Green Fluorescent Protein Chimeras Research Articles

16] Green fluorescent protein chimeras to probe protein-protein interactions

Publisher Summary Green fluorescent protein (GFP) from the jellyfish Aequorea victoria is autofluorescent. The fluorophore of GFP forms on the translation of the protein, without the action of any additional factors. GFP can be expressed in a wide variety of nonnative cell types. To date, GFP has been used in vivo as a marker for gene expression and as a fusion tag to monitor protein localization in living cells. GFP has exceptional physical and chemical properties besides spontaneous fluorescence. These properties include high thermal stability and resistance to detergents, organic solvents, and proteases. These properties endow GFP with enormous potential for biotechnical applications. Since the complementary deoxyribonucleic acid (cDNA) of GFP was cloned, a variety of GFP variants have been generated that broaden the spectrum of its application. Among those variants, S65T GFP is unique in having increased fluorescence intensity, faster fluorophore formation, and altered excitation and emission spectra than that of the wild-type protein. This chapter describes the use of S65T GFP to probe protein–protein interactions in vitro . This method requires fusing GFP to one of the target proteins to create a GFP chimera.

Visualization and functional analysis of a maxi-K channel (mSlo) fused to green fluorescent protein (GFP)

We have constructed a fusion protein between mSlo (a recombinant, high conductance, calcium-activated potassium channel or maxi-K), and GFP (green fluorescent protein). This construct represents a tag to not only monitor channel expression, but to locate the protein in living cells. The GFP was fused in frame to the carboxy-terminus of the mSlo core protein (mSloGFP fusion protein). Expression of this fusion protein in COS-7 cells resulted in robust fluorescence localized near the cell membrane. Fluorescing cells that were patch clamped exhibited whole cell currents with a direction consistent with potassium currents. Conversely, non-fluorescing cells showed no significant whole cell currents. Excised inside out patches revealed single channel currents and calculated conductances in the range of those expected for the maxi-K. The mSlo and mSlo-GFP channels reconstituted into lipid bilayers bound wild-type, recombinant CTX with high affinity and displayed a half-blocking concentration (KD ) of 7.4 and 7.6 nM, respectively (at +30 mV in 150 mM equimolar KCl). This resulted in single channel evaluation of the functional inhibition of CTX on these clones. As newly constructed GFP chimeras emerge for the study of physiological processes in living organisms, this work provides another area of insight illuminated by GFP.

Functional roles for fatty acylated amino-terminal domains in subcellular localization.

Several membrane-associating signals, including covalently linked fatty acids, are found in various combinations at the N termini of signaling proteins. The function of these combinations was investigated by appending fatty acylated N-terminal sequences to green fluorescent protein (GFP). Myristoylated plus mono/dipalmitoylated GFP chimeras and a GFP chimera containing a myristoylated plus a polybasic domain were localized similarly to the plasma membrane and endosomal vesicles, but not to the nucleus. Myristoylated, nonpalmitoylated mutant chimeric GFPs were localized to intracellular membranes, including endosomes and the endoplasmic reticulum, and were absent from the plasma membrane, the Golgi, and the nucleus. Dually palmitoylated GFP was localized to the plasma membrane and the Golgi region, but it was not detected in endosomes. Nonacylated GFP chimeras, as well as GFP, showed cytosolic and nuclear distribution. Our results demonstrate that myristoylation is sufficient to exclude GFP from the nucleus and associate with intracellular membranes, but plasma membrane localization requires a second signal, namely palmitoylation or a polybasic domain. The similarity in localization conferred by the various myristoylated and palmitoylated/polybasic sequences suggests that biophysical properties of acylated sequences and biological membranes are key determinants in proper membrane selection. However, dual palmitoylation in the absence of myristoylation conferred significant differences in localization, suggesting that multiple palmitoylation sites and/or enzymes may exist.

Using Inducible Vectors to Study Intracellular Trafficking of GFP-Tagged Steroid/Nuclear Receptors in Living Cells

Intracellular trafficking and localization of proteins can now be efficiently visualized by fusion of a polypeptide to the green fluorescent protein (GFP). Many spectral variants of this reagent are now available, providing powerful tools for studies in living cells. This approach is particularly useful for members of the steroid/nuclear receptor superfamily, since these molecules frequently undergo rapid subcellular redistribution on ligand activation. A major roadblock in the application of this technology concerns problems associated with transient transfections. This technique produces cell populations that are highly heterogeneous with respect to the newly introduced protein and usually contain the protein in a highly overexpressed state. In addition, long-term studies related to cell cycle and cellular differentiation are essentially impossible with this approach. These problems can be overcome by introduction of the GFP fusion into cells under appropriate induction control. We describe application of the tetracycline regulatory system to inducible control of a glucocorticoid receptor (GR)/GFP chimera. Intracellular concentrations of GFP–GR can be very effectively controlled in this system, providing an ideal environment in which to study subcellular trafficking of the receptor and interactions with a variety of intracellular targets.

Dynamics and Mobility of Nuclear Envelope Proteins in Interphase and Mitotic Cells Revealed by Green Fluorescent Protein Chimeras

Understanding how membrane proteins are targeted to and retained within the nuclear envelope (NE) and the fate of these proteins during NE disassembly/reassembly in mitosis is central for insight into the function of the NE in nuclear organization and dynamics. To address these issues we have attached green fluorescent protein (GFP) to a well-characterized protein of the inner nuclear membrane, lamin B receptor, believed to be one of the major chromatin docking protein in the NE. We have used this construct in a variety of applications, including dual-color GFP time-lapse imaging, to investigate the mechanisms underlying protein targeting to the NE and NE breakdown and reassembly during mitosis. In this review, we present a summary of the results from such studies and discuss the photobleaching and imaging methodology on which they were derived.

The "pro" sequence of the sporulation-specific sigma transcription factor sigma(E) directs it to the mother cell side of the sporulation septum.

sigma(E), a mother cell-specific transcription factor of sporulating Bacillus subtilis, is derived from an inactive precursor protein (pro-sigma(E)). Activation of sigma(E) occurs when a sporulation-specific protease (SpoIIGA) cleaves 27 amino acids from the pro-sigma(E) amino terminus. This reaction is believed to take place at the mother cell-forespore septum. Using a chimera of pro-sigma(E) and green fluorescent protein (GFP) to visualize the intracellular location of pro-sigma(E) by fluorescence microscopy, and lysozyme treatment to separate the mother cell and forespore compartments, we determined that the pro-sigma(E)::GFP signal, localized to the forespore septum prior to lysozyme treatment, is restricted to the mother cell compartment after treatment. Thus, pro-sigma(E)::GFP had been sequestered to the mother cell side of the septum. This segregation of pro-sigma(E)::GFP, and presumably pro-sigma(E), to the mother cell is likely to be the reason why sigma(E) activity is restricted to that compartment.

Insights into secretory traffic using green fluorescent protein chimeras

Insights into secretory traffic using green fluorescent protein chimeras

Glycosylphosphatidylinositol biosynthetic enzymes are localized to a stable tubular subcompartment of the endoplasmic reticulum in Leishmania mexicana

Glycosylphosphatidylinositols (GPI) are essential components in the plasma membrane of the protozoan parasite Leishmania mexicana, both as membrane anchors for the major surface macromolecules and as the sole class of free glycolipids. We provide evidence that L.mexicana dolichol-phosphate-mannose synthase (DPMS), a key enzyme in GPI biosynthesis, is localized to a distinct tubular subdomain of the endoplasmic reticulum (ER), based on the localization of a green fluorescent protein (GFP)-DPMS chimera and subcellular fractionation experiments. This tubular membrane (termed the DPMS tubule) is also enriched in other enzymes involved in GPI biosynthesis, can be specifically stained with the fluorescent lipid, BODIPY-C5-ceramide, and appears to be connected to specific subpellicular microtubules that underlie the plasma membrane. Perturbation of microtubules and DPMS tubule structure in vivo results in the selective accumulation of GPI anchor precursors, but not free GPIs. The DPMS tubule is closely associated morphologically with the single Golgi apparatus in non-dividing and dividing cells, appears to exclude luminal ER resident proteins and is labeled, together with the Golgi apparatus, with another GFP chimera containing the heterologous human Golgi marker beta1,2-N-acetylglucosaminyltransferase-I. The possibility that the DPMS-tubule is a stable transitional ER is discussed.

Hormone-dependent Translocation of Vitamin D Receptors Is Linked to Transactivation

Vitamin D receptor (VDR) acts as a transcription factor mediating genomic actions of calcitriol. Our earlier studies suggested that calcitriol induces translocation of cytoplasmic VDR, but the physiologic relevance of this finding remained uncertain. Previous studies demonstrated that the activation function 2 domain (AF-2) plays an essential role in VDR transactivation. To elucidate hormone-dependent VDR translocation and its role, we constructed green fluorescent protein (GFP) chimeras with full-length VDR (VDR-GFP), AF-2-truncated VDR (AF-2del-VDR-GFP), and ligand-binding domain (LBD)-truncated VDR (LBDdel-VDR-GFP). COS-7 cells were transiently transfected with these constructs. Western blot analysis, fluorescent microscopy, and transactivation assays showed that the generated chimeras are expressed and fluoresce and that VDR-GFP is transcriptionally active. After hormone treatment, cytoplasmic VDR-GFP translocated to the nucleus in a concentration-, time-, temperature-, and analog-specific manner. Hormone dose-response relationships for translocation and for transactivation were similar. Truncation of LBD and truncation of AF-2 each abolished hormone-dependent translocation and transactivation. Our data confirm a hormone-dependent VDR translocation, demonstrate that an intact AF-2 domain is required for this translocation, and indicate that translocation is part of the receptor activation process.

EGF-and NGF-stimulated translocation of cytohesin-1 to the plasma membrane of PC12 cells requires PI 3-kinase activation and a functional cytohesin-1 PH domain.

ADP-ribosylation factors (ARFs) are small GTP-binding proteins that function as regulators of eukaryotic vesicle trafficking. Cytohesin-1 is a member of a family of ARF guanine nucleotide-exchange factors that contain a C-terminal pleckstrin homology (PH) domain which has been proposed to bind the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3). Here we demonstrate that in vitro, recombinant cytohesin-1 binds, via its PH domain, the inositol head group of PIP3, inositol 1,3,4, 5-tetrakisphosphate (IP4), with an affinity greater than 200-fold higher than the inositol head group of either phosphatidylinositol 4, 5-bisphosphate or phosphatidylinositol 3,4-bisphosphate. Moreover, addition of glycerol or diacetylglycerol to the 1-phosphate of IP4 does not alter the ability to interact with cytohesin-1, data which is entirely consistent with cytohesin-1 functioning as a putative PIP3 receptor. To address whether cytohesin-1 binds PIP3 in vivo, we have expressed a chimera of green fluorescent protein (GFP) fused to the N terminus of cytohesin-1 in PC12 cells. Using laser scanning confocal microscopy we demonstrate that either EGF- or NGF-stimulation of transiently transfected PC12 cells results in a rapid translocation of GFP-cytohesin-1 from the cytosol to the plasma membrane. This translocation is dependent on the cytohesin-1 PH domain and occurs with a time course that parallels the rate of plasma membrane PIP3 production. Furthermore, the translocation requires the ability of either agonist to activate PI 3-kinase, since it is inhibited by wortmannin (100 nM), LY294002 (50 microM) and by coexpression with a dominant negative p85. This data therefore suggests that in vivo cytohesin-1 can interact with PIP3 via its PH domain.

Identification of centaurin-α1 as a potential in vivo phosphatidylinositol 3,4,5-trisphosphate-binding protein that is functionally homologous to the yeast ADP-ribosylation factor (ARF) GTPase-activating protein, Gcs1

Centaurin-α is a 46 kDa in vitro binding protein for the lipid second messenger PtdIns(3,4,5)P3. In this report we have addressed whether centaurin-α1, a human homologue of centaurin-α, binds PtdIns(3,4,5)P3in vivo and furthermore, identified a potential physiological function for centaurin-α1. Using confocal microscopy of live PC12 cells, transiently transfected with a chimera of green fluorescent protein (GFP) fused to the N-terminus of centaurin-α1 (GFP-centaurin-α1), we demonstrated the rapid plasma membrane recruitment of cytosolic GFP-centaurin-α1 following stimulation with either nerve growth factor or epidermal growth factor. This recruitment was dependent on the centaurin-α1 pleckstrin homology domains and was blocked by the PtdIns(4,5)P2 3-kinase (PI 3-kinase) inhibitors wortmannin (100 nM) and LY294002 (50 μM), and also by co-expression with a dominant negative p85. Functionally, we demonstrated that centaurin-α1 could complement a yeast strain deficient in the ADP-ribosylation factor (ARF) GTPase-activating protein Gcs1; a complementation that was blocked by mutagenesis of conserved cysteine residues within the ARF GTPase-activating protein analogous domain of centaurin-α1. Taken together, our data demonstrated that centaurin-α1 could potentially function as an ARF GTPase-activating protein that, on agonist stimulation, was recruited to the plasma membrane possibly through an ability to interact with PtdIns(3,4,5)P3.

Identification of centaurin-α1 as a potential in vivo phosphatidylinositol 3,4,5-trisphosphate-binding protein that is functionally homologous to the yeast ADP-ribosylation factor (ARF) GTPase-activating protein, Gcs1

Centaurin-alpha is a 46 kDa in vitro binding protein for the lipid second messenger PtdIns(3,4,5)P3. In this report we have addressed whether centaurin-alpha1, a human homologue of centaurin-alpha, binds PtdIns(3,4,5)P3 in vivo and furthermore, identified a potential physiological function for centaurin-alpha1. Using confocal microscopy of live PC12 cells, transiently transfected with a chimera of green fluorescent protein (GFP) fused to the N-terminus of centaurin-alpha1 (GFP-centaurin-alpha1), we demonstrated the rapid plasma membrane recruitment of cytosolic GFP-centaurin-alpha1 following stimulation with either nerve growth factor or epidermal growth factor. This recruitment was dependent on the centaurin-alpha1 pleckstrin homology domains and was blocked by the PtdIns(4,5)P2 3-kinase (PI 3-kinase) inhibitors wortmannin (100 nM) and LY294002 (50 microM), and also by co-expression with a dominant negative p85. Functionally, we demonstrated that centaurin-alpha1 could complement a yeast strain deficient in the ADP-ribosylation factor (ARF) GTPase-activating protein Gcs1; a complementation that was blocked by mutagenesis of conserved cysteine residues within the ARF GTPase-activating protein analogous domain of centaurin-alpha1. Taken together, our data demonstrated that centaurin-alpha1 could potentially function as an ARF GTPase-activating protein that, on agonist stimulation, was recruited to the plasma membrane possibly through an ability to interact with PtdIns(3,4,5)P3.

In Vivo Mitochondrial Import: A COMPARISON OF LEADER SEQUENCE CHARGE AND STRUCTURAL RELATIONSHIPS WITH THE IN VITRO MODEL RESULTING IN EVIDENCE FOR CO-TRANSLATIONAL IMPORT

The positive charges and structural properties of the mitochondrial leader sequence of aldehyde dehydrogenase have been extensively studied in vitro. The results of these studies showed that increasing the helicity of this leader would compensate for reduced import from positive charge substitutions of arginine with glutamine or the insertion of negative charged residues made in the native leader. In this in vivo study, utilizing the green fluorescent protein (GFP) as a passenger protein, import results showed the opposite effect with respect to helicity, but the results from mutations made within the native leader sequence were consistent between the in vitro and in vivo experiments. Leader mutations that reduced the efficiency of import resulted in a cytosolic accumulation of a truncated GFP chimera that was fluorescent but devoid of a mitochondrial leader. The native leader efficiently imported before GFP could achieve a stable, import-incompetent structure, suggesting that import was coupled with translation. As a test for a co-translational mechanism, a chimera of GFP that contained the native leader of aldehyde dehydrogenase attached at the N terminus and a C-terminal endoplasmic reticulum targeting signal attached to the C terminus of GFP was constructed. This chimera was localized exclusively to mitochondria. The import result with the dual signal chimera provides support for a co-translational mitochondrial import pathway.

Visualizing protein-protein interactions in the nucleus of the living cell.

A question of central importance to the molecular endocrinologist is how hormones function to orchestrate events within cells. The cascades of cellular responses that are triggered by endocrine signals require the formation of specific protein partnerships, and these protein-protein interactions must be coordinated in both space and time. For example, in the absence of ligand, the steroid hormone receptor for estradiol is associated with a multiprotein inhibitory complex (1). The binding of estradiol results in alterations in estrogen receptor conformation that allow it to dissociate from this complex, and the receptor becomes competent to interact with specific DNA elements in the regulatory regions of target genes. The efficient utilization of these regulatory elements by the receptor, however, requires that the receptor associate with other coregulatory proteins (2–4). Biochemical approaches such as coimmunoprecipitation and FarWestern blotting and in vivo approaches such as yeast two-hybrid assay have provided important information regarding the interactions between receptors and coregulatory proteins. These approaches, however, may sometimes implicate nonphysiological associations between proteins that do not normally occur in intact cells. Deciphering where and when specific protein partnerships form within the living cell will be critical to understanding these basic cellular events. The molecular cloning of the jellyfish green fluorescent protein (GFP) and its expression in a variety of cell types have had a major impact on our ability to monitor events within living cells (5–10). GFP retains its fluorescent properties when fused to other proteins, and this allows fluorescence microscopy to be used to monitor the dynamic behavior of the expressed GFP fusion proteins in their natural environment within the living cell. There are now many examples of proteins expressed as GFP chimeras that possess the same subcellular localization and biological function as their endogenous counterparts. For example, the dynamics of nuclear translocation for the glucocorticoid (11, 12) and androgen receptors (13) have been visualized using GFP fusions. GFP fusion proteins have also been used to monitor complex cellular events such as the sorting of proteins between organelles (14) and the dynamics of regulated protein secretion (15, 16). Recently, it was demonstrated that movement of b-arrestin2-GFP fusion proteins serves as a sensitive biosensor for G protein-coupled receptor activation (17). This illustrates the potential for GFP fusion proteins to act as indicators of many different intracellular events. Mutant forms of the GFP protein that emit lights of different colors have been generated that, when coexpressed in the same cell, can be readily distinguished by fluorescence microscopy. This allows the behavior of two independent proteins to be monitored in the intact cell, and the extent to which these proteins colocalize can be assessed. To determine whether these labeled proteins are physically interacting, however, would require resolution beyond the optical limit of the light microscope. Fortunately, this degree of spatial resolution can be achieved with the conventional light microscope using the technique of fluorescence resonance energy transfer (FRET). FRET microscopy involves the detection of increased (sensitized) emission from an acceptor fluorophore that occurs as the result of the direct transfer of excitation energy from an appropriately positioned fluorescent 0888-8809/99/$3.00/0 Molecular Endocrinology Copyright © 1999 by The Endocrine Society

Ligand-induced trafficking of the sphingosine-1-phosphate receptor EDG-1.

The endothelial-derived G-protein-coupled receptor EDG-1 is a high-affinity receptor for the bioactive lipid mediator sphingosine-1-phosphate (SPP). In the present study, we constructed the EDG-1-green fluorescent protein (GFP) chimera to examine the dynamics and subcellular localization of SPP-EDG-1 interaction. SPP binds to EDG-1-GFP and transduces intracellular signals in a manner indistinguishable from that seen with the wild-type receptor. Human embryonic kidney 293 cells stably transfected with the EDG-1-GFP cDNA expressed the receptor primarily on the plasma membrane. Exogenous SPP treatment, in a dose-dependent manner, induced receptor translocation to perinuclear vesicles with a tau1/2 of approximately 15 min. The EDG-1-GFP-containing vesicles are distinct from mitochondria but colocalize in part with endocytic vesicles and lysosomes. Neither the low-affinity agonist lysophosphatidic acid nor other sphingolipids, ceramide, ceramide-1-phosphate, or sphingosylphosphorylcholine, influenced receptor trafficking. Receptor internalization was completely inhibited by truncation of the C terminus. After SPP washout, EDG-1-GFP recycles back to the plasma membrane with a tau1/2 of approximately 30 min. We conclude that the high-affinity ligand SPP specifically induces the reversible trafficking of EDG-1 via the endosomal pathway and that the C-terminal intracellular domain of the receptor is critical for this process.

The dynamic dialogue between cells and matrices: implications of fibronectin's elasticity.

The extracellular matrix (ECM) provides vital structure and organization to all metazoans, but it is far from an inanimate, unchanging assemblage of collagens, proteoglycans, and glycoproteins. ECM undergoes constant remodeling, most obviously during development, wound healing, and other repair processes. Defects in regulation of the deposition and turnover of ECM play important roles in pathological processes such as osteoporosis, atherosclerosis, arthritis, and fibrotic diseases. The deposition and remodeling of ECM is accomplished by cells, and the paper of Ohashi et al. in a recent issue of the Proceedings (1) provides some of the first observations of these processes in real time in living cell cultures. Ohashi et al. transfected cells with a chimera of green fluorescent protein and fibronectin (FN). FN is a prominent constituent of ECM around and beneath many cells, and FN-rich matrices provide substrates for cell adhesion and migration during development, wound healing, and other situations, as well as affecting many cellular functions including proliferation, survival, and differentiation (2). Ohashi et al. show that the FN–GFP chimera assembles into the FN-rich matrix surrounding their transfected cells, and they then use fluorescence microscopy to observe alterations in that matrix over time. This approach offers many opportunities for future research, but the observations they report already reveal some intriguing features that have implications not only for ECM structure and function but more widely in cell adhesion and beyond.

Identification of the Ras GTPase-activating protein GAP1m as a phosphatidylinositol-3,4,5-trisphosphate-binding protein in vivo

GAP1m is a member of the GAP1 family of Ras GTPase-activating proteins (GAPs) [1]. In vitro, it has been shown to bind inositol 1,3,4,5-tetrakisphosphate (IP4), the water-soluble inositol head group of the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3) [2,3]. This has led to the suggestion that GAP1m might function as a PIP3 receptor in vivo[4]. Here, using rat pheochromocytoma PC12 cells transiently transfected with a plasmid expressing a chimera of green fluorescent protein fused to GAP1m (GFP–GAP1m), we show that epidermal growth factor (EGF) induces a rapid (less than 60 seconds) recruitment of GFP–GAP1m from the cytosol to the plasma membrane. This recruitment required a functional GAP1m pleckstrin homology (PH) domain, because a specific point mutation (R629C) in the PH domain that inhibits IP4 binding in vitro[5] totally blocked EGF-induced GAP1m translocation. Furthermore, the membrane translocation was dependent on PI 3-kinase, and the time course of translocation paralleled the rate by which EGF stimulates the generation of plasma membrane PIP3[6]. Significantly, the PIP3-induced recruitment of GAP1m did not appear to result in any detectable enhancement in its basal Ras GAP activity. From these results, we conclude that GAP1m binds PIP3in vivo, and it is recruited to the plasma membrane, but does not appear to be activated, following agonist stimulation of PI 3-kinase.

Differential Localization and Activity of the A- and B-Forms of the Human Progesterone Receptor Using Green Fluorescent Protein Chimeras

Differential Localization and Activity of the A- and B-Forms of the Human Progesterone Receptor Using Green Fluorescent Protein Chimeras

Visualization of caveolin-1, a caveolar marker protein, in living cells using green fluorescent protein (GFP) chimeras: The subcellular distribution of caveolin-1 is modulated by cell-cell contact

Caveolin-1, a suspected tumor suppressor, is a principal protein component of caveolae in vivo. Recently, we have shown that NIH 3T3 cells harboring anti-sense caveolin-1 exhibit a loss of contact inhibition and anchorage-independent growth. These observations may be related to the ability of caveolin-1 expression to positively regulate contact inhibition. In order to understand the postulated role of caveolin-1 in contact inhibition, it will be necessary to follow the distribution of caveolins in living cells in response to a variety of stimuli, such as cell density. Here, we visualize the distribution of caveolin-1 in living normal NIH 3T3 cells by creating GFP-fusion proteins. In many respects, the behavior of these GFP-caveolin-1 fusion proteins is indistinguishable from endogenous caveolin-1. These GFP-caveolin-1 fusion proteins co-fractionated with endogenous caveolin-1 using an established protocol that separates caveolae-derived membranes from the bulk of cellular membranes and cytosolic proteins, and co-localized with endogenous caveolin-2 in vivo as seen by immunofluorescence microscopy. We show here that as NIH 3T3 cells become confluent, the distribution of GFP-caveolin-1 and endogenous caveolin-1 shifts to areas of cell-cell contact, coincident with contact inhibition. However, unlike endogenous caveolin-1, the levels of GFP-caveolin-1 expression are unaffected by changes in cell density, serum starvation, or growth factor stimulation. These results are consistent with the idea that the levels of endogenous caveolin-1 are modulated by either transcriptional or translational control, and that this modulation is separable from density-dependent regulation of the distribution of caveolin-1. These studies provide a new living-model system for elucidating the dynamic mechanisms underlying the density-dependent regulation of the distribution of caveolin-1 and how this relates to contact inhibition.

GFP chimeras of E-MAP-115 (ensconsin) domains mimic behavior of the endogenous protein in vitro and in vivo.

E-MAP-115 (ensconsin) is a microtubule-associated protein (MAP) abundant in carcinoma and other epithelia-derived cells. We expressed chimeras of green fluorescent protein (GFP) conjugated to ensconsin's N-terminal MT-binding domain (EMTB), to study distribution, dynamics, and function of the MAP in living cells. We tested the hypothesis that behavior of expressed GFP-EMTB accurately matched behavior of endogenous ensconsin. Like endogenous MAP, GFP-EMTB was associated with microtubules in living or fixed cells, and microtubule association of either molecule was impervious to extraction with nonionic detergents. In cell lysates both GFP-EMTB and endogenous ensconsin were dissociated from microtubules by identical salt extraction conditions, and both molecules remained bound to a calcium-stable subset of Taxol-stabilized microtubules. These data show that microtubule association of ensconsin was affected neither by the absence of domains other than its microtubule-binding domain, nor by the presence of appended GFP. We took advantage of this finding to generate constructs in which additional GFP moieties were attached to EMTB, to obtain a more intensely fluorescent reporter of in vivo MAP binding. We show here that expression of chimeric proteins consisting of five GFP molecules attached to a single EMTB molecule produces brightly labeled microtubules without compromising the behavior of the MAP or the microtubules to which it is attached. Thus, we have demonstrated the utility of chimeric proteins containing GFP multimers as authentic reporters of ensconsin distribution and dynamics; expression of these GFP-EMTB chimeric molecules also provides a non-perturbing label of the microtubule system in living cells.