protein (GFP) under control of a bidirectional promoter and subsequently maintained in ex vivo culture. Control specimens were incubated with an identical virus bearing the GFP sequence only, or virus-free medium. Efficiency of gene transfer and localization was examined by fluorescence microscopy. En face confocal microscopy of the corneal endothelial surface was used to image recombinant E2F2 expression. 5-bromodeoxyuridine (BrdU) incorporation was used to examine progression to the S phase. Changes in density of the corneal endothelium were quantified by specular microscopy and counting of trypan-blue-stained cells. Apoptosis was tested with a TUNEL assay. Recombinant proteins were expressed predominantly in the endothelium and in a high proportion of endothelial cells in the first week after exposure to virus, diminishing thereafter. Compared with the control, transduction with E2F2 resulted in progression from the G(1) to the S phase in a significant number of cells and in increased cell density. Apoptosis was not found to any significant extent. Overexpression of the transcription factor E2F2 in nonmitotic human corneal endothelial cells results in short-term expression, cell-cycle progression, and increased monolayer cell density.

Plasma Membrane Residence of Hyaluronan Synthase Is Coupled to Its Enzymatic Activity

Hyaluronan is a multifunctional glycosaminoglycan up to 10(7) Da molecular mass produced by the integral membrane glycosyltransferase, hyaluronan synthase (HAS). When expressed in keratinocytes, N-terminally tagged green fluorescent protein-HAS2 and -HAS3 isoenzymes were found to travel through endoplasmic reticulum (ER), Golgi, plasma membrane, and endocytic vesicles. A distinct enrichment of plasma membrane HAS was found in cell protrusions. The total turnover time of HAS3 was 4-5 h as judged by the green fluorescent protein signal decay and hyaluronan synthesis inhibition in cycloheximide-treated cells. The transfer from ER to Golgi took about 1 h, and the dwell time on the plasma membrane was less than 2 h in experiments with a relief and introduction, respectively, of brefeldin A. Constructs of HAS3 with 16- and 45-amino-acid C-terminal deletions mostly stayed within the ER, whereas a D216A missense mutant was localized within the Golgi complex but not the plasma membrane. Both types of mutations were almost or completely inactive, similar to the wild type enzyme that had its entry to the plasma membrane experimentally blocked by brefeldin A. Inhibition of hyaluronan synthesis by UDP-glucuronic acid starvation using 4-methyl-umbelliferone also prevented HAS access to the plasma membrane. The results demonstrate that 1) a latent pool of HAS exists within the ER-Golgi pathway; 2) this pool can be rapidly mobilized and activated by insertion into the plasma membrane; and 3) inhibition of HAS activity through mutation or substrate starvation results in exclusion of HAS from the plasma membrane.

A novel insertion site inside the potyvirus P1 cistron allows expression of heterologous proteins and suggests some P1 functions

The P1 cistron encodes the first and most variable part of the polyprotein of potyviruses. A site tolerant to a pentapeptide insertion at the N-terminus of Potato virus A P1 (Genome Res. 12, 584–594) was used to express heterologous proteins (insertions up to 783 nucleotides) with or without flanking new proteolytic sites. Aequorea victoria green fluorescent protein (GFP) accumulated to high levels when proteolytically released from P1 and showed strong fluorescence in leaves systemically infected with vector virus. Deletions in GFP and adjacent viral sequences emerged 2–4 weeks after infection, revealing putative recombination hot spots. The inserts in P1 diminished infectivity host-specifically, reduced virus accumulation in protoplasts and systemically infected leaves, alleviated symptoms and reduced accumulation of mRNA and HCpro in cis in a virus-free system. This heterologous protein expression site is the first within a protein-encoding cistron and the third in the genome of potyviruses.

Inverse correlation between mRNA levels in GFP-silenced transgenic Nicotiana benthamiana and resistance to Potato virus X engineered to contain GFP sequence

Nicotiana benthamiana was transformed with a green fluorescent protein (GFP) gene driven by cauliflower mosaic virus 35S promoter. A GFP-silenced line and a nonsilenced line were selected after ultraviolet irradiation. GFP short-interfering RNAs (siRNAs) were detected in the silenced line but not in the nonsilenced line. T1 progeny of the silenced line varied in GFP suppression patterns and were grouped into three types (I, II, III) based on the GFP suppression pattern. With Northern blot analysis, different levels of GFP mRNA accumulated, from a very low level in type I and II to an intermediate level in type III, in contrast to a much higher level in the nonsilenced line. Plants were also inoculated with Potato virus X engineered to contain the GFP sequence to evaluate the levels of virus resistance. None to a few GFP spots were observed on inoculated leaves in types I and II, whereas numerous spots and systemic infection appeared in type III. These results showed that virus resistance was inversely correlated with the levels of mRNA, suggesting that the strength of RNA silencing determines the extent of virus resistance.

Expression of axin2 Is Regulated by the Alternative 5′-Untranslated Regions of Its mRNA

Axin2 is a negative regulator of Wnt/beta-catenin signaling with roles in early development and tumor suppression. We find that axin2 expression is regulated at both transcriptional and translational levels. The gene allows transcription of mRNAs with three alternative 5'-untranslated regions, and these are differentially expressed in various human cell types. These untranslated regions can differentially determine protein expression from messages by influencing mRNA stability and translational efficiency. We identify short upstream reading frames and structural motifs that are responsible for modulation of mRNA translational efficiencies. We show that the proportions of axin2 message expressing each 5'-untranslated region influence the amount of Axin2 protein expressed within cells. We discuss this complex regulation in the context of the function of Axin2 as a tumor suppressor.

Endosomal Targeting Sequences from Non-Classical Antigen Presenting Molecules Can Direct Antigens into the MIIC and Other Antigen Processing Compartments.

The ultimate goal of these studies is to enhance the development of MHC class II-restricted helper T cell responses that are required for effective and durable immunotherapy. In order to optimize the processing and presentation of MHC class II-restricted epitopes, we are using a differential targeting strategy that relies upon the unique endosomal targeting sequences from the individual isoforms of the CD1 family of non-classical antigen presenting molecules. Distinct endosomal compartments differ with respect to pH and resident proteases. MHC class II molecules can load antigen in each of the different endosomal compartments. This is in addition to the traditional antigen loading compartment, MIIC. Therefore, by forcing the expression of a tumor antigen across different endosomal compartments, we hope to maximize the number of successfully processed epitopes. To achieve this, we have developed a PCR-based strategy to link the leader, transmembrane, and endosomal targeting sequences from the four different human CD1 isoforms, CD1a-d, to DNA encoding for a surrogate antigen, green fluorescent protein (GFP). Using this strategy, we have demonstrated that fusion of individual CD1 tails to the sequence for GFP resulted in four distinct expression patterns in HELA cells transfected with these constructs. Using immunofluorescent staining and confocal laser microscopy, we determined the pattern of antigen expression in various endosomal compartments. GFP variably colocalized with recycling endosomes (ARF-6), early endosomes (CD71, transferrin receptor), late endosomes (CD63), and late endosomes/lysosmes/MIIC (CD107a, LAMP-1). Three of the four CD1 tails also resulted in co-expression of GFP with the MHC class II molecule, HLA-DR. The expression pattern for each CD1 tail is shown in Table 1. These experiments validate the feasibility of the CD1 targeting approach. Current studies are underway to apply this strategy to the leukemia antigen, WT1, using recently completed and sequenced WT1/CD1 fusion constructs.ARF-6CD71CD63CD107aCD1a−/+−−−CD1b−/+−+++CD1c+−/++++CD1d+−/+−+Table 1. Pattern of GFP/CD1 fusion expression in different endosomal compartments. Fusion of the endosomal targeting sequences from human CD1 molecules to the model antigen, GFP, results in different patterns of antigen expression within distinct endosomal compartments. The degree of colocalization between GFP and the indicated markers of endosomal compartments is represented as follows: (−), no colocalization; (−/+), infrequent colocalization; (+), frequent colocalization; (++), extensive colocalization.

A Context-dependent ClpX Recognition Determinant Located at the C Terminus of Phage Mu Repressor

The bacteriophage Mu immunity repressor is a conformationally sensitive sensor that can be interconverted between forms resistant to and sensitive to degradation by ClpXP protease. Protease-sensitive repressor molecules with an altered C-terminal sequence promote rapid degradation of the wild-type repressor by inducing its C-terminal end to become exposed. Here we determined that the last 5 C-terminal residues (CTD5) of the wild-type repressor contain the motif required for recognition by the ClpX molecular chaperone, a motif that is strongly dependent upon the context in which it is presented. Although attachment of the 11-residue ssrA degradation tag to the C terminus of green fluorescent protein (GFP) promoted its rapid degradation by ClpXP, attachment of 5-27 C-terminal residues of the repressor failed to promote degradation. Disordered peptides derived from 41 and 35 C-terminal residues of CcdA (CcdA41) and thioredoxin (TrxA35), respectively, activated CTD5 when placed as linkers between GFP and repressor C-terminal sequences. However, when the entire thioredoxin sequence was included as a linker to promote an ordered configuration of the TrxA35 peptide, the resulting substrate was not degraded. In addition, a hybrid tag, in which CTD5 replaced the 3-residue recognition motif of the ssrA tag, was inactive when attached directly to GFP but active when attached through the CcdA41 peptide. Thus, CTD5 is sufficient to act as a recognition motif but has requirements for its presentation not shared by the ssrA tag. We suggest that activation of CTD5 may require presentation on a disordered or flexible domain that confers ligand flexibility.

Coupling the iron-responsive element to GFP--an inducible system to study translation in a single living cell.

Local protein synthesis in a cell represents an elegant mechanism to achieve important biological phenomena such as cell migration, body axis formation during embryonic development and establishment of cell polarity. A prerequisite to studying translation in a restricted cellular compartment is the ability to unambiguously discriminate between proteins that arise through local protein synthesis and those that reach the site of interest by diffusion or transport. To tackle this problem, we set up a green fluorescent protein (GFP)-based reporter system that allows one to uncouple the translation of reporter gene mRNA from its subcellular localization. The system is based on the iron-responsive element, which regulates the translation of both endogenous ferritin and transferrin transcripts in response to changes in iron concentration. Translation of the reporter messenger RNA (mRNA) is thus dependent on iron in the medium; both its transcription and localization, however, are unaffected. Known targeting sequences can be used to direct the mRNA transcript to a subcellular compartment of interest. For instance, the full-length 3'-untranslated region of calcium/calmodulin-dependent protein kinase IIalpha mRNA can be added to the construct, after the stop codon of the GFP sequence, to selectively target the transcript into the dendrites of transiently transfected hippocampal neurons. This novel fluorescent assay will allow us to address a number of important biological questions in living mammalian cells.

Coupling the Iron-Responsive Element to GFP--An Inducible System to Study Translation in a Single Living Cell

Local protein synthesis in a cell represents an elegant mechanism to achieve important biological phenomena such as cell migration, body axis formation during embryonic development and establishment of cell polarity. A prerequisite to studying translation in a restricted cellular compartment is the ability to unambiguously discriminate between proteins that arise through local protein synthesis and those that reach the site of interest by diffusion or transport. To tackle this problem, we set up a green fluorescent protein (GFP)-based reporter system that allows one to uncouple the translation of reporter gene mRNA from its subcellular localization. The system is based on the iron-responsive element, which regulates the translation of both endogenous ferritin and transferrin transcripts in response to changes in iron concentration. Translation of the reporter messenger RNA (mRNA) is thus dependent on iron in the medium; both its transcription and localization, however, are unaffected. Known targeting sequences can be used to direct the mRNA transcript to a subcellular compartment of interest. For instance, the full-length 3′-untranslated region of calcium/calmodulin-dependent protein kinase IIα mRNA can be added to the construct, after the stop codon of the GFP sequence, to selectively target the transcript into the dendrites of transiently transfected hippocampal neurons. This novel fluorescent assay will allow us to address a number of important biological questions in living mammalian cells.

Safety Assessment of Recombinant Green Fluorescent Protein Orally Administered to Weaned Rats

Several proposed biotechnological applications of green fluorescent protein (GFP) are likely to result in its introduction into the food supply of domestic animals and humans. We fed pure GFP and diets containing transgenic canola expressing GFP to young male rats for 26 d to evaluate the potential toxicity and allergenicity of GFP. Animals (n = 8 per group) were fed either AIN-93G (control), control diet plus 1.0 mg of purified GFP daily, modified control diet with 200 g/kg canola (Brassica rapa cv Westar), or control diet with 200 g/kg transgenic canola containing one of two levels of GFP. Ingestion of GFP did not affect growth, food intake, relative weight of intestine or other organs, or activities of hepatic enzymes in serum. Comparison of the amino acid sequence of GFP to known food allergens revealed that the greatest number of consecutive amino acid matches between GFP and any food allergen was four, suggesting the absence of common allergen epitopes. Moreover, GFP was rapidly degraded during simulated gastric digestion. These data indicate that GFP is a low allergenicity risk and provide preliminary indications that GFP is not likely to represent a health risk.

The proline-rich region of the ecotropic Moloney murine leukaemia virus envelope protein tolerates the insertion of the green fluorescent protein and allows the generation of replication-competent virus.

Sequences encoding the green fluorescent protein (GFP) were inserted into the envelope protein (Env) of ecotropic Moloney murine leukaemia virus, MoMLV. Insertion of these sequences into the proline-rich region (PRR) of Env resulted in a chimeric GFP-Env protein that allowed retrovirus vector transduction of murine cells with titres similar to wild-type Env. However, N-terminal extension with GFP did not result in a functional Env protein. GFP sequences were then inserted into the Env PRR of E-MO virus, a MoMLV that carries epidermal growth factor sequences at the N terminus of its Env protein. The resulting virus, GFP-EMO1, replicates to the same titres as the parental virus. In a chronically infected cell culture, GFP-EMO1 was genetically stable. However, additional insertions of sequences that led to recombination or that may have been incompatible with virus replication were deleted and decreased virus titre. In summary, Env PRR can be used to tag individual virus particles with GFP, which leaves other regions available for modification in studies aimed at altering virus tropism.

A Synthetic Green Fluorescent Protein Gene for Plant Biotechnology.

Since Green Fluorescent Protein (GFP) from Aequorea jellyfish emits green fluorescence without any additional substrates or co-factors, it has emerged as a powerful new reporter for use in a variety of organisms. However, improvements such as the alteration of the excitation spectrum and the elimination of a cryptic intron site were necessary for its efficient use in plants. An engineered synthetic GFP with a S65T mutation (replacement of the serine in position 65 with a threonine) in the chromophore has provided up to 100-fold brighter fluorescent signals than the wild-type jellyfish GFP sequence without any toxic effects. The sGFP(S65T) has been widely used for studies such as localization analysis and promoter assays. This sGFP(S65T) is also an ideal candidate for the development of transformation methods for crops and trees in which it has previously proved difficult to obtain transformants via conventional methods. A non-invasive, quantitative detection technique for this GFP has been achieved in conjunction with a fluorescent imaging system.

Generation of siRNAs by T-DNA sequences does not require active transcription or homology to sequences in the plant.

Delivery into plants of T-DNAs containing promoter, terminator, or coding sequences generated small interfering RNAs (siRNAs) specific to each type of sequence. When both promoter and transcribed sequences were simultaneously present in the T-DNA, accumulation of siRNAs to transcribed sequences was favored over accumulation of siRNAs to the nontranscribed upstream promoter sequences. The generation of specific siRNA sequences occurred even in the absence of T-DNA homology to sequences in the plant. Delivery of T-DNA, with homology to the transgene limited to the nontranscribed cauliflower mosaic virus 35S promoter (35SP) and the transcribed nopaline synthase transcription termination (NosT)signal sequences, into transgenic plants expressing the green fluorescent protein (GFP), generated siRNAs in infiltrated tissues to both 35SP (35SsiRNAs) and NosT (NosTsiRNAs), but not to the GFP sequence (GFPsiRNAs). In infiltrated tissues, the 35SsiRNAs failed to trigger the transcriptional silencing of the transgene, accumulation of 35SsiRNAs could be prevented by the potyviral HC-Pro, and the NosTsiRNAs required an initial amplification to trigger efficient transgene silencing, which is mediated by transcripts from the exogenous T-DNA, and not from the transgene. In upper leaves, silencing correlated with the presence of GFPsiRNAs and the absence of 35SsiRNAs, confirming that its spread was posttranscriptionally mediated by the transgene mRNA.

Inhibition of tobacco mosaic virus replication in lateral roots is dependent on an activated meristem-derived signal.

Viral invasion of the root system of Nicotiana benthamiana was studied noninvasively with a tobacco mosaic virus (TMV) vector expressing the green-fluorescent protein (GFP). Lateral root primordia, which developed from the pericycle of primary roots, became heavily infected as they emerged from the root cortex. However, following emergence, a progressive wave of viral inhibition occurred that originated in the lateral-root meristem and progressed towards its base. Excision of source and sink tissues suggested that the inhibition of virus replication was brought about by the basipetal movement of a root meristem signal. When infected plants were inoculated with tobacco rattle virus (TRV) expressing the red-fluorescent protein, DsRed, TRV entered the lateral roots and suppressed the host response, leading to a reestablishment of TMV infection in lateral roots. By infecting GFP-expressing transgenic plants with TMV carrying the complementary GFP sequence it was possible to silence the host GFP, leading to the complete loss of fluorescence in lateral roots. The data suggest that viral inhibition in lateral roots occurs by a gene-silencing-like mechanism that is dependent on the activation of a lateral-root meristem.

Two point mutations produce infectious retrovirus bearing a green fluorescent protein-SU fusion protein.

Two second-site mutations in Moloney murine leukemia virus envelope surface protein (SU) were previously shown to rescue infection of two different SU mutants, a fusion-defective point mutant and a fusion-defective modified SU that exhibits weak subunit association. We report here that they also rescue infection of a third defective SU, one modified by insertion of the green fluorescent protein (GFP) between serine 6 and proline 7. GFP-SU assembled into virions and showed a strong association with the transmembrane protein (TM). However, these virions were noninfectious. GFP-SU expression was not maintained within cells, suggesting that the protein was toxic. Addition of the second-site mutations rendered the GFP-SU virus infectious and resulted in prolonged expression of the modified envelope protein. This virus showed a slight reduction in receptor binding but not in envelope protein processing, suggesting that addition of the GFP sequences results in subtle structural changes. Extrapolating these data, we see that the fundamental problem with the GFP-SU envelope protein appears to be a folding problem, suggesting that the second-site mutations rescue GFP-SU primarily by a mechanism that involves stabilizing the envelope protein structure.

The Tip of the Coiled-coil Rod Determines the Filament Formation of Smooth Muscle and Nonmuscle Myosin

Myosin II self-assembles to form thick filaments that are attributed to its long coiled-coil tail domain. The present study has determined a region critical for filament formation of vertebrate smooth muscle and nonmuscle myosin II. A monoclonal antibody recognizing the 28 residues from the C-terminal end of the coiled-coil domain of smooth muscle myosin II completely inhibited filament formation, whereas other antibodies recognizing other parts of the coiled-coil did not. To determine the importance of this region in the filament assembly in vivo, green fluorescent protein (GFP)-tagged smooth muscle myosin was expressed in COS-7 cells, and the filamentous localization of the GFP signal was monitored by fluorescence microscopy. Wild type GFP-tagged smooth muscle myosin colocalized with F-actin during interphase and was also recruited into the contractile ring during cytokinesis. Myosin with the nonhelical tail piece deleted showed similar behavior, whereas deletion of the 28 residues at the C-terminal end of the coiled-coil domain abolished this localization. Deletion of the corresponding region of GFP-tagged nonmuscle myosin IIA also abolished this localization. We conclude that the C-terminal end of the coiled-coil domain, but not the nonhelical tail piece, of myosin II is critical for myosin filament formation both in vitro and in vivo.