Regulation Of Protein Secretion Research Articles

Regulation of protein secretion by mycelial culture of the mushroom Termitomyces clypeatus

Termitomyces clypeatus secreted a 24-kDa xylanase constitutively in xylan medium, but required a gluconeogenic amino acid or Krebs cycle acid for the secretion of a 56-kDa amyloglucosidase in dextrin medium. Aspartate, glutamate, succinate and fumarate all increased secretion of amyloglucosidase from 50% to >90% and enzyme production by 10-fold with little effect on xylanase production. Glutamate or succinate stimulated in vitro release of intracellular amyloglucosidase from washed mycelia in the presence of cycloheximide. Amyloglucosidase accumulated in the absence of glutamate was a high-molecular-mass protein that did not migrate in PAGE. Cellular regulation by the fungus of the secretion of amyloglucosidase is indicated.

Secretion of Cartilage Oligomeric Matrix Protein Is Affected by the Signal Peptide

Cartilage oligomeric matrix protein (COMP) is a secreted glycoprotein found in the extracellular matrices of skeletal tissues. Mutations associated with two human skeletal dysplasias, pseudoachondroplasia and multiple epiphyseal dysplasia, disturb COMP secretion leading to intracellular accumulation of mutant COMP, especially in chondrocytes. Here we show that the manifestation of this secretory defect is dramatically influenced by the signal peptide that targets COMP for secretion. The comparison of wild type and mutant COMP secretion directed by the COMP or BM40 signal peptide in HEK-293 cells and rat chondrosarcoma cells revealed that the BM40 signal peptide substantially enhances secretion of mutant COMP that accumulates in endoplasmic reticulum-like structures when targeted by its own signal peptide. Additionally, we demonstrate that mutant COMP forms mixed pentamers with wild type COMP. Our findings suggest that the secretory defect in pseudoachondroplasia and multiple epiphyseal dysplasia is not specific for chondrocytes, nor does it require interaction of mutant COMP with other matrix proteins prior to transport from the cell. They also imply a previously unappreciated role for the signal peptide in the regulation of protein secretion beyond targeting to the endoplasmic reticulum.

Genome‐wide survey of non‐essential genes required for slowed DNA synthesis‐induced filamentous growth in yeast

We recently discovered that slowed DNA synthesis induces filamentous differentiation in S. cerevisiae. We screened the BY yeast deletion strains and identified four classes of non-essential genes that are required for both slowed DNA-induced filamentous growth and classic forms of filamentous growth: (a) genes encoding regulators of the actin cytoskeleton and cell polarity, ABP1, CAP2 and HUF1 (=YOR300W), in addition to the previously known BNI1, BUD2, PEA2, SPA2 and TPM1; (b) genes that are likely involved in cell wall biosynthesis, ECM25, GAS1 and PRS3; (c) genes encoding possible regulators of protein secretion, SEC66, RPL21A and RPL34B; (d) genes encoding factors for normal mitochondrial function, IML1 and UGO1. These results showed that pseudohyphal formation involves not the only previously known regulation of the actin cytoskeleton/cell polarity but also regulation of cell wall synthesis, protein secretion and mitochondrial function. Identification of multiple classes of genes that are required for both slowed DNA synthesis-induced and classic forms of filamentous growth confirms that slowed DNA synthesis-induced filamentous growth is bone fide filamentous differentiation.

The role of calcium on protein secretion of the albumen gland in Helisoma duryi (Gastropoda)

Abstract. The albumen gland of the freshwater pulmonate snail Helisoma duryi produces and secretes the perivitelline fluid, which coats fertilized eggs and provides nutrients to the developing embryos. It is known that perivitelline fluid secretion is stimulated by dopamine through the activation of a dopamine D1‐like receptor, which in turn stimulates cAMP production leading to the secretion of perivitelline fluid. This paper examines the glandular release of perivitelline fluid and provides evidence for the role of Ca2+ in the regulated secretion of perivitelline fluid based on protein secretion experiments and inositol 1,4,5‐trisphosphate assays. Dopamine‐stimulated protein secretion by the albumen gland is reduced in Ca2+‐free medium or in the presence of plasma membrane Ca2+ channel blockers, although the Ca2+ channel subtype involved is unclear. In addition, dopamine‐stimulated protein secretion does not directly involve phospholipase C‐generated signaling pathways and Ca2+ release from intracellular stores. Sarcoplasmic/endoplasmic reticulum Ca2+‐ATPase inhibitors had little effect on protein secretion when applied alone; however, they potentiated dopamine‐stimulated protein secretion. Dantrolene, an inhibitor of ryanodine receptors, 8‐(N,N‐diethylamino)‐octyl‐3,4,5‐trimethoxybenzoate hydrochloride, a nonspecific inhibitor of intracellular Ca2+ channels, and 2‐aminoethyldiphenylborate, an inhibitor of inositol 1,4,5‐trisphosphate receptors, did not suppress protein secretion, suggesting Ca2+ release from internal stores does not directly regulate protein secretion. Thus, the influx of Ca2+ from the extracellular space appears to be the major pathway mediating protein secretion by the albumen gland. The results are discussed with respect to the role of Ca2+ in controlling exocytosis of proteins from the albumen gland secretory cells.

Monitoring gene expression changes in bovine oviduct epithelial cells during the oestrous cycle.

The oviduct epithelium undergoes marked morphological and functional changes during the oestrous cycle. To study these changes at the level of the transcriptome we did a systematic gene expression analysis of bovine oviduct epithelial cells at oestrus and dioestrus using a combination of subtracted cDNA libraries and cDNA array hybridisation. A total of 3072 cDNA clones of two subtracted libraries were analysed by array hybridisation with cDNA probes derived from six cyclic heifers, three of them slaughtered at oestrus and three at dioestrus. Sequencing of cDNAs showing significant differences in their expression levels revealed 77 different cDNAs. Thirty-seven were expressed at a higher level at oestrus, for the other 40 genes expression levels were higher at dioestrus. The identified genes represented a variety of functional classes. During oestrus especially genes involved in the regulation of protein secretion and protein modification, and mRNAs of secreted proteins, were up-regulated, whereas during dioestrus particularly transcripts of genes involved in transcription regulation showed a slight up-regulation. The concentrations of seven selected transcripts were quantified by real-time RT-PCR to validate the cDNA array hybridisation data. For all seven transcripts, RT-PCR results were in excellent correlation (r>0.92) with the results obtained by array hybridisation. Our study is the first to analyse changes in gene expression profiles of bovine oviduct epithelial cells during different stages of the oestrous cycle, providing a starting point for the clarification of the key transcriptome changes in these cells.

Transcriptomic correlates of changes in bovine oviduct epithelial cell function during the estrous cycle

The oviduct plays a central role in mammalian reproduction, providing the microenvironment for the final maturation of gametes, capacitation of spermatozoa, transport of gametes, fertilization, and early cleavage-stage embryonic development. To fulfill these specialized tasks, the oviduct epithelium undergoes marked morphological and functional changes during the estrous cycle. In the ampulla of the bovine oviduct, ciliated cells predominate at diestrus whereas both ciliated and secretory cells are found at estrus. To identify molecular correlates of these marked morphological changes, we performed mRNA expression profiling of ipsilateral oviduct epithelial cells at estrus and diestrus using a combination of subtracted cDNA libraries and cDNA array hybridization. Two subtracted libraries were produced to enrich cDNAs of up-regulated genes at estrus and at diestrus, respectively. 1,536 cDNA clones of each library were analyzed by array hybridization with cDNA probes derived from six Simmental heifers, three of them slaughtered at estrus and three at diestrus. In total, 84 different cDNAs were identified by sequencing, of which 42 matched bovine genes or their homologs with known functions and 42 matched genes without a known function. Half of the genes (n=42) were expressed at a higher level at estrus, for the other (n=42) expression levels were higher at diestrus. The regulated genes or their products represented a variety of functional classes, including immune-related proteins, proteins involved in protein modifications and secretion, signaling molecules and regulators of transcription. At estrus especially genes involved in regulation of protein secretion and protein modification were up-regulated whereas at diestrus transcripts of genes involved in transcription regulation and transport processes predominated. The set of genes identified will be important for the evaluation of in vitro culture of oviduct epithelial cells and for the understanding of interactions between gametes or early embryos and their maternal environment.

Identification of SNARE and cell trafficking regulatory proteins in the salivary glands of the lone star tick, Amblyomma americanum (L.)

Prostaglandin E 2 (PGE 2) stimulates secretion of tick salivary gland proteins via a phosphoinositide signaling pathway and mobilization of intracellular Ca 2+ ( Qian et al., 1998; Yuan et al., 2000). Highly conserved intracellular SNARE (soluble NSF attachment protein receptors) complex proteins are associated with the mechanism of protein secretion in vertebrate and invertebrate neuronal and non-neuronal cells. Proteins in the salivary glands of partially fed female lone star ticks cross-react individually with antibodies to synaptobrevin-2 (vesicle (v)-SNARE), syntaxin-1A, syntaxin-2 and SNAP-25 (target (t)-SNAREs), cytosolic α/β SNAP and NSF ( N-ethylmaleimide-sensitive fusion protein), Ca 2+ sensitive synaptotagmin, vesicle associated synaptophysin, and regulatory cell trafficking GTPases Rab3A and nSec1. V-SNARE and t-SNARE proteins form an SDS-resistant, boiling sensitive core complex in the salivary glands. Antibodies to SNARE complex proteins inhibit PGE 2-stimulated secretion of anticoagulant protein in permeabilized tick salivary glands. We conclude that SNARE and cell trafficking regulatory proteins are present and functioning in the process of PGE 2-stimulated Ca 2+ regulated protein secretion in tick salivary glands.

Presynaptic muscarinic receptor mechanisms and submandibular responses to stimulation of the parasympathetic innervation in bursts in rats

Submandibular secretory responses to electrical stimulation of the parasympathetic innervation at variable frequencies were measured in anaesthetized rats. Selective blockade by pirenzepine and by methoctramine occurred at doses (50 and of 300 nmol kg −1, i.v., respectively) that did not inhibit the responses to exogenous acetylcholine. In the presence of methoctramine, the nerve-evoked fluid responses were increased by 100% at 1 Hz independently of the total number of impulses (10–300), suggesting that M2 receptor activation inhibits transmitter release. The magnitude of the increase was inversely related to frequency of stimulation. The protein concentrations in the fluid responses were not significantly affected by methoctramine. Pirenzepine had an inhibitory effect on the fluid secretory responses, which was dependent of frequency, as well as of number of impulses, suggesting that M1 receptor activation facilitates transmitter release. At 10 Hz given intermittently (for 1 s at 10-s intervals), pirenzepine reduced the fluid response by 25%. The protein release was substantially and significantly reduced by pirenzepine independent of frequency but only during long periods of stimulation (300 impulses). It is concluded that muscarinic M1 receptor activation normally has a facilitatory effect on transmitter release, and that the facilitation occurs during short, intense stimulation. Muscarinic M1 receptors are, however, likely to regulate protein secretion by other mechanisms. Muscarinic M2 receptors, on the other hand, normally inhibit cholinergic transmission at low frequencies. Similar to findings in the alimentary tract of several species, stimulation in bursts at high frequencies is a more efficient stimulation pattern than continuous low frequency stimulation. This pattern of stimulation thus takes advantage of transient facilitation and avoids the inhibition at less intense neuronal activity.

Stress and secretory immunity

Publisher Summary This chapter focuses on the relationship between stress and saliva secretory immunity in humans. Salivary gland function is largely under autonomic control; the parasympathetic nerves mainly govern salivary fluid secretion, whereas the sympathetic nerves regulate protein secretion. The primary salivary centers in the brain stem receive inhibitory and excitatory inputs from neural structures in the forebrain and brain stem. As well as governing typical salivary functions, these structures are also involved in generating bodily changes associated with stress. It is, therefore, reasonable to assume that salivary changes during stress are an integral part of a centrally coordinated stress response that encompasses many other bodily functions. The autonomic receptors in the salivary glands can be divided into two main groups: the classic autonomic receptor types, which respond to either noradrenaline or acetylcholine, and the nonadrenergic–noncholinergic (NANC) receptors that respond to other autonomic messenger substances, such as peptides, nitric oxide, and purines. Differential activation of these receptor types can cause additive, synergistic, or antagonistic intracellular responses, ultimately resulting in a protein release that is capable of being differentially regulated among glands.

Cloning, partial sequencing and expression of rabbit lacrimal gland beta-hexosaminidase.

Several proteins actively secreted by the lacrimal gland have now been identified, including lactoferrin, lysozyme, tear specific pre-albumin and secretory IgA in human tear fluid.1 In addition, some lysosomal hydrolases have also been detected in tears and lacrimal gland fluid.2,3 In our ongoing studies of rabbit lacrimal gland acinar cells in primary culture, we noted that β-hexosaminidase catalytic activity, which we had expected to serve as a marker for lysosomes, diverged from another potential lysosomal marker, cathepsin B. Both enzymes were distributed primarily to the soluble phase, consistent with release from disrupted vesicular and cisternal structures. However, the membrane-associated component of hexosaminidase activity, when compared to cathepsin B, showed a relatively greater steady-state content in membranes of the endoplasmic reticulum and Golgi complex, suggestive of a product undergoing significant bulk flow through the synthesis and assembly pathway.4,5 Since β-hexosaminidase has been reported to be present in human tears,6,7 we examined the possibility that β-hexosaminidase might serve as a marker of regulated protein secretion by rabbit lacrimal gland acinar cells (Gierow et al., submitted).8,9 KeywordsOcular SurfaceLacrimal GlandKeratoconjunctivitis SiccaConjunctival Epithelial CellOcular Surface InflammationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Rapid accumulation of phosphatidylinositol 4,5-bisphosphate and inositol 1,4,5-trisphosphate correlates with calcium mobilization in salt-stressed arabidopsis.

The phosphoinositide phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] is a key signaling molecule in animal cells. It can be hydrolyzed to release 1,2-diacyglycerol and inositol 1,4,5-trisphosphate (IP(3)), which in animal cells lead to protein kinase C activation and cellular calcium mobilization, respectively. In addition to its critical roles in constitutive and regulated secretion of proteins, PtdIns(4,5)P(2) binds to proteins that modify cytoskeletal architecture and phospholipid constituents. Herein, we report that Arabidopsis plants grown in liquid media rapidly increase PtdIns(4,5)P(2) synthesis in response to treatment with sodium chloride, potassium chloride, and sorbitol. These results demonstrate that when challenged with salinity and osmotic stress, terrestrial plants respond differently than algae, yeasts, and animal cells that accumulate different species of phosphoinositides. We also show data demonstrating that whole-plant IP(3) levels increase significantly within 1 min of stress initiation, and that IP(3) levels continue to increase for more than 30 min during stress application. Furthermore, using the calcium indicators Fura-2 and Fluo-3 we show that root intracellular calcium concentrations increase in response to stress treatments. Taken together, these results suggest that in response to salt and osmotic stress, Arabidopsis uses a signaling pathway in which a small but significant portion of PtdIns(4,5)P(2) is hydrolyzed to IP(3). The accumulation of IP(3) occurs during a time frame similar to that observed for stress-induced calcium mobilization. These data also suggest that the majority of the PtdIns(4,5)P(2) synthesized in response to salt and osmotic stress may be utilized for cellular signaling events distinct from the canonical IP(3) signaling pathway.

Cholesterol is required for the formation of regulated and constitutive secretory vesicles from the trans-Golgi network.

We studied the role of cholesterol in regulated protein secretion in neuroendocrine cells by manipulating the cholesterol content of AtT-20 cells. Depletion of cellular cholesterol levels caused a reversible block of immature secretory granule biogenesis at the level of the trans-Golgi-network, whereas increased cholesterol levels promoted immature secretory granule formation. Cholesterol depletion also blocked the formation of constitutive secretory vesicles, but did not inhibit the transport between the endoplasmic reticulum and the Golgi complex. Our results indicate that the assembly of cholesterol-based lipid microdomains is required for the biogenesis of both regulated and constitutive secretory vesicles from the trans-Golgi-network in neuroendocrine cells.

Crossing the finish line of development: regulated secretion of Dictyostelium proteins

The genesis of the spore coat of Dictyostelium represents an exquisite example of developmentally regulated protein secretion. The proteins that are destined to be assembled into the extracellular matrix of the spore coat are stored in unique prespore vesicles that are triggered to secrete their contents at terminal differentiation. The regulation of this process is being revealed by the identification of the individual proteins in these vesicles.

Popping a pill to regulate therapeutic protein delivery

The enormous potential for secreted protein therapeutics delivered using gene therapy approaches cannot be completely fulfilled until the kinetics of protein release can be controlled. Under natural conditions, some proteins, such as erythropoietin, are released steadily: others, such as insulin, need to be released in short bursts in response to stimuli. Following encouraging progress in the development of their ARGENT technology for slow, controlled protein delivery over a period of days 1 Stewart A. Orally activated protein therapy heads towards clinical trials. Mol. Med. Today. 1998; 4: 95 Google Scholar , ARIAD Gene Therapeutics (Cambridge, MA, USA) has now published proof-of-concept of a new technology that enables therapeutic protein delivery over a timescale of a few hours 2 Rivera V.M. et al. Regulation of protein secretion through controlled aggregation in the endoplasmic reticulum. Science. 2000; 287: 826-830 Crossref PubMed Scopus (272) Google Scholar .

Identification and cellular localisation of voltage-operated calcium channels in immature rat testis

Sertoli cells regulate the spermatogenic process mainly through the secretion of a complex fluid into the lumen of the seminiferous tubules behind the blood–testis barrier, containing many of the essential proteins necessary for maintenance and maturation of male germ cells. Thus, the study of Sertoli cell secretory processes is strictly correlated with the understanding of the regulatory mechanisms of spermatogenesis. In this work the authors have explored the voltage-sensitive calcium channel variety in the immature rat testis, their localisation and distribution within the seminiferous epithelium and peritubular and interstitial tissues as well as the possible role in the control of Sertoli cell secretion. The results reported in this paper, obtained by in situ hybridisation, immunohistology of rat testicular sections and Western blot analysis of Sertoli cell plasma membranes, show that mammalian Sertoli cells express mRNA encoding for several voltage-operated calcium channel subunits and express such proteins on their surface. Experiments performed on Sertoli cell monolayers cultured in the presence of specific toxins indicate that both N and P/Q-type Ca 2+ channels are involved in the regulation of protein secretion.

Regulation of protein secretion through controlled aggregation in the endoplasmic reticulum.

A system for direct pharmacologic control of protein secretion was developed to allow rapid and pulsatile delivery of therapeutic proteins. A protein was engineered so that it accumulated as aggregates in the endoplasmic reticulum. Secretion was then stimulated by a synthetic small-molecule drug that induces protein disaggregation. Rapid and transient secretion of growth hormone and insulin was achieved in vitro and in vivo. A regulated pulse of insulin secretion resulted in a transient correction of serum glucose concentrations in a mouse model of hyperglycemia. This approach may make gene therapy a viable method for delivery of polypeptides that require rapid and regulated delivery.

The Prespore Vesicles of Dictyostelium discoideum: PURIFICATION, CHARACTERIZATION, AND DEVELOPMENTAL REGULATION

The coordinate fusion of the prespore vesicles (PSVs) with the plasma membrane at the terminal stage of spore differentiation in Dictyostelium discoideum is an important example of developmentally regulated protein secretion. However, little is known about the composition of the vesicles, the molecular signals regulating secretion, or the mechanics of the membrane fusion. Taking a biochemical approach, we purified PSVs from different developmental stages. These preparations are highly enriched for their specific cargo of spore coat proteins while devoid of markers for other cellular compartments. Electron microscopic observations show that the PSV preparations are homogenous, with the soluble spore coat protein PsB/SP85 distributed throughout the lumen and the acid mucopolysaccharide localized in the central core. During development the PSVs increase in size and density concomitant with an increase in their protein cargo. The PSVs contain approximately 80 proteins, and we have identified a PSV-specific GTP-binding protein that may be involved in regulating vesicle fusion. The PSVs are not clathrin-coated and do not contain the SpiA spore coat protein. The PSV preparations are ideal for a global proteome analysis to identify proteins involved in signal reception, vesicle movement, docking, and fusion in this developmentally regulated organelle.

Multimerin Processing by Cells With and Without Pathways for Regulated Protein Secretion

Multimerin is a massive, soluble, homomultimeric, factor V-binding protein found in platelet alpha-granules and in vascular endothelium. Unlike platelets, endothelial cells contain multimerin within granules that lack the secretory granule membrane protein P-selectin, and in culture, they constitutively secrete most of their synthesized multimerin. To further evaluate multimerin's posttranslational processing and storage, we expressed human endothelial cell prepromultimerin in a variety of cell lines, with and without pathways for regulated secretion. The recombinant multimerin produced by these different cells showed variations in its glycosylation, proteolytic processing, and multimer profile, and human embryonic kidney 293 cells recapitulated multimerin's normal processing for constitutive secretion by human endothelial cells. When multimerin was expressed in a neuroendocrine cell line capable of regulated protein secretion, it was efficiently targeted for regulated secretion. However, the multimerin stored in these cells was proteolyzed more extensively than normally occurs in platelets, suggesting that endoproteases similar to those expressed by megakaryocytes are required to produce platelet-type multimerin. The impact of the tissue-specific differences in multimerin's posttranslational processing on its functions is not yet known. Multimerin's sorting and targeting for regulated secretion may be important for its functions and its association with factor V in secretion granules.

Multimerin Processing by Cells With and Without Pathways for Regulated Protein Secretion

AbstractMultimerin is a massive, soluble, homomultimeric, factor V-binding protein found in platelet α-granules and in vascular endothelium. Unlike platelets, endothelial cells contain multimerin within granules that lack the secretory granule membrane protein P-selectin, and in culture, they constitutively secrete most of their synthesized multimerin. To further evaluate multimerin's posttranslational processing and storage, we expressed human endothelial cell prepromultimerin in a variety of cell lines, with and without pathways for regulated secretion. The recombinant multimerin produced by these different cells showed variations in its glycosylation, proteolytic processing, and multimer profile, and human embryonic kidney 293 cells recapitulated multimerin's normal processing for constitutive secretion by human endothelial cells. When multimerin was expressed in a neuroendocrine cell line capable of regulated protein secretion, it was efficiently targeted for regulated secretion. However, the multimerin stored in these cells was proteolyzed more extensively than normally occurs in platelets, suggesting that endoproteases similar to those expressed by megakaryocytes are required to produce platelet-type multimerin. The impact of the tissue-specific differences in multimerin's posttranslational processing on its functions is not yet known. Multimerin's sorting and targeting for regulated secretion may be important for its functions and its association with factor V in secretion granules.

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