Exploration of Pericyte-Derived Factors Implicated in Lung Cancer Brain Metastasis Protection: A Pilot Messenger RNA Sequencing Using the Blood-Brain Barrier In Vitro Model.

Metastatic brain tumors have poor prognoses and pose unmet clinical problems for the patients. The blood-brain barrier (BBB) implication is supposed to play a major role in brain metastasis. However, the role of pericytes remains to be elucidated in the brain metastasis. This pilot study described the expression profile of interactions between pericytes, endothelial cells, and cancer cells. We applied an in vitro BBB model with rat primary cultured BBB-related cells (endothelial cells and pericytes), and performed the gene expression analyses of pericytes under the lung cancer cells coculture conditions. Pericytes demonstrated inhibition of the cancer cell proliferation significantly (p < 0.05). RNA was extracted from the pericytes, complementary DNA library was prepared, and RNA-seq was performed. The sequence read data were analyzed on the Management and Analysis System for Enormous Reads and Tag Count Comparison-Graphical User Interface platforms. No statistically or biologically significant differentially expressed genes (DEGs) were detected in the explanatory analyses. Lot-specific DEG detection demonstrated significant decreases in the expression of two genes (Wwtr1 and Acin1), and enrichment analyses using Metascape software revealed the inhibition of apoptotic processes in fibroblasts. Our results suggest that the expression profiles of brain pericytes are partially implicated in the prevention of lung cancer metastasis to the brain. Pericytes exerted an anti-metastatic effect in the BBB model, and their neurohumoral factors remain to be elucidated.

Different aggregation states of a nuclear localization signal-tagged 25-kDa C-terminal fragment of TAR RNA/DNA-binding protein 43 kDa.

The mechanism and cause of motor neuronal cell death in amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disorder, are unknown; gain of function of oligomers and aggregation of misfolded proteins, including carboxyl-terminal fragments (CTFs) of TAR RNA/DNA-binding protein 43 kDa (TDP-43), have been proposed as important causative factors in the onset of ALS. We recently reported that a nuclear localization signal (NLS)-tagged 25-kDa CTF of TDP-43 (TDP25) could decrease the cell-death proportion compared with that promoted by TDP25. Here, we show oligomeric states of NLS-TDP25 and its detailed localization property using super-resolution fluorescence microscopy, FRET, fluorescence recovery after photobleaching, and fluorescence correlation spectroscopy analysis. NLS-TDP25 efficiently formed a nucleolar cap structure via RNA binding in the presence of actinomycin D, but TDP25 did not. Although cytoplasmic inclusion bodies including TDP25 had a disordered and immobile structure, NLS-TDP25 in the nucleolus was ordered and dynamic. In the diffuse state, TDP25 formed fewer oligomers and interacted with the molecular chaperone, HSP70; however, NLS-TDP25 formed oligomers. These results suggested that NLS-tagged TDP25 can change its structure to use ordered oligomeric but nontoxic state. Moreover, the structure of ordered oligomers as well as nuclear sequestration may be important in mediating cytotoxicity in ALS pathology.

Prefoldin protects neuronal cells from polyglutamine toxicity by preventing aggregation formation.

Huntington disease is caused by cell death after the expansion of polyglutamine (polyQ) tracts longer than ∼40 repeats encoded by exon 1 of the huntingtin (HTT) gene. Prefoldin is a molecular chaperone composed of six subunits, PFD1-6, and prevents misfolding of newly synthesized nascent polypeptides. In this study, we found that knockdown of PFD2 and PFD5 disrupted prefoldin formation in HTT-expressing cells, resulting in accumulation of aggregates of a pathogenic form of HTT and in induction of cell death. Dead cells, however, did not contain inclusions of HTT, and analysis by a fluorescence correlation spectroscopy indicated that knockdown of PFD2 and PFD5 also increased the size of soluble oligomers of pathogenic HTT in cells. In vitro single molecule observation demonstrated that prefoldin suppressed HTT aggregation at the small oligomer (dimer to tetramer) stage. These results indicate that prefoldin inhibits elongation of large oligomers of pathogenic Htt, thereby inhibiting subsequent inclusion formation, and suggest that soluble oligomers of polyQ-expanded HTT are more toxic than are inclusion to cells.

Determination of dissociation constant of the NFκB p50/p65 heterodimer using fluorescence cross-correlation spectroscopy in the living cell.

Two-laser-beam fluorescence cross-correlation spectroscopy (FCCS) is promising technique that provides quantitative information about the interactions of biomolecules. The p50/p65 heterodimer is the most abundant and well understood of the NFκB dimers in most cells. However, the quantitative value of affinity, namely the K(d), for the heterodimer in living cells is not known yet. To quantify the heterodimerization of the IPT domain of p50/p65 in the living cell, we used two-laser-beam FCCS. The K(d) values of mCherry2- and EGFP-fused p50 and p65 were determined to be 0.46 µM in the cytoplasm and 1.06 µM in the nucleus of the living cell. These results suggest the different binding affinities of the p50/p65 heterodimer in the cytoplasm and nucleus of the living cell and different complex formation in each region.

A modified FCCS procedure applied to Ly49A-MHC class I cis-interaction studies in cell membranes.

The activity of natural killer (NK) cells is regulated by a fine-tuned balance between activating and inhibitory receptors. Dual-color fluorescence cross-correlation spectroscopy (FCCS) was used to directly demonstrate a so-called cis-interaction between a member of the inhibitory NK cell receptor family Ly49 (Ly49A), and its ligand, the major histocompatibility complex (MHC) class I, within the plasma membrane of the same cell. By a refined FCCS model, calibrated by positive and negative control experiments on cells from the same lymphoid cell line, concentrations and diffusion coefficients of free and interacting proteins could be determined on a collection of cells. Using the intrinsic intercellular variation of their expression levels for titration, it was found that the fraction of Ly49A receptors bound in cis increase with increasing amounts of MHC class I ligand. This increase shows a tendency to be more abrupt than for a diffusion limited - three dimensional bimolecular reaction, which most likely reflects the two-dimensional confinement of the reaction. For the Ly49A- MHC class I interaction it indicates that within a critical concentration range the local concentration level of MHC class I can provide a distinct regulation mechanism of the NK cell activity.

Dysregulation of erythropoiesis and altered erythroblastic NMDA receptor-mediated calcium influx in Lrfn2-deficient mice.

LRFN2 encodes a synaptic adhesion-like molecule that physically interacts with N-methyl-D-aspartate (NMDA) receptor 1 and its scaffold proteins. Previous studies in humans and mice have demonstrated its genetic association with neurodevelopmental disorders such as learning deficiency and autism. In this study, we showed that Lrfn2-deficient (KO) mice exhibit abnormalities of erythropoietic systems due to altered NMDA receptor function. In mature Lrfn2 KO male mice, peripheral blood tests showed multilineage abnormalities, including normocytic erythrocythemia, and reduced platelet volume. Colony forming unit assay using bone marrow cells revealed decreases in the counts of erythrocyte progenitors (CFU-E) as well as granulocytes and monocyte progenitors (CFU-GM). Whole bone marrow cell staining showed that serum erythropoietin (EPO) level was decreased and EPO receptor-like immunoreactivity was increased. Flow cytometry analysis of bone marrow cells revealed increased early erythroblast count and increased transferrin receptor expression in late erythroblasts. Further, we found that late erythroblasts in Lrfn2 KO exhibited defective NMDA receptor-mediated calcium influx, which was inhibited by the NMDA receptor antagonist MK801. These results indicate that Lrfn2 has biphasic roles in hematopoiesis and is associated with the functional integrity of NMDA receptors in hematopoietic cells. Furthermore, taken together with previous studies that showed the involvement of NMDA receptors in hematopoiesis, the results of this study indicate that Lrfn2 may regulate erythropoiesis through its regulatory activity on NMDA receptors.

Increased In Vitro Intercellular Barrier Function of Lung Epithelial Cells Using Adipose-Derived Mesenchymal Stem/Stromal Cells.

With the emergence of coronavirus disease-2019, researchers have gained interest in the therapeutic efficacy of mesenchymal stem/stromal cells (MSCs) in acute respiratory distress syndrome; however, the mechanisms of the therapeutic effects of MSCs are unclear. We have previously reported that adipose-derived MSCs (AD-MSCs) strengthen the barrier function of the pulmonary vessels in scaffold-based bioengineered rat lungs. In this study, we evaluated whether AD-MSCs could enhance the intercellular barrier function of lung epithelial cells in vitro using a transwell coculture system. Transepithelial electrical resistance (TEER) measurements revealed that the peak TEER value was significantly higher in the AD-MSC coculture group than in the AD-MSC non-coculture group. Similarly, the permeability coefficient was significantly decreased in the AD-MSC coculture group compared to that in the AD-MSC non-coculture group. Immunostaining of insert membranes showed that zonula occuldens-1 expression was significantly high at cell junctions in the AD-MSC coculture group. Moreover, cell junction-related gene profiling showed that the expression of some claudin genes, including claudin-4, was upregulated in the AD-MSC coculture group. Taken together, these results showed that AD-MSCs enhanced the barrier function between lung epithelial cells, suggesting that both direct adhesion and indirect paracrine effects strengthened the barrier function of lung alveolar epithelium in vitro.

Levofloxacin or gonadotropin releasing hormone agonist treatment decreases intrauterine microbial colonization in human endometriosis.

OBJECTIVE: We examined the hypothesis that antibiotic treatment with or without gonadotropin releasing hormone agonist (GnRHa) may decrease intrauterine infection with consequent decrease in tissue inflammation, cell proliferation and angiogenesis in human endometriosis. STUDY DESIGN: This is a prospective non-randomized observational study. Endometrial/endometriotic samples were collected during surgery from 53 women with endometriosis and 47 control women who were treated with levofloxacin (LVFX, 500 mg, once per os) or GnRHa (1.88 mg/IM for 3 months) before surgery. Endometrial samples were analyzed by broad-range polymerase-chain reaction (PCR) amplification of bacteria targeting V5-V6 region of 16S rRNA gene. Immunohistochemical analysis was performed using antibodies against CD138 (Syndecan-1, a marker of plasma cells), CD68 (marker of macrophages), Ki-67 (cell proliferation marker), and CD31 (vascular cells marker). RESULTS: 16S rDNA metagenome assay indicated that treatment with either of LVFX or GnRHa + LVFX significantly decreased some components of major bacterial genera comparing to untreated group. In women with endometriosis, treatment with either of LVFX or GnRHa + LVFX significantly decreased Gardnerella, Prevotella, Acidibactor, Atopobium, Megasphaera, and Bradyrhizobium (p < 0.05 for each) comparing to untreated group. Cochran-Mantel-Haenszel test indicated that occurrence rate of chronic endometritis was significantly decreased after GnRHa + LVFX treatment comparing to GnRHa treatment group (p = 0.041). These findings were coincided with significantly decreased CD68-stained macrophage infiltration, Ki-67- stained cell proliferation and CD31-stained micro-vessel density in endometria and endometriotic lesions with histology proven improvement in the morphological appearance of ovarian endometrioma. CONCLUSIONS: These findings suggest that clinical administration of a broad-spectrum antibiotic with or without GnRHa may be effective in improving uterine infection with decrease of tissue inflammation, cell proliferation, and angiogenesis in human endometriosis.

STPR, a 23-amino acid tandem repeat domain, found in the human function-unknown protein ZNF821.

The STPR motif is composed of 23-amino acid repeats aligned contiguously. STPR was originally reported as the DNA-binding domain of the silkworm protein FMBP-1. ZNF821, the human protein that contains the STPR domain, is a zinc finger protein of unknown function. In this study, we prepared peptides of silkworm FMBP-1 STPR (sSTPR) and human ZNF821 STPR (hSTPR) and compared their DNA binding behaviors. This revealed that hSTPR, like sSTPR, is a double-stranded DNA-binding domain. Sequence-independent DNA binding affinities and α-helix-rich DNA-bound structures were comparable between the two STPRs, although the specific DNA sequence of hSTPR is still unclear. In addition, a subcellular expression experiment showed that the hSTPR domain is responsible for the nuclear localization of ZNF821. ZNF821 showed a much slower diffusion rate in the nucleus, suggesting the possibility of interaction with chromosomal DNA. STPR sequences are found in many proteins from vertebrates, insects, and nematodes. Some of the consensus amino acid residues would be responsible for DNA binding and concomitant increases in α-helix structure content.

Fluorescence cross-correlation spectroscopy of plant proteins.

Fluorescence cross-correlation spectroscopy (FCCS) is a technique that physically evaluates the molecular interaction between two fluorophore-tagged molecules such as proteins and oligonucleotides in a quantitative manner. Because it simply makes use of the coincidental movement of two molecules, it could avoid the complexity that sometimes occurs in other fluorescent techniques such as fluorescence resonance energy transfer. The present chapter describes procedures for FCCS of plant proteins expressed and measured in HeLa cells. Determination in plant cells is also mentioned briefly.

Differential Levels of Regulatory T Cells and T-Helper-17 Cells in Women With Early and Advanced Endometriosis.

CONTEXT: Regulatory T (Treg) cells and T-helper-17 (Th17) cells may be involved in endometriosis. Information on the pattern of change in the percentages of Treg and Th17 cells in the peripheral blood (PB) and peritoneal fluid (PF) of women with early and advanced endometriosis is unclear. OBJECTIVE: To investigate the pattern of change in the percentages of Treg and Th17 cells in the PB and PF of women with early and advanced endometriosis. METHODS: We recruited 31 women with laparoscopically and histologically confirmed, revised American Society of Reproductive Medicine stage I-II endometriosis, 39 women with stage III-IV endometriosis, and 36 control subjects without visible endometriosis. PB and PF samples were collected and T-cell subpopulations analyzed by flow cytometry using specific monoclonal antibodies recognizing CD4+, CD25+, FOXP3+, and IL-17A+ markers. PF concentrations of TGF-ß and IL-17 were measured by ELISA. RESULTS: The percentages of CD25+FOXP3+ Treg cells within the CD4+ T-cell population were significantly higher in the PF of women with advanced endometriosis than in either early endometriosis or in control subjects (P < 0.05 for both). A persistently lower percentage of CD4+IL-17A+ Th17 cells was found in both PB and PF of women with early and advanced endometriosis. Compared with IL-17 levels, PF levels of TGF-ß were significantly higher in women with endometriosis (P = 0.01). CONCLUSION: Our findings reconfirmed the current speculation that endometriosis is related to alteration of Treg and Th17 cells in the pelvis causing survival and implantation of ectopic endometrial lesions.

Whole genome characterisation of G11P[25] and G9P[19] rotavirus A strains from adult patients with diarrhoea in Nepal.

Rotavirus A (RVA) causes acute diarrhoea in children and less frequently in adults. However, the knowledge about the genotype distribution of RVA strains circulating in adults is limited particularly in developing countries. This study aimed to characterise the RVA strains detected from adult patients with diarrhoea in Nepal. A total of 47 RVA positive stool samples from adult patients with diarrhoea in Kathmandu, Nepal during 2007-2008 were examined for the G and P genotypes by sequencing. Nearly half (49%) of the samples were genotyped as G9P[8] (n = 23), G1P[8], G2P[4] (n = 5 each), G12P[8] (n = 4), G12P[6] (n = 3), G1P[6] (n = 2), G3P[8] and G9P[6] (n = 1 each). Interestingly, two G11P[25] and one G9P[19] strains detected were further subjected to Illumina MiSeq next generation sequencing to determine their whole genome sequences. The genotype constellations of RVA/Human-wt/NPL/TK2615/2008/G11P[25] and RVA/Human-wt/NPL/TK2620/2008/G11P[25] were I12-R1-C1-M1-A1-N1-T1-E1-H1, whereas that of RVA/Human-wt/NPL/TK1797/2007/G9P[19] was I5-R1-C1-M1-A8-N1-T1-E1-H1. The 11 genes of TK2615 and TK2620 were virtually identical, and they were either porcine-like or unique except the VP2 and NSP1 genes which were of human RVA origin. The two G11P[25] strains were also very similar to KTM368, another G11P[25] isolated from a child in Nepal in 2004. On the other hand, no gene of TK1797 was likely to be of human RVA origin. The observation that porcine-like RVAs were detected from adult patients justifies further studies to explore the role of adults in the interspecies transmission of animal RVA to humans.

Molecular detection of intrauterine microbial colonization in women with endometriosis.

OBJECTIVE: Increased intrauterine microbial colonization by bacteria culture method and occurrence of endometritis have been reported in women with endometriosis. Here we investigated microbial colonization in intrauterine environment and cystic fluid of women with and without endometriosis by molecular approach. STUDY DESIGN: This is a case-controlled biological study with a total of 32 women each with and without endometriosis. Among them, 16 each in these two groups of women received treatment with gonadotropin-releasing hormone agonist (GnRHa). Pattern of microbial colonization in endometrial swabs and endometrioma/non-endometrioma cystic fluid was examined using broad-range polymerase-chain reaction (PCR) amplification of bacteria targeting 16S rRNA gene (rDNA). After quantification of index PCR product, 16S rDNA metagenome sequence analysis was done by Illumina Miseq system. RESULTS: A wide proportion (0.01-97.8%) of multiple bacteria was detected in both endometrial swabs and cystic fluid collected from women with and without endometriosis. 16S metagenome assay indicated that proportion of Lactobacillacae was significantly decreased (p<0.01) and of Streptococcaceae, Staphylococaceae, Enterobacteriaceae was significantly increased (p<0.05 for each) in GnRHa-treated women with endometriosis than in GnRHa-untreated women. While bacteria culture method failed to detect a single colony, 16S metagenome assay could detect significantly higher percentage of Streptococcaceae (p<0.01) and Staphylococaceae (p<0.05) in the cystic fluid derived from women with ovarian endometrioma comparing to that in cystic fluid collected from non-endometrioma cysts. CONCLUSION: These findings indicate the occurrence of sub-clinical infection in intrauterine environment and in the cystic fluid of ovarian endometrioma. Additional side effect of GnRHa treatment in promoting silent intrauterine and/or ovarian infection should be considered.

In vivo fluorescence correlation spectroscopy analyses of FMBP-1, a silkworm transcription factor.

Fibroin modulator-binding protein 1 (FMBP-1) is a silkworm transcription factor that has a unique DNA-binding domain called the one score and three amino acid peptide repeat (STPR). Here we used fluorescence correlation spectroscopy (FCS) to analyze the diffusion properties of an enhanced green fluorescent protein-tagged FMBP-1 protein (EGFP-FMBP-1) expressed in posterior silk gland (PSG) cells of Bombyx mori at the same developmental stage as natural FMBP-1 expression. EGFP-FMBP-1 clearly localized to cell nuclei. From the FCS analyses, we identified an immobile DNA-bound component and three discernible diffusion components. We also used FCS to observe the movements of wild-type and mutant EGFP-FMBP-1 proteins in HeLa cells, a simpler experimental system. Based on previous in vitro observation, we also introduced a single amino acid substitution in order to suppress stable FMBP-1-DNA binding; specifically, we replaced the ninth Arg in the third repeat within the STPR domain with Ala. This mutation completely disrupted the slowest diffusion component as well as the immobile component. The diffusion properties of other FMBP-1 mutants (e.g. mutants with N-terminal or C-terminal truncations) were also analyzed. Based on our observations, we suggest that the four identifiable movements might correspond to four distinct FMBP-1 states: (a) diffusion of free protein, (b) and

Fluorescence Cross-correlation Spectroscopy (FCCS) to Observe Dimerization of Transcription Factors in Living Cells.

Fluorescence cross-correlation spectroscopy (FCCS) is an established spectroscopic method to observe the interaction between the different fluorescent molecules. Using FCCS, researchers can assess the interaction of target molecules in the aqueous condition, and can apply the technique in cultured cells. Here, we describe the method of FCCS to demonstrate direct observation of dimerization between transcription factors in a living cell.

Current research on protein-protein interactions among auxin-signaling factors in regulation of plant growth and development.

In many signaling pathways, various factors have been isolated by molecular genetic studies and wholegenome analysis. Understanding the interactions among these factors is crucial to understanding the signaling process as a whole. Recent studies on auxin signaling in the regulation of plant growth and development have discovered primary factors interacting with each other, and elucidated a very simple pathway modulating gene expression in response to auxin. However, these studies of auxin-signaling led to the question of how such a simple pathway generates multiple types of regulation in various processes of different cells throughout the life of a plant. Here, we provide an overview of recent progress in auxin biology focusing on protein-protein interactions in the signal transduction pathway and discuss various possibilities and approaches to resolve the issue.

Direct observation of dimerization between different CREB1 isoforms in a living cell.

Cyclic AMP-responsive element binding protein 1 (CREB1) plays multiple functions as a transcription factor in gene regulation. CREB1 proteins are also known to be expressed in several spliced isoforms that act as transcriptional activators or repressors. The activator isoforms, possessing the functional domains for kinase induction and for interaction with other transcriptional regulators, act as transcriptional activators. On the other hand, some isoforms, lacking those functional domains, are reported to be repressors that make heterodimers with activator isoforms. The complex and ingenious function for CREB1 arises in part from the variation in their spliced isoforms, which allows them to interact with each other. To date, however, the dimerization between the activator and repressor isoforms has not yet been proved directly in living cells. In this study, we applied fluorescence cross-correlation spectroscopy (FCCS) to demonstrate direct observation of dimerization between CREB1 activator and repressor. The FCCS is a well established spectroscopic method to determine the interaction between the different fluorescent molecules in the aqueous condition. Using differently labeled CREB1 isoforms, we successfully observed the interaction of CREB1 activator and repressor via dimerization in the nuclei of cultured cells. As a result, we confirmed the formation of heterodimer between CREB1 activator and repressor isoforms in living cells.

What Makes each Aux/IAA Gene Unique in its Gene Family, Expression Pattern or Properties of the Gene Product?

In the auxin signal transduction, two protein families, Aux/IAAs and auxin response factors, play a crucial role just downstream of auxin F-box receptors. Distinct and overlapping phenotypes of the dominant Aux/IAA mutants suggest some functional differentiation of the Aux/IAA genes in auxin signaling. Taking advantage of unique phenotypes of the msg2/iaa19 mutants, we carried out promoter-exchange experiments, where cDNA of the msg2, axr2/iaa7 or slr/iaa14 gene was driven by the MSG2 or AXR2 promoter. The cDNAs were translationally fused to the green fluorescent protein gene to measure levels of expressed protein. Results showed that many abnormal phenotypes of the dominant Aux/IAA mutants were governed by their promoter activity, but some were dependent on their gene products. The latter result highlights the possible importance of Aux/IAA protein level controled by auxin F-box receptors.

Specificity and similarity of functions of the Aux/IAA genes in auxin signaling of Arabidopsis revealed by promoter-exchange experiments among MSG2/IAA19, AXR2/IAA7, and SLR/IAA14.

As indicated by various and some overlapped phenotypes of the dominant mutants, the Aux/IAA genes of Arabidopsis (Arabidopsis thaliana) concomitantly exhibit a functional similarity and differentiation. To evaluate the contributions of their expression patterns determined by promoter activity and molecular properties of their gene products to Aux/IAA function, we examined phenotypes of transgenic plants expressing the green fluorescent protein (GFP)-tagged msg2-1/iaa19, axr2-1/iaa7, or slr-1/iaa14 cDNA by the MSG2 or AXR2 promoter. When driven by the MSG2 promoter (pMSG2), each GFP-tagged cDNA caused the msg2-1 phenotype, that is, the wild-type stature in the mature-plant stage, long and straight hypocotyls in the dark, reduced lateral root formation, relatively mild agravitropic traits in hypocotyls, and a normal gravitropic response in roots. However, development of one or two cotyledonary primordia was often arrested in embryogenesis of the pMSG2::axr2-1::GFP and pMSG2::slr-1::GFP plants, resulting in monocotyledonary or no cotyledonary seedlings. Such defects in embryogenesis were never seen in pMSG2::msg2-1::GFP or the msg2-1, axr2-1, or slr-1 mutant. The MSG2 promoter-GUS staining showed that expression of MSG2 started specifically in cotyledonary primordia of the triangular-stage embryos. When driven by the AXR2 promoter (pAXR2), each GFP-tagged mutant cDNA caused, in principle, aberrant aboveground phenotypes of the corresponding dominant mutant. However, either the axr2-1::GFP or slr-1::GFP cDNA brought about dwarf, agravitropic stems almost identical to those of axr2-1, and the pAXR2::msg2-1::GFP and pAXR2::slr-1::GFP hypocotyls exhibited complete loss of gravitropism as did axr2-1. These results showed functional differences among the msg2-1, axr2-1, and slr-1 proteins, though some phenotypes were determined by the promoter activity.

Fluorescence cross-correlation analyses of the molecular interaction between an Aux/IAA protein, MSG2/IAA19, and protein-protein interaction domains of auxin response factors of arabidopsis expressed in HeLa cells.

Since auxin may elicit numerous developmental responses by the use of a combination of auxin response factors (ARFs) and their Aux/IAA repressors, it is important to determine the interaction between the two protein families in a quantitative manner. We transiently expressed the C-terminal protein-protein interaction domains (CTDs) of Arabidopsis ARFs, MP/ARF5 and NPH4/ARF7, and MSG2/IAA19, fused to fluorescent proteins in HeLa cells, and determined their molecular interactions with fluorescence cross-correlation spectroscopy (FCCS). Almost complete association was found between MSG2 and MP-CTD and between MSG2 and NPH4-CTD. Approximately 20% association was found for MSG2 homodimers, NPH4-CTD homodimers and MP-CTD/NPH4-CTD heterodimers. Homotypic binding of MP-CTD may be weaker than that of MSG2. MSG2 was localized in cytoplasmic compartments in HeLa cells, whereas it was localized in the nuclei in plant cells. The fact that the heterotypic interaction between MSG2 and ARF-CTDs is stronger than each of the homotypic interactions appears to be the molecular basis for tight control of the transcriptional activity of ARFs by auxin. These results also show that FCCS is useful to examine protein-protein interactions especially for transcriptional regulators.