Statistical analysis of the autocorrelation function in fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) is a powerful method to measure concentration, mobility, and stoichiometry in solution and in living cells, but quantitative analysis of FCS data remains challenging due to the correlated noise in the autocorrelation function (ACF) of FCS. We demonstrate here that least-squares fitting of the conventional ACF is incompatible with the χ2 goodness-of-fit test and systematically underestimates the true fit parameter uncertainty. To overcome this challenge, a simple method to fit the ACF is introduced that allows proper calculation of goodness-of-fit statistics and that provides more tightly constrained parameter estimates than the conventional least-squares fitting method, achieving the theoretical minimum uncertainty. Because this method requires significantly more data than the standard method, we further introduce an approximate method that requires fewer data. We demonstrate both these new methods using experiments and simulations of diffusion. Finally, we apply our method to FCS data of the peripheral membrane protein HRas, which has a slow-diffusing membrane-bound population and a fast-diffusing cytoplasmic population. Despite the order-of-magnitude difference of the diffusion times, conventional FCS fails to reliably resolve the two species, whereas the new method identifies the correct model and provides robust estimates of the fit parameters for both species.

Significance of endolymphatic sac surgery with and/or without simultaneous cochlea implant surgery in respect of vertigo control and speech perception in patients with Menière's disease.

PURPOSE: The focus on treating patients with Menière's Disease (MD) lies on the reduction of vertigo attacks and the preservation of sensory function. Endolympathic hydrops is considered as an epiphenomenon in MD, which can potentially be altered by endolymphatic sac surgery (ESS). Purpose of the study was to investigate the influences on vertigo control through manipulation of the perilymphatic system with or without ESS. METHODS: Retrospective data analysis of 86 consecutive patients with MD according to current diagnostic criteria after endolymphatic sac surgery alone (ESSalone; n = 45), cochlear implantation (CI) alone (CIalone; n = 12), and ESS with CI (ESS + CI; n = 29), treated at a tertiary referral center. MAIN OUTCOME MEASURES: vertigo control, speech perception pre- and postoperatively. RESULTS: Gender, side, and preoperative treatment were similar in all groups. Age was younger in the ESSalone-group with 56.2 ± 13.0 years (CIalone = 64.2 ± 11.4 years; ESS + CI = 63.1 ± 9.7 years). Definitive MD was present in all the CIalone, in 79.3% of the ESS + CI and in 59.6% of the ESSalone-patients. Likewise, vertigo control rate was 100% in the CIalone, 89.7% in the ESS + CI and 66.0% in the ESSalone-group. CONCLUSIONS: Vertigo control was improved in all three groups, however, superior in groups treated with CI, potentially contributed by the manipulation of both the endo- and perilymphatic systems. A more systematic characterization of the patients with larger case numbers and documentation of follow up data would be needed to evaluate a clinical effect more properly.

Analysis of Arc/Arg3.1 Oligomerization In Vitro and in Living Cells.

Arc (also known as Arg3.1) is an activity-dependent immediate early gene product enriched in neuronal dendrites. Arc plays essential roles in long-term potentiation, long-term depression, and synaptic scaling. Although its mechanisms of action in these forms of synaptic plasticity are not completely well established, the activities of Arc include the remodeling of the actin cytoskeleton, the facilitation of AMPA receptor (AMPAR) endocytosis, and the regulation of the transcription of AMPAR subunits. In addition, Arc has sequence and structural similarity to retroviral Gag proteins and self-associates into virus-like particles that encapsulate mRNA and perhaps other cargo for intercellular transport. Each of these activities is likely to be influenced by Arc's reversible self-association into multiple oligomeric species. Here, we used mass photometry to show that Arc exists predominantly as monomers, dimers, and trimers at approximately 20 nM concentration in vitro. Fluorescence fluctuation spectroscopy revealed that Arc is almost exclusively present as low-order (monomer to tetramer) oligomers in the cytoplasm of living cells, over a 200 nM to 5 µM concentration range. We also confirmed that an α-helical segment in the N-terminal domain contains essential determinants of Arc's self-association.

Comparison of speech perception in bimodal cochlear implant patients with respect to the cochlear coverage.

BACKGROUND: The hearing success of patients with bimodal fitting, utilizing both a cochlear implant (CI) and a hearing aid (HA), varies considerably: While some patients benefit from bimodal CI and HA, others do not. OBJECTIVES: This retrospective study aimed to investigate speech perception in bimodally fitted patients and compare it with the cochlear coverage (CC). METHODS: The CC was calculated with the OTOPLAN software, measuring the cochlear duct length on temporal bone CT scans of 39 patients retrospectively. The patients were categorized into two groups: CC ≤ 65% (CC500) and CC > 65% (CC600). Monaural speech intelligibility for monosyllables at a sound pressure level (SPL) of 65 dB in a free-field setting was assessed before and after CI at various time points. The two groups, one with preoperative HA and one with postoperative CI, were compared. Additionally, speech intelligibility was correlated with CC in the entire cohort before CI and at the last available follow-up (last observation time, LOT). RESULTS: Overall, there was no significant difference in speech intelligibility between CC500 and CC600 patients, with both groups demonstrating a consistent improvement after implantation. While CC600 patients tended to exhibit earlier improvement in speech intelligibility, CC500 patients showed a slower initial improvement within the first 3 months but demonstrated a steeper learning curve thereafter. At LOT, the two patient groups converged, with no significant differences in expected speech intelligibility. There was no significant relationship between unimodal/unilateral free-field speech intelligibility and CC. Interestingly, patients with a CC of 70-75% achieved the highest speech intelligibility. CONCLUSION: Despite of the lack of a significant correlation between CC and speech perception, patients appeared to reach their maximum in unimodal/unilateral speech perception primarily at a coverage level of 70-75%. Nevertheless, further investigation is warranted, as CC500 was associated with shorter cochlear duct length, and different types of electrodes were used in both groups.

Autocorrelation function of finite-length data in fluorescence correlation spectroscopy.

The experimental autocorrelation function of fluorescence correlation spectroscopy calculated from finite-length data is a biased estimator of the theoretical correlation function. This study presents a new theoretical framework that explicitly accounts for the data length to allow for unbiased analysis of experimental autocorrelation functions. To validate our theory, we applied it to experiments and simulations of diffusion and characterized the accuracy and precision of the resulting parameter estimates. Because measurements in living cells are often affected by instabilities of the fluorescence signal, autocorrelation functions are typically calculated on segmented data to improve their robustness. Our reformulated theory extends the range of usable segment times down to timescales approaching the diffusion time. This flexibility confers unique advantages for live-cell data that contain intensity variations and instabilities. We describe several applications of short segmentation to analyze data contaminated with unwanted fluctuations, drifts, or spikes in the intensity that are not suited for conventional fluorescence correlation analysis. These results demonstrate the potential of our theoretical framework to significantly expand the experimental systems accessible to fluorescence correlation spectroscopy.

PI4P-Dependent Targeting of ATG14 to Mature Autophagosomes.

Degradation of autophagosomal cargo requires the tethering and fusion of autophagosomes with lysosomes that is mediated by the scaffolding protein autophagy related 14 (ATG14). Here, we report that phosphatidylinositol 4-kinase 2A (PI4K2A) generates a pool of phosphatidylinositol 4-phosphate (PI4P) that facilitates the recruitment of ATG14 to mature autophagosomes. We also show that PI4K2A binds to ATG14, suggesting that PI4P may be synthesized in situ in the vicinity of ATG14. Impaired targeting of ATG14 to autophagosomes in PI4K2A-depleted cells is rescued by the introduction of PI4P but not its downstream product phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Thus, PI4P and PI(4,5)P2 have independent functions in late-stage autophagy. These results provide a mechanism to explain prior studies indicating that PI4K2A and its product PI4P are necessary for autophagosome-lysosome fusion.

Differentiating Luminal and Membrane-Associated Nuclear Envelope Proteins.

The nuclear envelope (NE) consists of two concentric nuclear membranes separated by the lumen, an ∼40-nm-wide fluid layer. NE proteins are implicated in important cellular processes ranging from gene expression to nuclear positioning. Although recent progress has been achieved in quantifying the assembly states of NE proteins in their native environment with fluorescence fluctuation spectroscopy, these studies raised questions regarding the association of NE proteins with nuclear membranes during the assembly process. Monitoring the interaction of proteins with membranes is important because the binding event is often associated with conformational changes that are critical to cellular signaling pathways. Unfortunately, the close physical proximity of both membranes poses a severe experimental challenge in distinguishing luminal and membrane-associated NE proteins. This study seeks to address this problem by introducing new, to our knowledge, fluorescence-based assays that overcome the restrictions imposed by the NE environment. We found that luminal proteins violate the Stokes-Einstein relation, which eliminates a straightforward use of protein mobility as a marker of membrane association within the NE. However, a surprising anomaly in the temperature-dependent mobility of luminal proteins was observed, which was developed into an assay for distinguishing between soluble and membrane-bound NE proteins. We further introduced a second independent tool for distinguishing both protein populations by harnessing the previously reported undulations of the nuclear membranes. These membrane undulations introduce local volume changes that produce an additional fluorescence fluctuation signal for luminal, but not for membrane-bound, proteins. After testing both methods using simple model systems, we apply the two assays to investigate a previously proposed model of membrane association for the luminal domain of SUN2, a constituent protein of the linker of nucleoskeleton and cytoskeleton complex. Finally, we investigate the effect of C- and N-terminal tagging of the luminal ATPase torsinA on its ability to associate with nuclear membranes.

Identifying Heteroprotein Complexes in the Nuclear Envelope.

The nucleus is delineated by the nuclear envelope (NE), which is a double membrane barrier composed of the inner and outer nuclear membranes as well as a ∼40-nm wide lumen. In addition to its barrier function, the NE acts as a critical signaling node for a variety of cellular processes, which are mediated by protein complexes within this subcellular compartment. Although fluorescence fluctuation spectroscopy is a powerful tool for characterizing protein complexes in living cells, it was recently demonstrated that conventional fluorescence fluctuation spectroscopy methods are not suitable for applications in the NE because of the presence of slow nuclear membrane undulations. We previously addressed this challenge by developing time-shifted mean-segmented Q (tsMSQ) analysis and applied it to successfully characterize protein homo-oligomerization in the NE. However, many NE complexes, such as the linker of the nucleoskeleton and cytoskeleton complex, are formed by heterotypic interactions, which single-color tsMSQ is unable to characterize. Here, we describe the development of dual-color (DC) tsMSQ to analyze NE heteroprotein complexes built from proteins that carry two spectrally distinct fluorescent labels. Experiments performed on model systems demonstrate that DC tsMSQ properly identifies heteroprotein complexes and their stoichiometry in the NE by accounting for spectral cross talk and local volume fluctuations. Finally, we applied DC tsMSQ to study the assembly of the linker of the nucleoskeleton and cytoskeleton complex, a heteroprotein complex composed of Klarsicht/ANC-1/SYNE homology and Sad1/UNC-84 (SUN) proteins, in the NE of living cells. Using DC tsMSQ, we demonstrate the ability of the SUN protein SUN2 and the Klarsicht/ANC-1/SYNE homology protein nesprin-2 to form a heterocomplex in vivo. Our results are consistent with previously published in vitro studies and demonstrate the utility of the DC tsMSQ technique for characterizing NE heteroprotein complexes.

Sensitive Detection of Protein Binding to the Plasma Membrane with Dual-Color Z-Scan Fluorescence.

Delicate and transitory protein engagement at the plasma membrane (PM) is crucial to a broad range of cellular functions, including cell motility, signal transduction, and virus replication. Here, we describe a dual-color (DC) extension of the fluorescence z-scan technique, which has proven successful for quantification of peripheral membrane protein binding to the PM in living cells. We demonstrate that the coexpression of a second, distinctly colored fluorescent protein provides a soluble reference species that delineates the extent of the cell cytoplasm and lowers the detection threshold of z-scan PM-binding measurements by an order of magnitude. DC z-scan generates an intensity profile for each detection channel that contains information on the axial distribution of the peripheral membrane and reference protein. Fit models for DC z-scan are developed and verified using simple model systems. Next, we apply the quantitative DC z-scan technique to investigate the binding of two peripheral membrane protein systems for which previous z-scan studies failed to detect binding: human immunodeficiency virus type 1 (HIV-1) matrix (MA) protein and lipidation-deficient mutants of the fibroblast growth factor receptor substrate 2α. Our findings show that these mutations severely disrupt PM association of fibroblast growth factor receptor substrate 2α but do not eliminate it. We further detected binding of HIV-1 MA to the PM using DC z-scan. Interestingly, our data indicate that HIV-1 MA binds cooperatively to the PM with a dissociation coefficient of Kd ∼16 µM and Hill coefficient of n ∼2.

Protein oligomerization and mobility within the nuclear envelope evaluated by the time-shifted mean-segmented Q factor.

Analysis of fluorescence fluctuation experiments by the mean-segmented Q (MSQ) method was recently used to successfully characterize the oligomeric state and mobility of proteins within the nuclear envelope (NE) of living cells. However, two significant shortcomings of MSQ were recognized. Non-ideal detector behavior due to dead-time and afterpulsing as well as the lack of error analysis currently limit the potential of MSQ. This paper presents time-shifted MSQ (tsMSQ), a new formulation of MSQ that is robust with respect to dead-time and afterpulsing. In addition, a protocol for performing error analysis on tsMSQ data is introduced to assess the quality of fit models and estimate the uncertainties of fit parameters. Together, these developments significantly simplify and improve the analysis of fluorescence fluctuation data taken within the NE. To demonstrate these new developments, tsMSQ was used to characterize the oligomeric state and mobility of the luminal domains of two inner nuclear membrane SUN proteins. The results for the luminal domain of SUN2 obtained through tsMSQ without correction for non-ideal detector effects agree with a recent study that was conducted using the original MSQ formulation. Finally, tsMSQ was applied to characterize the oligomeric state and mobility of the luminal domain of the germline-restricted SUN3.

Effect of Heat Exposure on Activity Degradation of Enzymes in Mango Varieties Sindri, SB Chaunsa, and Tommy Atkins during Drying.

Mango has been described as a valuable source of nutrients and enzymes that are beneficial to human health. Drying at different temperatures not only affects the nutritional properties but can also contribute to the degradation of valuable enzymes in dried fruit. The novelty of this paper is to investigate the quality of hot air dried mango in terms of activity retention of the heat-sensitive enzymes (HSE). For this, HSE was first screened in fresh mango flesh of the variety Samar Bahisht (SB) Chaunsa. Later, the combined effect of different drying temperatures (40 °C, 50 °C, 60 °C, 70 °C, and 80 °C) and air velocities (1.0 ms-1 and 1.4 ms-1) on the activity retention of HSE in dried mango slices of the varieties Sindri, SB Chaunsa, and Tommy Atkins were investigated. The results showed that the drying temperature had a significant impact on the degradation of HSE, while at the same time some influence of the air velocity was also observed. Drying at 40 °C and an air velocity of 1.4 ms-1 retained more HSE compared to those samples dried at higher temperatures. The least retention of HSE was found in samples dried at 80 °C.

Myristoylation-Dependent Palmitoylation of the Receptor Tyrosine Kinase Adaptor FRS2α.

An early step in signaling from activated receptor tyrosine kinases (RTKs) is the recruitment of cytosolic adaptor proteins to autophosphorylated tyrosines in the receptor cytoplasmic domains. Fibroblast growth factor receptor substrate 2α (FRS2α) associates via its phosphotyrosine-binding domain (PTB) to FGF receptors (FGFRs). Upon FGFR activation, FRS2α undergoes phosphorylation on multiple tyrosines, triggering recruitment of the adaptor Grb2 and the tyrosine phosphatase Shp2, resulting in stimulation of PI3K/AKT and MAPK signaling pathways. FRS2α also undergoes N-myristoylation, which was shown to be important for its localization to membranes and its ability to stimulate downstream signaling events (Kouhara et al., 1997). Here we show that FRS2α is also palmitoylated in cells and that cysteines 4 and 5 account for the entire modification. We further show that mutation of those two cysteines interferes with FRS2α localization to the plasma membrane (PM), and we quantify this observation using fluorescence fluctuation spectroscopy approaches. Importantly, prevention of myristoylation by introduction of a G2A mutation also abrogates palmitoylation, raising the possibility that signaling defects previously ascribed to the G2A mutant may actually be due to a failure of that mutant to undergo palmitoylation. Our results demonstrate that FRS2α undergoes coupled myristoylation and palmitoylation. Unlike stable cotranslational modifications, such as myristoylation and prenylation, palmitoylation is reversible due to the relative lability of the thioester linkage. Therefore, palmitoylation may provide a mechanism, in addition to phosphorylation, for dynamic regulation of FRS2 and its downstream signaling pathways.

Critical Role of the Human T-Cell Leukemia Virus Type 1 Capsid N-Terminal Domain for Gag-Gag Interactions and Virus Particle Assembly.

The retroviral Gag protein is the main structural protein responsible for virus particle assembly and release. Like human immunodeficiency virus type 1 (HIV-1) Gag, human T-cell leukemia virus type 1 (HTLV-1) has a structurally conserved capsid (CA) domain, including a ß-hairpin turn and a centralized coiled-coil-like structure of six α helices in the CA amino-terminal domain (NTD), as well as four α-helices in the CA carboxy-terminal domain (CTD). CA drives Gag oligomerization, which is critical for both immature Gag lattice formation and particle production. The HIV-1 CA CTD has previously been shown to be a primary determinant for CA-CA interactions, and while both the HTLV-1 CA NTD and CTD have been implicated in Gag-Gag interactions, our recent observations have implicated the HTLV-1 CA NTD as encoding key determinants that dictate particle morphology. Here, we have conducted alanine-scanning mutagenesis in the HTLV-1 CA NTD nucleotide-encoding sequences spanning the loop regions and amino acids at the beginning and ends of α-helices due to their structural dissimilarity from the HIV-1 CA NTD structure. We analyzed both Gag subcellular distribution and efficiency of particle production for these mutants. We discovered several important residues (i.e., M17, Q47/F48, and Y61). Modeling implicated that these residues reside at the dimer interface (i.e., M17 and Y61) or at the trimer interface (i.e., Q47/F48). Taken together, these observations highlight the critical role of the HTLV-1 CA NTD in Gag-Gag interactions and particle assembly, which is, to the best of our knowledge, in contrast to HIV-1 and other retroviruses.IMPORTANCE Retrovirus particle assembly and release from infected cells is driven by the Gag structural protein. Gag-Gag interactions, which form an oligomeric lattice structure at a particle budding site, are essential to the biogenesis of an infectious virus particle. The CA domain of Gag is generally thought to possess the key determinants for Gag-Gag interactions, and the present study has discovered several critical amino acid residues in the CA domain of HTLV-1 Gag, an important cancer-causing human retrovirus, which are distinct from that of HIV-1 as well as other retroviruses studied to date. Altogether, our results provide important new insights into a poorly understood aspect of HTLV-1 replication that significantly enhances our understanding of the molecular nature of Gag-Gag interaction determinants crucial for virus particle assembly.

Quantitative modeling of self-oligomerization of proteins in the nuclear envelope by fluorescence fluctuation analysis.

Analysis of fluorescence fluctuation data through the time-shifted mean-segmented Q (tsMSQ) analysis method has recently been shown to successfully identify protein oligomerization and mobility in the nuclear envelope by properly accounting for local volume fluctuations of the nuclear envelope within living cells. However, by its nature, tsMSQ produces correlated data which poses unique challenges for applying goodness of fit tests and obtaining parameter uncertainties from individual measurements. In this paper, we overcome these challenges by introducing bootstrap tsMSQ which involves randomly resampling the fluorescence intensity data to eliminate the correlations in the tsMSQ data. This analysis technique was verified in both the cytoplasm and the lumen of the nuclear envelope with well-characterized proteins that served as model systems. Uncertainties and goodness of fit tests of individual measurements were compared to estimates obtained from sampling multiple experiments. We further applied bootstrapping to fluorescence fluctuation data of the luminal domain of the SUN domain-containing protein 2 in order to characterize its self-oligomerization within the nuclear envelope. Analysis of the concentration-dependent brightness suggests a monomer-trimer transition of the protein.

Comobility of GABARAP and Phosphatidylinositol 4-Kinase 2A on Cytoplasmic Vesicles.

We previously reported that recruitment of the type IIA phosphatidylinositol 4-kinase (PI4K2A) to autophagosomes by GABARAP, a member of the Atg8 family of autophagy-related proteins, is important for autophagosome-lysosome fusion. Because both PI4K2A and GABARAP have also been implicated in the intracellular trafficking of plasma membrane receptors in the secretory/endocytic pathway, we characterized their interaction in cells under nonautophagic conditions. Fluorescence fluctuation spectroscopy measurements revealed that GABARAP exists predominantly as a cytosolic monomer in live cells, but is recruited to small cytoplasmic vesicles upon overexpression of PI4K2A. C-Terminal lipidation of GABARAP, which is essential for its autophagic activities, is not necessary for its recruitment to these PI4K2A-containing transport vesicles. However, a GABARAP truncation mutant lacking C-terminal residues 103-117 fails to bind to PI4K2A, is not recruited to cytoplasmic vesicles, and does not codistribute with PI4K2A on subcellular organelles. These observations suggest that the PI4K2A-GABARAP interaction plays a role in membrane trafficking both under autophagic and nonautophagic conditions.

Disparate Contributions of Human Retrovirus Capsid Subdomains to Gag-Gag Oligomerization, Virus Morphology, and Particle Biogenesis.

The capsid domain (CA) of the retroviral Gag protein is a primary determinant of Gag oligomerization, which is a critical step for immature Gag lattice formation and virus particle budding. Although the human immunodeficiency virus type 1 (HIV-1) CA carboxy-terminal domain (CTD) is essential for CA-CA interactions, the CA CTD has been suggested to be largely dispensable for human T-cell leukemia virus type 1 (HTLV-1) particle biogenesis. To more clearly define the roles of the HTLV-1 CA amino-terminal domain (NTD) and CA CTD in particle biogenesis, we generated and analyzed a panel of Gag proteins with chimeric HIV-1/HTLV-1 CA domains. Subcellular distribution and protein expression levels indicated that Gag proteins with a chimeric HIV-1 CA NTD/HTLV-1 CA CTD did not result in Gag oligomerization regardless of the parent Gag background. Furthermore, chimeric Gag proteins with the HTLV-1 CA NTD produced particles phenotypically similar to HTLV-1 immature particles, highlighting the importance of the HTLV-1 CA NTD in HTLV-1 immature particle morphology. Taken together, these observations support the conclusion that the HTLV-1 CA NTD can functionally replace the HIV-1 CA CTD, but the HIV-1 CA NTD cannot replace the HTLV-1 CA CTD, indicating that the HTLV-1 CA subdomains provide distinct contributions to Gag-Gag oligomerization, particle morphology, and biogenesis. Furthermore, we have shown for the first time that HIV-1 and HTLV-1 Gag domains outside the CA (e.g., matrix and nucleocapsid) impact Gag oligomerization as well as immature particle size and morphology.IMPORTANCE A key aspect in virus replication is virus particle assembly, which is a poorly understood process for most viruses. For retroviruses, the Gag structural protein is the primary driver of virus particle biogenesis, and the CA CTD is the primary determinant of Gag-Gag interactions for HIV-1. In this study, the HTLV-1 capsid amino-terminal domain was found to provide distinct contributions to Gag-Gag oligomerization, particle morphology, and biogenesis. This study provides information that will aid efforts for discovery of therapeutic targets for intervention.

Quantitative Brightness Analysis of Protein Oligomerization in the Nuclear Envelope.

Brightness analysis of fluorescence fluctuation experiments has been used to successfully measure the oligomeric state of proteins at the plasma membrane, in the nucleoplasm, and in the cytoplasm of living cells. Here we extend brightness analysis to the nuclear envelope (NE), a double membrane barrier separating the cytoplasm from the nucleoplasm. Results obtained by applying conventional brightness analysis to fluorescently tagged proteins within the NE exhibited an unusual concentration dependence. Similarly, the autocorrelation function of the fluorescence fluctuations exhibited unexpected changes with protein concentration. These observations motivated the application of mean-segmented Q analysis, which identified the existence of a fluctuation process distinct from molecular diffusion in the NE. We propose that small changes in the separation of the inner and outer nuclear membrane are responsible for the additional fluctuation process, as suggested by results obtained for luminal and nuclear membrane-associated EGFP-tagged proteins. Finally, we applied these insights to study the oligomerization of the luminal domains of two nuclear membrane proteins, nesprin-2 and SUN2, which interact transluminally to form a nuclear envelope-spanning linker molecular bridge known as the linker of the nucleoskeleton and cytoskeleton complex.

Characterization of Ternary Protein Systems In Vivo with Tricolor Heterospecies Partition Analysis.

Tools and assays that characterize protein-protein interactions are of fundamental importance to biology, because protein assemblies play a critical role in the control and regulation of nearly every cellular process. The availability of fluorescent proteins has facilitated the direct and real-time observation of protein-protein interactions inside living cells, but existing methods are mostly limited to binary interactions between two proteins. Because of the scarcity of techniques capable of identifying ternary interactions, we developed tricolor heterospecies partition analysis. The technique is based on brightness analysis of fluorescence fluctuations from three fluorescent proteins that serve as protein labels. We identified three fluorescent proteins suitable for tricolor brightness experiments. In addition, we developed the theory of identifying interactions in a ternary protein system using tricolor heterospecies partition analysis. The theory was verified by experiments on well-characterized protein systems. A graphical representation of the heterospecies partition data was introduced to visualize interactions in ternary protein systems. Lastly, we performed fluorescence fluctuation experiments on cells expressing a coactivator and two nuclear receptors and applied heterospecies partition analysis to explore the interactions of this ternary protein system.

Association of Endophilin B1 with Cytoplasmic Vesicles.

Endophilins are SH3- and BAR domain-containing proteins implicated in membrane remodeling and vesicle formation. Endophilins A1 and A2 promote the budding of endocytic vesicles from the plasma membrane, whereas endophilin B1 has been implicated in vesicle budding from intracellular organelles, including the trans-Golgi network and late endosomes. We previously reported that endophilins A1 and A2 exist almost exclusively as soluble dimers in the cytosol. Here, we present results of fluorescence fluctuation spectroscopy analyses indicating that, in contrast, the majority of endophilin B1 is present in multiple copies on small, highly mobile cytoplasmic vesicles. Formation of these vesicles was enhanced by overexpression of wild-type dynamin 2, but suppressed by expression of a catalytically inactive dynamin 2 mutant. Using dual-color heterospecies partition analysis, we identified the epidermal growth factor receptor on endophilin B1 vesicles. Moreover, a proportion of endophilin B1 vesicles also contained caveolin, whereas clathrin was almost undetectable on those vesicles. These results raise the possibility that endophilin B1 participates in dynamin 2-dependent formation of a population of transport vesicles distinct from those generated by A-type endophilins.

In situ quantification of protein binding to the plasma membrane.

This study presents a fluorescence-based assay that allows for direct measurement of protein binding to the plasma membrane inside living cells. An axial scan through the cell generates a fluorescence intensity profile that is analyzed to determine the membrane-bound and cytoplasmic concentrations of a peripheral membrane protein labeled by the enhanced green fluorescent protein (EGFP). The membrane binding curve is constructed by mapping those concentrations for a population of cells with a wide range of protein expression levels, and a fit of the binding curve determines the number of binding sites and the dissociation coefficient. We experimentally verified the technique, using myosin-1C-EGFP as a model system and fit its binding curve. Furthermore, we studied the protein-lipid interactions of the membrane binding domains from lactadherin and phospholipase C-δ1 to evaluate the feasibility of using competition binding experiments to identify specific lipid-protein interactions in living cells. Finally, we applied the technique to determine the lipid specificity, the number of binding sites, and the dissociation coefficient of membrane binding for the Gag matrix domain of human T-lymphotropic virus type 1, which provides insight into early assembly steps of the retrovirus.