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PubMed2006

pTcINDEX: a stable tetracycline-regulated expression vector for Trypanosoma cruzi.

BACKGROUND: Trypanosoma cruzi is a protozoan pathogen of major medical importance in Latin America. It is also an early diverging eukaryote that displays many unusual biochemical features. The completion of the T. cruzi genome project has highlighted the need to extend the range of techniques available to study gene function. To this end we report the development of a stable tetracycline-dependent expression vector applicable to this parasite and describe in detail the parameters of the system. RESULTS: We first produced T. cruzi cell lines that constitutively expressed bacteriophage T7 RNA polymerase and the tetracycline repressor protein from a multicopy episome. An integrative vector with an inducible expression site under the control of a tetracycline-regulatable T7 promoter (pTcINDEX) was targeted to the transcriptionally silent ribosomal RNA spacer region of these parasites and transformants selected using a T7 RNA polymerase-dependent hygromycin resistance gene. To test the system we used two marker proteins, luciferase and red fluorescent protein (RFP), and an endogenous parasite protein (a mitochondrial superoxide dismutase). In each case we found that induction was both time and dose-dependent. Luciferase mRNA could be induced by at least 100-fold, and luciferase activity up to 60-fold, within 24 hours of the addition of tetracycline. When we examined RFP induction by confocal microscopy and fluorescence activated cell sorter, we observed very high levels of expression (>1000-fold increase in fluorescence intensity), although this was not synchronous throughout clonal populations. Induction of superoxide dismutase resulted in an 18-fold increase in cellular activity. The observation that a tagged version of the enzyme was correctly targeted to the mitochondrion demonstrates that our expression system may also provide a high-throughput strategy for subcellular localisation. CONCLUSION: Our results show that pTcINDEX represents a valuable addition to the genetic tools available for T. cruzi. The vector system is sufficiently flexible that it should have widespread uses including inducible expression of tagged proteins, generation of conditional knockout cell lines and the application of dominant-negative approaches.

Animals↗
PubMed2003

Establishment of red fluorescent protein-tagged HeLa tumor metastasis models: determination of DsRed2 insertion effects and comparison of metastatic patterns after subcutaneous, intraperitoneal, or intravenous injection.

Metastasis is the leading cause of death in patients with cervical cancer. In this report, we establish novel fluorescent HeLa tumor metastasis models to determine whether HeLa transfected with the enhanced red fluorescent protein (DsRed2) gene in vitro and xenotransplanted through subcutaneous, intraperitoneal, or intravenous route into SCID mice would permit the detection of tumor micro-metastasis in vivo. Our results showed that DsRed2 insertions did not interfere the tumorigenic properties of HeLa cells. We also demonstrated that DsRed2-transduced HeLa cells maintained stable high-level DsRed2 expressions during their growth in vivo. DsRed2 fluorescence clearly demarcated the primary seeding place and readily allowed for the visualization of distant micro-metastasis and local invasion at the single-cell level. Lung metastasis, the major cause of cervical carcinoma related death, was found in all three models. However, intravenous injections of the HeLa-DsRed2 cells established tumor foci in the lung, while subcutaneous and intraperitoneal injections only established lung metastasis at single-cell levels. The DsRed2 tagged HeLa cancer model allowed detection and investigation of physiologically relevant patterns of cancer invasion and metastasis in vivo.

Animals↗
PubMed2004

The molecular properties and applications of Anthozoa fluorescent proteins and chromoproteins.

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its fluorescent homologs from Anthozoa corals have become invaluable tools for in vivo imaging of cells and tissues. Despite spectral and chromophore diversity, about 100 cloned members of the GFP-like protein family possess common structural, biochemical and photophysical features. Anthozoa GFP-like proteins are available in colors and properties unlike those of A. victoria GFP variants and thus provide powerful new fluorophores for molecular labeling and intracellular detection. Although Anthozoa GFP-like proteins provide some advantages over GFP, they also have certain drawbacks, such as obligate oligomerization and slow or incomplete fluorescence maturation. In the past few years, effective approaches for eliminating some of these limitations have been described. In addition, several Anthozoa GFP-like proteins have been developed into novel imaging agents, such as monomeric red and dimeric far-red fluorescent proteins, fluorescent timers and photoconvertible fluorescent labels. Future studies on the structure of this diverse set of proteins will further enhance their use in animal tissues and as intracellular biosensors.

Animals↗
PubMed2000

"Fluorescent timer": protein that changes color with time.

We generated a mutant of the red fluorescent protein drFP583. The mutant (E5) changes its fluorescence from green to red over time. The rate of color conversion is independent of protein concentration and therefore can be used to trace time-dependent expression. We used in vivo labeling with E5 to measure expression from the heat shock-dependent promoter in Caenorhabditis elegans and from the Otx-2 promoter in developing Xenopus embryos. Thus, E5 is a "fluorescent timer" that can be used to monitor both activation and down-regulation of target promoters on the whole-organism scale.

Animals↗
PubMed2004

Evaluation of RNA interference in developing porcine granulosa cells using fluorescence reporter genes.

Gene silencing using small interfering RNA (siRNA) may be useful for functional analyses of unidentified genes expressed during cell differentiation. The present study was performed to evaluate RNA interference (RNAi) in porcine granulosa cells stimulated with bovine FSH, by using two fluorescence reporter genes: a plasmid encoding green fluorescent protein (GFP) and a plasmid encoding red fluorescent protein (RFP). The siRNA targeting GFP mRNA sequence (GFP-siRNA) with both plasmids was introduced into cultured cells by lipofection. GFP- and RFP-expressing cells were observed under fluorescence microscopy and analyzed by flow cytometry. Strong fluorescence was observed after introduction of both plasmids into cells. The intensity of green fluorescence generated by GFP was greatly suppressed by introduction of GFP-siRNA, showing an approximate 70% decrease in the ratio of green to red fluorescence. Consequently, we concluded that gene silencing by siRNA can be used to analyze the functions of genes of interest during differentiation of porcine granulosa cells.

Animals↗
PubMed2002

Localization of lens intrinsic membrane protein MP19 and mutant protein MP19(To3) using fluorescent expression vectors.

PURPOSE: [corrected] MP19 is the second most abundant major intrinsic protein of the lens fiber cell membrane. A specific heritable mutation at amino acid 15 in the MP19 protein, termed MP19To3, results in total cataract and microphthalmia in the mouse. The goals of this study were to determine the specific localization of MP19 in the cell membrane and to determine whether the mutant MP19To3 protein migrates to the cell membrane in a similar fashion to normal MP19. METHODS: MP19 and MP19To3 cDNAs were cloned into two different sets of expression vectors. The first set was composed of two vectors, pEGFP-N1 and pDsRed2-N1. The first vector expressed green fluorescent protein and the second expressed a red fluorescent protein when transfected into mammalian cells. The two lens membrane protein cDNAs were separately cloned into the vectors so that the cDNA was at the 5'-end of the fluorescent protein coding DNA. These vectors expressed each of the lens proteins fused to the fluorescent protein upon transfection into mammalian cell cultures. The second vector set was a single vector, pcDNA4/TO which must be induced in the transfected cells by tetracycline in order to express the cloned cDNAs. Each of the membrane cDNAs coupled to the fluorescent protein coding region was cut out of the first vector set and cloned into pcDNA4/TO and stable clones were isolated. Each of the prepared plasmids was transfected into human and chick embryo lens epithelial cells and human T-RexTM-293 cells. The fluorescent cells were viewed using confocal and episcopic-fluorescence microscopy. RESULTS: Each of the transfected plasmids expressed fluorescent protein in all three cell lines. MP19 was observed to transport to the cell membrane. When compared to the distribution of another, separate fusion protein consisting of a signal peptide that targets to cell membranes fused to EGFP, MP19 did not distribute uniformly on the membrane, but appeared to localize into "spots" or pools of fluorescent material around the cell membrane. In contrast, MP19To3 protein appeared to not distribute to the cell membrane; it instead appeared to collect in a particular subcellular compartment within the cell. CONCLUSIONS: The distribution of MP19 and MP19To3 in the cell appeared to be quite distinct. MP19 was observed to distribute to the cell membrane while MP19To3 did not. The fact that the MP19To3 did not traffic to the membrane, instead appearing to be trapped within a subcellular compartment within the cell sheds further light on the cause of the cataract and microphthalmia observed in the MP19To3 mutation, and further sheds information on the pathway of MP19 transport to the cell membrane.

Animals↗
PubMed2002

Lentiviral vectors for sustained transgene expression in human bone marrow-derived stromal cells.

Bone marrow-derived mesenchymal stromal cells (MSCs) have attracted attention as potential platforms for the systemic delivery of therapeutic proteins in vivo following gene transfer using oncogenic retroviruses. However, the major limitations of this strategy include low levels of gene transfer and a general lack of long-term transgene expression. We have investigated the expression of several transgenes in MSCs following HIV-1 lentiviral vector-mediated gene transfer. Vectors containing a variety of strong promoters driving enhanced green fluorescence protein (EGFP) and coral (Discosoma sp.)-derived red fluorescent protein (DsRed) reporter genes pseudotyped with the vesicular stomatitis virus-G (VSV-G) glycoprotein were able to transduce cultured MSCs with high efficiency. Transduction efficiencies and transgene expression levels in MSCs were found to be higher with lentiviral vectors than with a vector based on the murine stem cell virus pseudotyped with VSV-G. Transgene expression was maintained in culture for at least 5 months. HIV-1-based lentiviral vectors were able to transduce clonogenic mesenchymal progenitor cells, which were capable of maintaining transgene expression by their MSC progeny, over several cell divisions and during differentiation into adipocytes, indicating that terminal adipocyte cell differentiation was unaffected by lentivirus-mediated reporter gene transfer. Collectively these results suggest that lentivirus-mediated gene transfer strategies provide an efficient tool for ex vivo modification of MSCs that does not interfere with differentiation.

Bone Marrow Cells↗
PubMed2005

Macromolecular translocation inhibitor II (Zn(2+)-binding protein, parathymosin) interacts with the glucocorticoid receptor and enhances transcription in vivo.

Macromolecular translocation inhibitor II (MTI-II), which was first identified as an in vitro inhibitor of binding between the highly purified glucocorticoid receptor (GR) and isolated nuclei, is an 11.5-kDa Zn(2+)-binding protein that is also known as ZnBP or parathymosin. MTI-II is a small nuclear acidic protein that is highly conserved in rats, cows, and humans and widely distributed in mammalian tissues, yet its physiological function is unknown. To elucidate its in vivo function in relation to GR, we transiently transfected mammalian cells with an expression plasmid encoding MTI-II. Unexpectedly, we found that the expression of MTI-II enhances the transcriptional activity of GR. The magnitude of the transcriptional enhancement induced by MTI-II is comparable with that induced by the steroid receptor coactivator SRC-1. In contrast, MTI-II had little effect on the transcriptional activity of estrogen receptor. Immunoprecipitation analysis showed that in the presence of glucocorticoid hormone, GR coprecipitates with MTI-II, and, vice versa, MTI-II coprecipitates with GR. The expression of various deletion mutants of MTI-II revealed that the central acidic domain is essential for the enhancement of GR-dependent transcription. Microscopic analysis of MTI-II fused to green fluorescent protein and GR fused to red fluorescent protein in living HeLa cells showed that MTI-II colocalizes with GR in discrete subnuclear domains in a hormone-dependent manner. Coexpression of MTI-II with the coactivator SRC-1 or p300 further enhances GR-dependent transcription. Immunoprecipitation analysis showed that in the presence of glucocorticoid hormone, p300 and CREB-binding protein are coprecipitated with MTI-II. Furthermore, the knockdown of endogenous MTI-II by RNAi reduces the transcriptional activity of GR in cells. Moreover, expression of MTI-II enhances the glucocorticoid-dependent transcription of the endogenous glucocorticoid-inducible enzyme in cells. Taken together, these results indicate that MTI-II enhances GR-dependent transcription via a direct interaction with GR in vivo. Thus, MTI-II is a new member of the GR-coactivator complex.

Animals↗
PubMed2001

Three-color imaging using fluorescent proteins in living zebrafish embryos.

The zebrafish embryo is especially valuable for cell biological studies because of its optical clarity. In this system, use of an in vivo fluorescent reporter has been limited to green fluorescent protein (GFP). We have examined other fluorescent proteins alone or in conjunction with GFP to investigate their efficacy as markers for multi-labeling purposes in live zebrafish. By injecting plasmid DNA containing fluorescent protein expression cassettes, we generated single-, double-, or triple-labeled embryos using GFP, blue fluorescent protein (BFP, a color-shifted GFP), and red fluorescent protein (DsRed, a wild-type protein structurally related to GFP). Fluorescent imaging demonstrates that GFP and DsRed are highly stable proteins, exhibiting no detectable photoinstability, and a high signal-to-noise ratio. BFP demonstrated detectable photoinstability and a lower signal-to-noise ratio than either GFP or DsRed. Using appropriate filter sets, these fluorescent proteins can be independently detected even when simultaneously expressed in the same cells. Multiple labels in individual zebrafish cells open the door to a number of biological avenues of investigation, including multiple, independent tags of transgenic fish lines, lineage studies of wild-type proteins expressed using polycistronic messages, and the detection of protein-protein interactions at the subcellular level using fluorescent protein fusions.

Animals↗
PubMed2003

5'-AMP-activated protein kinase controls insulin-containing secretory vesicle dynamics.

Changes in 5'-AMP-activated protein kinase (AMPK) activity have recently been implicated in the control of insulin secretion by glucose (da Silva Xavier, G., Leclerc, I., Varadi, A., Tsuboi, T., Moule, S. K., and Rutter, G. A. (2003) Biochem. J. 371, 761-774). Here, we examine the possibility that activation of AMPK may regulate distal steps in insulin secretion, including vesicle movement and fusion with the plasma membrane. Vesicle dynamics were imaged in single pancreatic MIN6 beta-cells expressing lumen-targeted pH-insensitive yellow fluorescent protein, neuropeptide Y.Venus, or monomeric red fluorescent protein by total internal reflection fluorescence and Nipkow disc confocal microscopy. Overexpression of a truncated, constitutively active form of AMPK (AMPKalpha1, 1-312, T172D; AMPK CA), inhibited glucose-stimulated (30 versus 3.0 mM) vesicle movements, and decreased the number of vesicles docked or fusing at the plasma membrane, while having no effect on the kinetics of individual secretory events. Expression of the activated form of AMPK also prevented dispersal of the cortical actin network at high glucose concentrations. Monitored in permeabilized cells, where the effects of AMPK CA on glucose metabolism and ATP synthesis were bypassed, AMPK CA inhibited Ca2+ and ATP-induced insulin secretion, and decreased ATP-dependent vesicle movements. These findings suggest that components of the vesicle transport network, including vesicle-associated motor proteins, may be targets of AMPK in beta-cells, dephosphorylation of which is required for vesicle mobilization at elevated glucose concentrations.

AMP-Activated Protein Kinases↗
PubMed2005

Analysis of the subcellular localization of the proteins Rep, Rep' and Cap of porcine circovirus type 1.

Porcine circovirus type 1 (PCV1) encodes two major ORFs. The cap gene comprises the major structural protein of PCV, the rep gene specifies Rep and Rep', which are both essential for initiating the replication of the viral DNA. Rep corresponds to the full-length protein, whereas Rep' is a truncated splice product that is frame-shifted in its C-terminal sequence. In this study, the cellular localization of PCV1-encoded proteins was investigated by immune fluorescence techniques using antibodies against Rep, Rep' and Cap and by expression of viral proteins fused to green and red fluorescence proteins. Rep and Rep' protein co-localized in the nucleus of infected cells as well as in cells transfected with plasmids expressing Rep and Rep' fused to fluorescence proteins, but no signal was seen in the nucleoli. Rep and Rep' carry three potential nuclear localization signals in their identical N-termini, and the contribution of these motifs to nuclear import was experimentally dissected. In contrast to the rep gene products, the localization of the Cap protein varied. While the Cap protein was restricted to the nucleoli in plasmid-transfected cells and was also localized in the nucleoli at an early stage of PCV1 infection, it was seen in the nucleoplasm and the cytoplasm later in infection, suggesting that a shuttling between distinct cellular compartments occurs.

Amino Acid Sequence↗
PubMedDate not supplied

[A natural fluorescent protein that changes its fluorescence during maturation].

The gene of a new red fluorescent protein zoan2RFP from a coral polyp Zoanthus sp., a homologue of the known green fluorescent protein from the Aequorea victoria jellyfish, was cloned. At early maturation stages, zoan2RFP exhibits a green fluorescence, which then turns into the red one. A similar phenomenon was recently reported for the E5 mutant of the red fluorescent coral protein DsRed. Zoan2RFP differs from E5 by faster maturation kinetics and the complete disappearance of green fluorescence in the mature protein. Naturally occurring proteins of this type can be considered as intermediate forms between the green and red fluorescent proteins, which are formed during the microevolution of fluorescent proteins.

Amino Acid Sequence↗
PubMed2004

Application of RNA interference to chicken embryos using small interfering RNA.

Gene silencing using small interfering RNA (siRNA) is not established in avian species. The present study was performed to evaluate RNA interference (RNAi) in the chicken embryo by using a dual fluorescence reporter assay, a plasmid encoding green fluorescent protein (GFP) and a plasmid encoding red fluorescent protein (RFP). The siRNA targeting the GFP mRNA sequence (GFP-siRNA) with both plasmids was introduced into cultured cells and whole embryos by lipofection and microelectroporation, respectively. GFP- and RFP-expressed cells and embryos were observed under fluorescent microscopy and analyzed by flow cytometer, and their mRNAs were analyzed by reverse transcription-PCR (RT-PCR). The strong fluorescence was observed by introducing both plasmids into cells. The intensity of the green fluorescence generated by GFP was greatly suppressed by introducing GFP-siRNA. RT-PCR analysis showed that introducing GFP-siRNA also decreased GFP mRNA levels. In contrast to GFP, the intensity of the red fluorescence generated by RFP and the RFP mRNA levels remained unchanged. In whole embryos, also, introducing GFP-siRNA specifically suppressed GFP expression, and the suppression was maintained for at least 72 h. Consequently, it was concluded that the gene silencing using siRNA is applicable to analyzing the function of genes of interest during avian embryogenesis.

Animals↗
PubMed2006

Dynamic monitoring of oncolytic adenovirus in vivo by genetic capsid labeling.

BACKGROUND: Conditionally replicative adenoviruses represent a promising strategy to address the limited efficacy and safety issues associated with conventional cancer treatment. Despite rapid translation into human clinical trials and demonstrated safety, the fundamental properties of oncolytic adenovirus replication and spread and host-vector interactions in vivo have not been completely evaluated. METHODS: We developed a noninvasive dynamic monitoring system to detect adenovirus replication. We constructed capsid-labeled E1/E3-deleted and wild-type adenoviruses (Ad-wt) by fusing the minor capsid protein IX with red fluorescent proteins mRFP1 and tdimer2(12), resulting in Ad-IX-mRFP1, Ad-IX-tdimer2(12), and Ad-wt-IX-mRFP1. Virus DNA replication, encapsidation, cytopathic effect, thermostability, and binding to primary receptor (coxsackie adenovirus receptor) were analyzed using real-time quantitative polymerase chain reaction, cell viability (MTS) assay, and fluorescence microscopy. Athymic mice (n = 4) carrying xenograft tumors that were derived from A549 lung adenocarcinoma cells were intratumorally inoculated with Ad-wt-IX-mRFP1, and adenovirus replication was dynamically monitored with a fluorescence noninvasive imaging system. Correlations between fluorescence signal intensity and viral DNA synthesis and replication were calculated using Pearson's correlation coefficient (r). RESULTS: The red fluorescence label had little effect on viral DNA replication, encapsidation, cytopathic effect, thermostability, and coxsackie adenovirus receptor binding. The fluorescent signal correlated with viral DNA synthesis and infectious progeny production both in vitro and in vivo (in A549 cells, r = .99 and r = .65; in tumors, r = .93 and r = .92, respectively). The replication efficiency of Ad-wt-IX-mRFP1 in vivo was variable, and replication and viral spreading and persistence were limited, consistent with clinical observations. CONCLUSIONS: Genetic capsid labeling provides a promising approach for the dynamic assessment of oncolytic adenovirus function in vivo.

Adenocarcinoma↗
PubMed2004

Expression and trafficking of fluorescent viral membrane proteins in baculovirus-transduced BHK cells.

Baculovirus vectors show promise as a novel tool for gene delivery into mammalian cells and gene transfer with wild-type baculovirus has been demonstrated both in vitro and in vivo. To study expression and intracellular trafficking of foreign viral membrane proteins in baculovirus-transduced mammalian cells, the envelope proteins, E1 and E2, of rubella virus (RV) were chosen as a model. The enhanced green fluorescent protein (EGFP) and a red fluorescent protein (RFP) were fused to the C-terminus of E1 and E2, respectively. The proteins were cloned under a cytomegalovirus (CMV) promoter and expressed as fluorescent fusion proteins in baculovirus-transduced baby hamster kidney (BHK) cells. Expression of the chimeric proteins in these cells showed that E1 was retained within the ER and cis-Golgi when expressed alone. In contrast, E2 was efficiently transported to the trans-Golgi network (TGN). However, when expressed together, E1 co-localized with E2 in TGN and to some extent in the lysosomes. The recombinant baculovirus vectors were able to transduce the BHK cells efficiently and the fluorescent fusion constructs allowed easy detection of the trafficking events in the transduced mammalian cells. Consequently, this technique should have wide applications when intracellular analysis of protein synthesis and maturation is under study.

Animals↗
PubMed2003

Homodimerization of neuropeptide y receptors investigated by fluorescence resonance energy transfer in living cells.

Up to now neuropeptide Y (NPY) receptors, which belong to the large family of G-protein-coupled receptors and are involved in a broad range of physiological processes, are believed to act as monomers. Studies with the Y(1)-receptor antagonist and Y(4)-receptor agonist GR231118, which binds with a 250-fold higher affinity than its monomer, led to the first speculation that NPY receptors can form homodimers. In the present work we used the fluorescence resonance energy transfer (FRET) to study homodimerization of the hY(1)-, hY(2)-, and hY(5)-receptors in living cells. For this purpose, we generated fusion proteins of NPY receptors and green fluorescent protein or spectral variants of green fluorescent protein (cyan, yellow, and red fluorescent protein), which can be used as FRET pairs. Two different FRET techniques, fluorescence microscopy and fluorescence spectroscopy, were applied. Both techniques clearly showed that the hY(1)-, hY(2)-, and hY(5)-NPY receptor subtypes are able to form homodimers. By using transiently transfected cells, as well as a stable cell line expressing the hY(2)-GFP fusion protein, we could demonstrate that the Y-GFP fusion proteins are still functional and that dimerization varies from 26 to 44% dependent on the receptor. However, homodimerization is influenced neither by NPY nor by Galpha protein binding.

Animals↗
PubMed2003

LEDGF/p75 is essential for nuclear and chromosomal targeting of HIV-1 integrase in human cells.

We have reported that human immunodeficiency virus type 1 (HIV-1) integrase (IN) forms a specific nuclear complex with human lens epithelium-derived growth factor/transcription co-activator p75 (LEDGF/p75) protein. We now studied the IN-LEDGF/p75 interaction and nuclear import of IN in living cells using fusions of IN and LEDGF/p75 with enhanced green fluorescent protein and far-red fluorescent protein HcRed1. We show that both the N-terminal zinc binding domain and the central core domains of IN are involved in the interaction with LEDGF/p75. Both domains are essential for nuclear localization of IN as well as for the association of IN with condensed chromosomes during mitosis. However, upon overexpression of LEDGF/p75, the core domain fragment of IN was recruited to the nuclei and mitotic chromosomes with a distribution pattern characteristic of the full-length protein, indicating that it harbors the main determinant for interaction with LEDGF/p75. Although the C-terminal domain of IN was dispensable for nuclear/chromosomal localization, a fusion of the C-terminal IN fragment with enhanced green fluorescent protein was found exclusively in the nucleus, with a diffuse nuclear/nucleolar distribution, suggesting that the C-terminal domain may also play a role in the nuclear import of IN. In contrast to LEDGF/p75, its alternative splice variant, p52, did not interact with HIV-1 IN in vitro and in living cells. Finally, RNA interference-mediated knock-down of endogenous LEDGF/p75 expression abolished nuclear/chromosomal localization of IN. We conclude, therefore, that the interaction with LEDGF/p75 accounts for the karyophilic properties and chromosomal targeting of HIV-1 IN.

Active Transport, Cell Nucleus↗
PubMed2006

The truncated prelamin A in Hutchinson-Gilford progeria syndrome alters segregation of A-type and B-type lamin homopolymers.

Hutchinson-Gilford progeria syndrome (HGPS) is a dominant autosomal premature aging syndrome caused by the expression of a truncated prelamin A designated progerin (Pgn). A-type and B-type lamins are intermediate filament proteins that polymerize to form the nuclear lamina network apposed to the inner nuclear membrane of vertebrate somatic cells. It is not known if in vivo both type of lamins assemble independently or co-assemble. The blebbing and disorganization of the nuclear envelope and adjacent heterochromatin in cells from patients with HGPS is a hallmark of the disease, and the ex vivo reversal of this phenotype is considered important for the development of therapeutic strategies. Here, we investigated the alterations in the lamina structure that may underlie the disorganization caused in nuclei by Pgn expression. We studied the polymerization of enhanced green fluorescent protein- and red fluorescent protein-tagged wild-type and mutated lamins in the nuclear envelope of living cells by measuring fluorescence resonance energy transfer (FRET) that occurs between the two fluorophores when tagged lamins interact. Using time domain fluorescence lifetime imaging microscopy that allows a quantitative analysis of FRET signals, we show that wild-type lamins A and B1 polymerize in distinct homopolymers that further interact in the lamina. In contrast, expressed Pgn co-assembles with lamin B1 and lamin A to form a mixed heteropolymer in which A-type and B-type lamin segregation is lost. We propose that such structural lamina alterations may be part of the primary mechanisms leading to HGPS, possibly by impairing functions specific for each lamin type such as nuclear membrane biogenesis, signal transduction, nuclear compartmentalization and gene regulation.

Animals↗