Z-scan Fluorescence Fluctuation Spectroscopy Research Articles

Challenges and Opportunities for Characterizing the Assembly of Nuclear Envelope Proteins by Fluorescence Fluctuation Spectroscopy

The nuclear envelope (NE), consisting of two membranes separated by a thin perinuclear space, has proven to be a challenging environment for measuring protein dynamics in living cells. First, exogenously expressed, fluorescently tagged proteins must be properly localized to the NE. Furthermore, undulations of the nuclear membranes complicate the application of fluorescence fluctuation spectroscopy (FFS) to proteins located within the perinuclear space by adding a slowly fluctuating signal. While this signal is absent for proteins associated with the nuclear membranes, distinguishing soluble from membrane-associated protein complexes by FFS presents a significant challenge since the diffusion time depends strongly on molecular size within the perinuclear space. Some proteins, such as the AAA+ ATPase TorsinA, may exist in both a membrane-associated and soluble form depending on the conditions in the cell. Determining the extent to which a protein associates with the membrane can provide valuable insight into its function within the cell. While diffusion time alone is insufficient to differentiate these populations, we explore whether the dependence of diffusion time on temperature can provide insight into a protein's localization within the NE. In addition, we explore how spatial correlations can be used to study the dynamics of proteins within the NE and the membrane undulations we previously reported. Finally, z-scan FFS provides a means to quantify the localization of proteins to the NE. These examples highlight the ways in which FFS techniques can be used to overcome the challenges of quantifying protein behavior in the NE of living cells. This work has been supported by a grant from the National Institutes of Health (R01 GM64589).

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.

Brightness Characterization of SUN1 and SUN2 by Z-Scan Fluorescence Fluctuation Spectroscopy

Fluorescence fluctuation spectroscopy (FFS) has been used to quantify the stoichiometry of soluble proteins in the cytoplasm of cells by brightness analysis. The development of z-scan FFS broadened the capability of brightness analysis to include proteins distributed across stratified layers, such as the cytoplasm and the plasma membrane. Here, we use z-scan FFS to study proteins that reside in the nuclear envelope/perinuclear space (PNS). Experimentally, we place a 20 amino acid signal-sequence in front of EGFP to drive translation into the lumen of the NE. The brightness of EGFP is determined by performing z-scan FFS measurements where corrections are applied for both the thin-layer geometry and coexcitation of adjacent layers. We then generated a tandem dimeric EGFP protein to establish a model for calibrating brightness and stoichiometry. Finally, we utilize our technique to study the assembly of the inner-nuclear membrane proteins SUN1 and SUN2 within the PNS. SUN proteins are transmembrane proteins that interact with nuclear lamins in the nucleoplasm and with cytoskeletal-interacting, outer nuclear-membrane nesprin proteins within the PNS. Together, SUN and nesprin proteins form the linker of nucleoskeleton and cytoskeleton complex, which is critical for nuclear positioning and movement. The crystal structure of SUN2 reveals a trimer composed of SUN domains and a stalk of coiled-coils. Each SUN trimer binds three nesprin proteins. Currently, evidence for this structure in living cells does not exist nor does stoichiometric information for SUN1. Here, we show that the lumenal regions of both SUN1 and SUN2 oligomerize in a regulated manner within the PNS. SUN2 oligomerizes into trimers while SUN1 forms even higher order oligomers. Taken together, our results establish z-scan FFS as a powerful method for the biochemical and biophysical analysis of nuclear membrane proteins within living cells.

Fluorescence Fluctuation Spectroscopy in the Perinuclear Space

The importance of the nuclear envelope extends well beyond separating the cytoplasm and nucleoplasm. The perinuclear space (PS) located within the nuclear envelope contains proteins involved in many cellular functions including cellular signaling, force transduction, and mechanical properties of the cell. Protein oligomerization plays a central role in the control of these biological processes. Currently, there is no technique that allows for quantitative, real time exploration of the oligomeric state of proteins inside the PS. We previously used fluorescence fluctuation spectroscopy (FFS) with brightness analysis to measure the oligomeric state of protein complexes in the cytoplasm, the nucleus, and the plasma membrane. To extend these measurements to the PS we performed z-scan FFS to account for the layered structure of the nuclear envelope within the cell. However, the conventional analysis tools that allowed us to quantify protein complexes in the cytoplasm and at the plasma membrane failed in the PS. To overcome this obstacle we explored an alternative analysis approach and tested it on fluorescent proteins that were co-translationally targeted to the PS. We compared the recovered diffusion and brightness of these proteins in the PS and in the cytoplasm. We extended our investigation to functional proteins, including SUN2, and demonstrate that measurements of the oligomeric state of protein complexes in the PS are feasible. This work has been supported by a grant from the National Institutes of Health (R01 GM64589).

Scan and Conquer: A Novel Approach to the Analysis of Interactions by Molecular Brightness Determination

Scan and Conquer: A Novel Approach to the Analysis of Interactions by Molecular Brightness Determination

Brightness Characterization of Internal Endomembrane Proteins by Z-Scan Fluorescence Fluctuation Spectroscopy

Traditionally, fluorescence fluctuation spectroscopy (FFS) has been used to quantify the stoichiometry of soluble proteins in the nucleus and the cytoplasm of mammalian cells by brightness analysis. The development of z-scan FFS broadened the capability of brightness analysis to include proteins distributed across stratified layers, such as the cytoplasm and the plasma membrane. In this work, we further extend z-scan FFS to study proteins that reside on or within internal endomembranes, including the endoplasmic reticulum (ER) membrane/lumen and the nuclear envelope/perinuclear space. Experimentally, we place a 20 amino acid ER signal sequence in front of EGFP (SS-EGFP); the complex is translated into the lumen of the ER where of the signal sequence is cleaved, leaving EGFP to diffuse within the ER and nuclear envelope. The brightness of SS-EGFP is determined by performing z-scan FFS measurements where corrections are applied for both the thin layer geometry and coexcitation of adjacent layers. Additionally, we create and test a tandem dimeric EGFP protein (SS-EGPF2) to establish a model for calibrating brightness and stoichiometry. Finally, we test the limits of our technique and apply z-scan FFS to characterize the brightness of other proteins found within the endomembrane system. This research was supported by grants from the National Institutes of Health (R01GM064589) and the National Science Foundation (PHY-0346782).

Interrelationship between Cytoplasmic Retroviral Gag Concentration and Gag–Membrane Association

The early events in the retrovirus assembly pathway, particularly the timing and nature of Gag translocation from the site of protein translation to the inner leaflet of the plasma membrane, are poorly understood. We have investigated the interrelationship between cytoplasmic Gag concentration and plasma membrane association using complementary live-cell biophysical fluorescence techniques in real time with both human T-cell leukemia virus type 1 (HTLV-1) and human immunodeficiency virus type 1 (HIV-1) Gag proteins. In particular, dual-color, z-scan fluorescence fluctuation spectroscopy in conjunction with total internal reflection fluorescence and conventional, epi-illumination imaging were utilized. Our results demonstrate that HTLV-1 Gag is capable of membrane targeting and particle assembly at low (i.e., nanomolar) cytoplasmic concentrations and that there is a critical threshold concentration (approaching micromolar) prior to the observation of HIV-1 Gag associated with the plasma membrane. These observations imply fundamental differences between HIV-1 and HTLV-1 Gag trafficking and membrane association.

Brightness Characterization of Cytoplasmic & Membrane-Bound Protein Mixtures by Z-Scan Fluorescence Fluctuation Spectroscopy

Fluorescence fluctuation spectroscopy (FFS) has been employed to quantify the stoichiometry of soluble proteins in the nucleus and cytoplasm of mammalian cells. However, some proteins have both cytoplasmic and membrane-bound components which render traditional FFS methods inapplicable due to coexcitation of the spatially distributed protein mixture. In this work, we apply z-scan FFS to characterize the brightness of both the membrane-bound and cytoplasmic populations of a protein. Experimentally, we choose HRas-EGFP as our membrane-bound/cytoplasmic model system. We use a combination of stationary FFS measurements to determine the brightness of membrane-bound and cytoplasmic HRas-EGFP and z-scan intensity profiles to correct for brightness bias due to thin layer geometry and coexcitation of membrane and cytoplasmic layers. Our data show HRas-EGFP to be monomeric at both the upper and lower plasma membrane and also in the cytoplasm. We further examine the brightness statistics of proteins by comparing the experimental brightness distribution of a membrane-bound protein system with that of a soluble protein system. This work is extended by creating a dimeric membrane protein system in order to establish a model for calibrating brightness and stoichiometry. The z-scan FFS measurement technique along with a reliable monomer/dimer calibration system will be important for future membrane protein stoichiometry studies. This research was supported by grants from the National Institutes of Health (GM64589) and the National Science Foundation (PHY 0346782).

Characterization of Cytoplasmic Gag-Gag Interactions by Dual-Color Z-Scan Fluorescence Fluctuation Spectroscopy

Fluorescence fluctuation spectroscopy (FFS) quantifies the interactions of fluorescently-labeled proteins inside living cells by brightness analysis. However, the study of cytoplasmic proteins that interact with the plasma membrane is challenging with FFS. If the cytoplasmic section is thinner than the axial size of the observation volume, cytoplasmic and membrane-bound proteins are coexcited, which leads to brightness artifacts. This brightness bias, if not recognized, leads to erroneous interpretation of the data. We have overcome this challenge by introducing dual-color z-scan FFS and the addition of a distinctly colored reference protein. Here, we apply this technique to study the cytoplasmic interactions of the Gag proteins from human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus type 1 (HTLV-1). The Gag protein plays a crucial role in the assembly of retroviruses and is found in both membrane and cytoplasm. Dual-color z-scans demonstrate that brightness artifacts are caused by a dim nonpunctate membrane-bound fraction of Gag. We perform an unbiased brightness characterization of cytoplasmic Gag by avoiding the membrane-bound fraction and reveal previously unknown differences in the behavior of the two retroviral Gag species. HIV-1 Gag exhibits concentration-dependent oligomerization in the cytoplasm, whereas HTLV-1 Gag lacks significant cytoplasmic Gag-Gag interactions.

Characterization of Cytoplasmic / Membrane-Bound Protein Mixtures by Z-Scan Fluorescence Fluctuation Spectroscopy

Fluorescence fluctuation spectroscopy (FFS) has been employed to quantify the stoichiometry of soluble proteins in cells. However, some proteins have both soluble (cytoplasmic) and membrane-bound fractions. In such conditions, coexcitation of the membrane-bound and cytoplasmic proteins makes standard FFS unsuitable. In this work, we apply z-scan FFS to characterize the intensity profile of proteins with both a cytoplasmic and membrane-bound population. Analysis of the intensity profile determines unequivocally the intensity contributions from each membrane layer and the cytoplasm, as well as, the length of cytoplasm layer. Experimentally, we choose Matrix-EGFP (the MA domain of HTLV-1 Gag) as our model system and apply a dual color z-scan technique. Cells are cotransfected with Matrix-EGFP and mCherry and their emitted signal is split into two different detection channels based on color. Simultaneous fitting of both channels with separate model geometries aids in correct determination of parameters. The use of the dual color technique is possible because we demonstrate a co-localization of the observation volume and agreement in the point spread function shape factors for both the red and green channels. We extend our work by applying z-scan FFS to determine the brightness (stoichiometry) of the cytoplasmic protein fraction. Yet determining the brightness of the membrane-bound protein is significantly more challenging because of slower diffusion times, inhomogeneity in protein distribution, and possible brightness bias due to geometry and orientation effects. Here, we investigate the potential of z-scan FFS to address these factors and measure the unbiased brightness of membrane proteins. This study provides insight into the fundamental considerations for correct application of FFS in the study of proteins with both cytoplasmic and membrane-bound fractions. This work is supported by NIH grant GM064589 and NIH grant GM075401.

Brightness Analysis of Cytoplasmic HTLV-1 and HIV-1 Gag in the Presence of Membrane-Bound Gag Complexes by Dual-Color Z-Scan Fluorescence Fluctuation Spectroscopy

Fluorescence fluctuation spectroscopy (FFS) brightness analysis is capable of quantifying protein interactions occurring in the cytoplasm of living cells. Conventional FFS implicitly requires that the fluorescent molecules of interest visit all parts of the excitation volume with equal probability. This condition can be violated when studying protein systems in cells which exhibit inhomogeneous cellular distribution. An example of such a system is retroviral Gag structural proteins, which are known to have cytoplasmic and membrane-bound states with low-order and high-order oligomeric states, respectively. The presence of high-order, fluorescently-labeled Gag oligomers at the membrane can impact brightness analysis of the cytoplasmic fraction of Gag. The inhomogeneous distribution of Gag complexes at the membrane allows for accurate brightness analysis of the cytoplasmic fraction provided the measurement position is carefully selected. Measurement sites selected by eye using fluorescence epi-illumination exhibited brightness artifacts characteristic of sites impacted by the presence of a Gag membrane fraction. To achieve greater sensitivity to the membrane complexes, we developed dual-color z-scan FFS. We demonstrate that dual-color z-scan FFS is capable of identifying measurement sites impacted by the inhomogeneous distribution of Gag in the cytoplasm and at the membrane. This measure of quality control allows for the accurate determination of the cytoplasmic oligomerization of the HTLV-1 and HIV-1 Gag species. Our brightness analysis of wild-type and mutant Gag species reveals previously unknown differences in the cytoplasmic behavior of the two retroviral Gag species. HIV-1 Gag exhibits concentration-dependent oligomerization in the cytoplasm, while HTLV-1 Gag exhibits minimal cytoplasmic interactions. This work is supported by NIH Grant AI81673, NIH Grant GM064589, and a Cancer Center Cancer Biology Training Grant (T32CA09138).

Brightness Characterization of Cellular and In Vitro Expressed Proteins

Most biophysical characterizations of proteins and their interactions are performed in vitro. Because the cell interior is considerably more complex than the aqueous solution of in vitro experiments, differences in the strength and behavior of protein interactions in these two environments are anticipated. Characterization of such differences requires parallel studies of the same proteins and labels expressed in cells and in vitro. Cell-free expression systems offer a convenient method for comparing cell experiments with in vitro measurements. Because fluorescence fluctuation spectroscopy (FFS) determines the brightness of soluble proteins, it allows a direct comparison of protein interactions in the two systems. However, both environments present distinct obstacles to unbiased brightness measurement. The restricted geometry and size of the cellular environment influences the amplitude of the fluctuations. This effect is especially pronounced in thin cellular regions, where it leads to a biased brightness. We demonstrate that z-scan FFS abolishes this artifact for a thin slab geometry. We further extend the technique to multi-layer geometries that take differential protein localization into account. The main challenge for FFS experiments in the cell-free environment is the presence of fluorescent and non-fluorescent aggregates that are by-products of the expression reaction. Additionally, as cell-free experiments typically use small sample volumes, evaporation and surface-adsorption affect sample concentration. We introduce experimental protocols that limit these issues and permit unbiased FFS analysis. With these advances, we are able to successfully perform parallel brightness experiments on cellular and in vitro expressed proteins. This work was supported by a grant from the National Institutes of Health (GM64589).

Brightness Analysis by Z-Scan Fluorescence Fluctuation Spectroscopy for the Study of Protein Interactions within Living Cells

Fluorescence fluctuation spectroscopy (FFS) quantifies interactions of fluorescently labeled proteins inside living cells by brightness analysis. Conventional FFS implicitly requires that the sample thickness exceeds the size of the observation volume. This condition is not always fulfilled when measuring cells. Cytoplasmic sections, especially, can be thinner than the axial size of the observation volume. The finite sample thickness introduces a brightness bias which, if not recognized, leads to an erroneous interpretation of the data. To avoid this artifact, we introduce z-scan FFS which consists of a fluorescence intensity z scan through the sample followed by an FFS measurement. To model the experimental z-scan data, a new PSF model had to be introduced. We use the intensity z scan together with the PSF model to determine the geometry of the sample and then extract the brightness from the FFS data. Cells expressing EGFP serve as a model system for testing the experimental approach. We demonstrate that z-scan FFS abolishes the brightness artifact and use the method to determine the oligomerization of cytoplasmic nuclear transport factor 2.