Time-shifted mean-segmented Q data of a luminal protein measured at the nuclear envelope by fluorescence fluctuation microscopy.

Fluorescence fluctuation microscopy is a widely used method to determine the mobility and oligomeric state of proteins in the live cell environment. Existing analysis methods rely on statistical evaluation of data segments with the implicit assumption that no significant signal fluctuations occur on the time scale of a data segment. Recent work on extending fluorescence fluctuation methods to the nuclear envelope of living cells identified a slow fluctuation process that is associated with the undulations of the nuclear membranes, which lead to intensity fluctuations due to local volume changes at the nuclear envelope. This environment violates the above-mentioned assumption and is associated with biased evaluation of fluorescence fluctuation data by traditional analysis methods, such as the autocorrelation function. This challenge was overcome by the introduction of the time-shifted mean-segmented Q function, which relies on a sliding scale of data segment lengths. Here, we share experimental fluorescence fluctuation data taken at the nuclear envelope and demonstrate the calculation of the time-shifted mean-segmented Q function from the raw data. The data and analysis should be valuable for researchers interested in fluorescence fluctuation techniques and provides an opportunity to examine the influence of slow fluctuations on existing data analysis methods. The data is related to the research article titled "Protein oligomerization and mobility within the nuclear envelope evaluated by the time-shifted mean-segmented Q factor" [1].

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.

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.

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.

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.

Investigating LINC Complex Protein Homo-oligomerization in the Nuclear Envelopes of Living Cells Using Fluorescence Fluctuation Spectroscopy.

Linkers of nucleoskeleton and cytoskeleton (LINC) complexes are conserved nuclear envelope (NE) spanning molecular bridges which mechanically integrate the nucleus with the cytoskeleton and mediate force transmission into the nucleoplasm. Despite their critical roles in fundamental cellular processes such as meiotic chromosome and nuclear positioning, the mechanism of LINC complex assembly in cells remains unclear. To begin to address this deficit, we recently developed z-scan fluorescence fluctuation spectroscopy (FFS) and brightness analysis as a method for quantifying the oligomeric states of fluorescent protein-tagged NE proteins including nesprins and SUN proteins. Since the homo-oligomerization of SUN2 is critical for its ability to interact with nesprins within the perinuclear space, the knowledge obtained through quantitative brightness experiments reveals important insights into the in vivo mechanisms of LINC complex assembly. Here we describe the procedure we use to determine the brightness of proteins in the NE of living cells. In addition to the measurement procedure, we discuss the instrumentation requirements and present the results of applying this procedure to measure the brightness of nesprin-2 and SUN2.

Fluorescence fluctuation spectroscopy reveals differential SUN protein oligomerization in living cells.

Linker-of-nucleoskeleton-and-cytoskeleton (LINC) complexes are conserved molecular bridges within the nuclear envelope that mediate mechanical force transmission into the nucleoplasm. The core of a LINC complex is formed by a transluminal interaction between the outer and inner nuclear membrane KASH and SUN proteins, respectively. Mammals encode six KASH proteins and five SUN proteins. Recently, KASH proteins were shown to bind to the domain interfaces of trimeric SUN2 proteins in vitro. However, neither the existence of SUN2 trimers in living cells nor the extent to which other SUN proteins conform to this assembly state have been tested experimentally. Here we extend the application of fluorescence fluctuation spectroscopy to quantify SUN protein oligomerization in the nuclear envelopes of living cells. Using this approach, we demonstrate for the first time that SUN2 trimerizes in vivo and we demonstrate that the in vivo oligomerization of SUN1 is not limited to a trimer. In addition, we provide evidence to support the existence of potential regulators of SUN protein oligomerization in the nuclear envelope. The differential SUN protein oligomerization illustrated here suggests that SUN proteins may have evolved to form different assembly states in order to participate in diverse mechanotransduction events.

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.

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.

Z-scan fluorescence profile deconvolution of cytosolic and membrane-associated protein populations.

This study introduces a technique that characterizes the spatial distribution of peripheral membrane proteins that associate reversibly with the plasma membrane. An axial scan through the cell generates a z-scan intensity profile of a fluorescently labeled peripheral membrane protein. This profile is analytically separated into membrane and cytoplasmic components by accounting for both the cell geometry and the point spread function. We experimentally validated the technique and characterized both the resolvability and stability of z-scan measurements. Furthermore, using the cellular brightness of green fluorescent protein, we were able to convert the fluorescence intensities into concentrations at the membrane and in the cytoplasm. We applied the technique to study the translocation of the pleckstrin homology domain of phospholipase C delta 1 labeled with green fluorescent protein on ionomycin treatment. Analysis of the z-scan fluorescence profiles revealed protein-specific cell height changes and allowed for comparison between the observed fluorescence changes and predictions based on the cellular surface area-to-volume ratio. The quantitative capability of z-scan fluorescence profile deconvolution offers opportunities for investigating peripheral membrane proteins in the living cell that were previously not accessible.