Jolene Johnson

Observing protein interactions and their stoichiometry in living cells by brightness analysis of fluorescence fluctuation experiments.

A single fluorescently labeled protein generates a short burst of light whenever it passes through a tiny observation volume created within a biological cell. The average amplitude of the burst is related to the stoichiometry of the fluorescently labeled protein complex. Fluorescence fluctuation spectroscopy quantifies the burst amplitude by introducing the brightness parameter. Brightness provides a spectroscopic marker for observing protein interactions and their stoichiometry directly inside cells. Not all fluorescent proteins are suitable for brightness experiments. Here we discuss how brightness properties of the fluorophore influence brightness measurements and how to identify a well-behaved fluorescent protein. Protein interactions and stoichiometry are determined from a brightness titration. Experimental details of brightness titration measurements are described together with the necessary calibration and control experiments.

Biophysical analysis of HTLV-1 particles reveals novel insights into particle morphology and Gag stoichiometry

BackgroundHuman T-lymphotropic virus type 1 (HTLV-1) is an important human retrovirus that is a cause of adult T-cell leukemia/lymphoma. While an important human pathogen, the details regarding virus replication cycle, including the nature of HTLV-1 particles, remain largely unknown due to the difficulties in propagating the virus in tissue culture. In this study, we created a codon-optimized HTLV-1 Gag fused to an EYFP reporter as a model system to quantitatively analyze HTLV-1 particles released from producer cells.ResultsThe codon-optimized Gag led to a dramatic and highly robust level of Gag expression as well as virus-like particle (VLP) production. The robust level of particle production overcomes previous technical difficulties with authentic particles and allowed for detailed analysis of particle architecture using two novel methodologies. We quantitatively measured the diameter and morphology of HTLV-1 VLPs in their native, hydrated state using cryo-transmission electron microscopy (cryo-TEM). Furthermore, we were able to determine HTLV-1 Gag stoichiometry as well as particle size with the novel biophysical technique of fluorescence fluctuation spectroscopy (FFS). The average HTLV-1 particle diameter determined by cryo-TEM and FFS was 71 ± 20 nm and 75 ± 4 nm, respectively. These values are significantly smaller than previous estimates made of HTLV-1 particles by negative staining TEM. Furthermore, cryo-TEM reveals that the majority of HTLV-1 VLPs lacks an ordered structure of the Gag lattice, suggesting that the HTLV-1 Gag shell is very likely to be organized differently compared to that observed with HIV-1 Gag in immature particles. This conclusion is supported by our observation that the average copy number of HTLV-1 Gag per particle is estimated to be 510 based on FFS, which is significantly lower than that found for HIV-1 immature virions.ConclusionsIn summary, our studies represent the first quantitative biophysical analysis of HTLV-1-like particles and reveal novel insights into particle morphology and Gag stochiometry.

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.

Differentiation of oral Actinomyces species by 16S ribosomal DNA polymerase chain reaction-restriction fragment length polymorphism

16S rDNA polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to generate restriction profiles of the reference strains, including the American Type Culture Collection type strains, of oral Actinomyces spp., i.e., A. israelii, A. gerencseriae, A. naeslundii genospecies 1 and 2, A. odontolyticus, A. meyeri and A. georgiae, and 23 Actinomyces strains isolated from human dental plaque. The 16S rRNA gene sequences from isolated genomic DNA samples were amplified by PCR. The PCR products were purified and characterized by single digestion with four restriction endonucleases, i.e., MnlI, HaeIII, CfoI, or HpaII. Among them, MnlI was found to discriminate the respective reference strains. The clinical isolates were assigned to one of the species, i.e., A. gerencseriae, A. naeslundii genospecies 1 and 2 and A. odontolyticus, on the basis of their restriction profiles by single digestion with MnlI. Thus, 16S rDNA PCR-RFLP, using MnlI, is a rapid and reliable method for the differentiation of oral Actinomyces spp.

Chapter Four - Brightness Analysis

Brightness analysis provides a powerful tool for the study of protein interactions both in solution and in living cells. We provide a brief survey of some widely used techniques for extracting brightness from fluorescent fluctuation spectroscopy experiments. While all the techniques are equivalent under ideal conditions, we touch upon their relative strengths and discuss in detail a specific scenario wherein the photon-counting histogram (PCH) separates the brightness of rare, bright particles from a dominant background. In a practical vein for ensuring quantitative and unbiased brightness data, we address a number of potential issues stemming from both theoretical assumptions and experimental realities. Two additional issues arising from geometry are examined in greater detail. An oil-immersion objective skews the geometry of the excitation volume as a function of penetration depth. The bias can be characterized and corrected or avoided through the use of a water-immersion objective. Brightness measurements in thin sample geometries, frequently encountered in cells, may be biased. We use z-scan FFS to characterize sample geometry and correct any resulting bias in the brightness.

New Insights into HTLV-1 Particle Structure, Assembly, and Gag-Gag Interactions in Living Cells

Human T-cell leukemia virus type 1 (HTLV-1) has a reputation for being extremely difficult to study in cell culture. The challenges in propagating HTLV-1 has prevented a rigorous analysis of how these viruses replicate in cells, including the detailed steps involved in virus assembly. The details for how retrovirus particle assembly occurs are poorly understood, even for other more tractable retroviral systems. Recent studies on HTLV-1 using state-of-the-art cryo-electron microscopy and fluorescence-based biophysical approaches explored questions related to HTLV-1 particle size, Gag stoichiometry in virions, and Gag-Gag interactions in living cells. These results provided new and exciting insights into fundamental aspects of HTLV-1 particle assembly—which are distinct from those of other retroviruses, including HIV-1. The application of these and other novel biophysical approaches promise to provide exciting new insights into HTLV-1 replication.

Characterization of Brightness and Stoichiometry of Bright Particles by Flow-Fluorescence Fluctuation Spectroscopy

Characterization of bright particles at low concentrations by fluorescence fluctuation spectroscopy (FFS) is challenging, because the event rate of particle detection is low and fluorescence background contributes significantly to the measured signal. It is straightforward to increase the event rate by flow, but the high background continues to be problematic for fluorescence correlation spectroscopy. Here, we characterize the use of photon-counting histogram analysis in the presence of flow. We demonstrate that a photon-counting histogram efficiently separates the particle signal from the background and faithfully determines the brightness and concentration of particles independent of flow speed, as long as undersampling is avoided. Brightness provides a measure of the number of fluorescently labeled proteins within a complex and has been used to determine stoichiometry of protein complexes in vivo and in vitro. We apply flow-FFS to determine the stoichiometry of the group specific antigen protein within viral-like particles of the human immunodeficiency virus type-1 from the brightness. Our results demonstrate that flow-FFS is a sensitive method for the characterization of complex macromolecular particles at low concentrations.

Molecular brightness analysis reveals phosphatidylinositol 4-kinase IIβ association with clathrin-coated vesicles in living cells

Mammalian cells express two classes of phosphatidylinositol 4-kinase (PI4K), designated as Types II and III, that phosphorylate phosphatidylinositol to generate PI4P. A number of studies have indicated that these enzymes are important for Golgi trafficking and both early and late stages of endocytosis. In this study, we focus on PI4KIIβ, a protein that is evenly distributed between membrane and soluble fractions, and is believed to participate in stimulus-dependent phosphoinositide signaling. Using molecular brightness analysis, we found that EGFP-tagged PI4KIIβ exists as two distinct species in the cytoplasm: a soluble monomer and a high-order complex enriched with multiple copies of PI4KIIβ. This observation was confirmed by an autocorrelation analysis that identified two species with distinct mobilities. We further demonstrate that the high-order complex enriched with PI4KIIβ is sensitive to inhibition of palmitoylation, indicating that it is associated with membranes, very likely vesicles. Indeed, we show that the high-order PI4KIIβ complex is sensitive to expression of dynamin 2 (K44A), a dominant-negative inhibitor of endocytosis. Using dual-color heterospecies partition analysis, we directly detected that PI4KIIβ comoves with clathrin light chain on vesicles. This analysis allows us to isolate the comobile species in the presence of strong background contribution from the monomeric pool of PI4KIIβ. Our results strongly suggest that PI4KIIβ is involved in an early stage of endocytosis and is associated with clathrin-coated vesicles. Moreover, we establish molecular brightness as a powerful tool for characterizing cellular cytosolic vesicles that are otherwise difficult to characterize by other techniques.

Analysis of the HTLV-1 Gag assembly pathway by biophysical fluorescence

Much of the mechanistic details for how HTLV-1 Gag orchestrates virus particle assembly and release are poorly understood. Here, we monitored the behavior of both membrane-bound and cytoplasmic HTLV-1 Gag in real-time in living cells incubated on a fluorescence microscope. We used both fluorescence fluctuation spectroscopy (FFS, conventional and z-scan) and fluorescence imaging (epi-illumination, total internal reflection fluorescence (TIRF)) to investigate the relationship between cytoplasmic and membrane bound Gag, using a Gag-YFP model system. FFS determines the brightness, mobility, and concentration (conventional) and localization (z-scan) of fluorescent particles from the intensity bursts generated by individual particles passing through a small observation volume, which yields information about protein stoichiometry, interactions, transport, and distribution. By coupling the single-molecule FFS technique with imaging techniques capable of monitoring Gag localization (epi-illumination) and membrane-specific localization (TIRF), we achieved new insights into the earliest events in HTLV-1 Gag assembly, and differences to HIV-1 Gag. We found that HTLV-1 Gag membrane-targeting occurred at all cytoplasmic concentrations measured, while appreciable membrane-targeting for HIV-1 required Gag cytoplasmic concentration to exceed a threshold. In addition, z-scan FFS revealed that a substantial population of membrane-bound HTLV-1 Gag exists not as puncta, but as a diffuse, low-order, dynamic “sheet.” These observations, coupled with previous observations of cytoplasmic Gag interactions and mobility, point to differences in membrane targeting of HTLV-1 and HIV-1 Gag. In summary, the suite of biophysical fluorescence techniques, applied HTLV-1 Gag, provide unparalleled information concerning HTLV-1 Gag trafficking processes in vivo, elucidating assembly pathway differences between HTLV-1 and other retroviruses.

Characterizing Fluorescently Labeled Viral-Like Particles by Fluorescence Imaging

Previous studies with fluorescence fluctuation spectroscopy (FFS) on viral-like particles (VLP) formed from fluorescently labeled Gag proteins demonstrated that the VLP samples contain more than a single brightness species. This observation holds both for human immunodeficiency virus type 1 (HIV-1) VLPs and for human T-cell leukemia virus type 1 (HTLV-1) VLPs. Thus, the FFS results imply that the Gag stoichiometry of VLPs is heterogeneous.While FFS analysis identified two brightness species for the VLP samples, the technique cannot differentiate between discrete populations and a broad distribution of Gag stoichiometries. In an effort to characterize the underlying brightness distribution, we combine FFS studies with fluorescence imaging of a VLP population. We first perform a brightness characterization of the VLPs by FFS. Next, the particles from the same sample are immobilized on the surface of a glass coverslip and fluorescence images of the immobile VLPs are collected. The intensity of individual particles is determined by image analysis. We found that the intensity of particles is heterogeneous, which confirms the existence of a distribution of Gag stoichiometries. We characterize the histogram of intensity values and compare it with the FFS results. In addition, we explore the use of particle tracking to determine the diffusion coefficient and the size of the particles by imaging mobile VLPs in a thin sample layer with the goal of relating brightness and size of particles. Combining FFS and fluorescence imaging offers a promising tool for the characterization of protein stoichiometry in viral particles and the heterogeneity. This work was supported by a grant from the National Institutes of Health (R01 GM64589).