Hacene Boukari

Direct measurement of association and dissociation rates of DNA binding in live cells by fluorescence correlation spectroscopy

Measurement of live-cell binding interactions is vital for understanding the biochemical reactions that drive cellular processes. Here, we develop, characterize, and apply a new procedure to extract information about binding to an immobile substrate from fluorescence correlation spectroscopy (FCS) autocorrelation data. We show that existing methods for analyzing such data by two-component diffusion fits can produce inaccurate estimates of diffusion constants and bound fractions, or even fail altogether to fit FCS binding data. By analyzing live-cell FCS measurements, we show that our new model can satisfactorily account for the binding interactions introduced by attaching a DNA binding domain to the dimerization domain derived from a site-specific transcription factor (the vitellogenin binding protein (VBP)). We find that our FCS estimates are quantitatively consistent with our fluorescence recovery after photobleaching (FRAP) measurements on the same VBP domains. However, due to the fast binding interactions introduced by the DNA binding domain, FCS generates independent estimates for the diffusion constant (6.7 +/- 2.4 microm2/s) and the association (2 +/- 1.2 s(-1)) and dissociation (19 +/- 7 s(-1)) rates, whereas FRAP produces only a single, but a consistent, estimate, the effective-diffusion constant (4.4 +/- 1.4 microm2/s), which depends on all three parameters. We apply this new FCS method to evaluate the efficacy of a potential anticancer drug that inhibits DNA binding of VBP in vitro and find that in vivo the drug inhibits DNA binding in only a subset of cells. In sum, we provide a straightforward approach to directly measure binding rates from FCS data.

T‐Cell Antigen Receptor‐Induced Signaling Complexes: Internalization Via a Cholesterol‐Dependent Endocytic Pathway

T‐cell antigen receptor engagement causes the rapid assembly of signaling complexes. The adapter protein SLP‐76, detected as SLP‐yellow fluorescent protein, initially clustered with the TCR and other proteins, then translocated medially on microtubules. As shown by total internal reflection fluorescence microscopy and the inhibition of SLP‐76 movement at 16°C, this movement required endocytosis. Immunoelectron microscopy showed SLP‐76 staining of smooth pits and tubules. Cholesterol depletion decreased the movement of SLP‐76 clusters, as did coexpression of the ubiquitin‐interacting motif domain from eps15. These data are consistent with the internalization of SLP‐76 via a lipid raft‐dependent pathway that requires interaction of the endocytic machinery with ubiquitinylated proteins. The endocytosed SLP‐76 clusters contained phosphorylated SLP‐76 and phosphorylated LAT. The raft‐associated, transmembrane protein LAT likely targets SLP‐76 to endocytic vesicles. The endocytosis of active SLP‐76 and LAT complexes suggests a possible mechanism for downregulation of signaling complexes induced by TCR activation.

Solute diffusion and interactions in cross-linked poly(ethylene glycol) hydrogels studied by Fluorescence Correlation Spectroscopy

Controlled diffusion and release of soluble molecules is one of the key challenges in developing three-dimensional (3D) scaffolds for tissue engineering and drug delivery applications in part because current methods to measure dynamic transport properties are difficult to perform directly, are strongly affected by the experimental setup, and therefore can be a subject to various artifacts. In this work we present a method for direct measurement of translational diffusion of solutes, namely Fluorescence Correlation Spectroscopy (FCS), by characterizing the diffusion of model proteins through a 3D cross-linked poly(ethylene glycol) (PEG) hydrogel scaffold. We examined both the dynamics of hydrogel structure (e.g., cross-linking and swelling) as well as protein size and their effect on protein diffusivity. For example, we demonstrated that protein diffusivity was closely related to protein size as smaller proteins (e.g., lysozyme) diffused faster than larger proteins (e.g., γ-globulin or Ig). We validated the FCS protein diffusivity results by comparison to standard bulk diffusion assays. Additionally, due to the nature of FCS measurements, we were able to probe for hydrogel-protein interactions during cross-linking that may contribute to the obstructed protein diffusion in the 3D scaffold. We determined that such interactions in this system were not covalent (i.e., were independent of the cross-linking chemistry) but may be due to weaker hydrogen bonding or ionic interactions. Also, these interactions were protein specific and contributed up to 25% of the total decrease in protein diffusivity in the hydrogel as compared to diffusivity in water. Though interactions between various proteins and PEG have been reported, this is the first study that has explored these effects in detail in cross-linked PEG hydrogels using FCS; our findings question the assumption that PEG hydrogels are completely inert to protein interactions when applied as drug delivery matrices and tissue engineering scaffolds.

Bax activates endophilin B1 oligomerization and lipid membrane vesiculation

Endophilins participate in membrane scission events that occur during endocytosis and intracellular organelle biogenesis through the combined activity of an N-terminal BAR domain that interacts with membranes and a C-terminal SH3 domain that mediates protein binding. Endophilin B1 (Endo B1) was identified to bind Bax, a Bcl-2 family member that promotes apoptosis, through yeast two-hybrid protein screens. Although Endo B1 does not bind Bax in healthy cells, during apoptosis, Endo B1 interacts transiently with Bax and promotes cytochrome c release from mitochondria. To explore the molecular mechanism of action of Endo B1, we have analyzed its interaction with Bax in cell-free systems. Purified recombinant Endo B1 in solution displays a Stokes radius indicating a tetrameric quarternary structure. However, when incubated with purified Bax, it assembles into oligomers more than 4-fold greater in molecular weight. Although Endo B1 oligomerization is induced by Bax, Bax does not stably associate with the high molecular weight Endo B1 complex. Endo B1 oligomerization requires its C-terminal Src homology 3 domain and is not induced by Bcl-xL. Endo B1 combined with Bax reduces the size and changes the morphology of giant unilamellar vesicles by inducing massive vesiculation of liposomes. This activity of purified Bax protein to induce cell-free assembly of Endo B1 may reflect its activity in cells that regulates apoptosis and/or mitochondrial fusion.

Thin films of oriented collagen fibrils for cell motility studies.

Collagen films with oriented fibrils mimic tissues that have been remodeled by fibroblasts, which naturally tend to orient collagen fibrils in vivo. We have prepared thin films of ordered fibrils of collagen I, a major component of the extracellular matrix. The films were prepared by modifying a technique previously used to produce collagen I films for studies of cell morphology and intracellular signaling. By modifying the drying step, we were able to produce thin monolayers of collagen fibrils with consistent orientations over macroscopic (>100 microm) distances. We quantified the degree of orientation of the collagen fibrils using Fourier analysis of optical microscopy images. We also conducted experiments with vascular endothelial cells, and found that cell orientation and migration are well-correlated with fibril orientation. Using polarized cells, we showed oriented thin collagen film induces natural migration along the fibrils without using any sort of attractor. Taken together, these results demonstrate additional functionality and physiological relevance for a class of films being successfully applied in a variety of cell biology experiments.

Fluorescence Correlation Spectroscopy and Its Application to the Characterization of Molecular Properties and Interactions

Fluorescence correlation spectroscopy (FCS) utilizes temporal fluctuations in fluorescence emission to extract quantitative measures of inter- or intramolecular dynamics or molecular motions of probe molecules, which occur on submicrosecond to second timescales. In typical experiments, one can readily obtain the probes diffusion coefficient and concentration from small volumes of sample. Recent FCS applications have yielded information on interactions of the probe with changing or structured solvent, binding with other molecules, photophysical or conformational changes in the probe, polymerization, and other changes in the dynamics of the probe. In cross-correlation mode FCS promises to attract more applications as the technique can monitor interactions in a system with two or more probes with different fluorophores.

TOPOLOGY OF THE HETEROGENEOUS NATURE OF THE EXTRACELLULAR MATRIX ON STOCHASTIC MODELING OF TUMOR-INDUCED ANGIOGENESIS

We have modeled tumor-induced angiogenesis; our model includes the phenomena of the migratory response of endothelial cells (ECs) to tumor angiogenic factors, and the interaction of ECs with the extracellular matrix (ECM). ECs switch between growth, differentiation, motility, or apoptotic behavior in response to the local topology and composition of the ECM. Assuming the ECM medium as a statistically inhomogeneous medium (some area support sprout growth, some not), we show that the ECM can be a natural barrier to angiogenesis. We study vascular network formation for several ECM distributions and topologies, and we find an analogy with percolation. A threshold exists, under which sprouts cannot reach the tumor. During the growth of the vascular network, a competition exists between the attraction exerted by tumor and the preferred path created by the ECM. We also examined the influence of branching on the tumor vascularization. Branching is a natural phenomenon which helps the tumor become vascularized. By increasing the number of sprouts, the vascular network increases the probability of reaching the tumor, as it can explore more pathways. Our simulations show after two branching events, the vascular network is very likely to reach the tumor.

Effects of multiple scattering on fluorescence correlation spectroscopy measurements of particles moving within optically dense media

Abstract. Fluorescence correlation spectroscopy (FCS) is increasingly being used to assess the movement of particles diffusing in complex, optically dense surroundings, in which case measurement conditions may complicate data interpretation. It is considered how a single-photon FCS measurement can be affected if the sample properties result in scattering of the incident light. FCS autocorrelation functions of Atto 488 dye molecules diffusing in solutions of polystyrene beads are measured, which acted as scatterers. Data indicated that a scattering-linked increase in the illuminated volume, as much as two fold, resulted in minimal increase in diffusivity. To analyze the illuminated beam profile, Monte-Carlo simulations were employed, which indicated a larger broadening of the beam along the axial than the radial directions, and a reduction of the incident intensity at the focal point. The broadening of the volume in the axial direction has only negligible effect on the measured diffusion time, since intensity fluctuations due to diffusion events in the radial direction are dominant in FCS measurements. Collectively, results indicate that multiple scattering does not result in FCS measurement artifacts and thus, when sufficient signal intensity is attainable, single-photon FCS can be a useful technique for measuring probe diffusivity in optically dense media.

The Characterization of Biomolecular Interactions Using Fluorescence Fluctuation Techniques

Fluorescence correlation spectroscopy (FCS) has become one of the most popular methods available for investigating the physical properties of biomolecular complexes ever since it was first proposed by the groups of Elliot Elson and Watt Webb in the 1970s (Aragon and Pecora, 1974; Magde et al., 1974; Webb, 1976; Icenogle and Elson, 1983; Elson, 2001). Over the years, a number of thorough reviews have appeared of the now extensive literature (Madge, 1976; Webb, 1976; Berland et al., 1995; Rigler, 1995; Schwille, 2001; Hess et al., 2002; Thompson et al., 2002; Haustein and Schwille, 2003; Muller et al., 2003; Elson, 2004; Haustein and Schwille, 2004).

Imaging and tracking HIV viruses in human cervical mucus

Abstract. We describe a systematic approach to image, track, and quantify the movements of HIV viruses embedded in human cervical mucus. The underlying motivation for this study is that, in HIV-infected adults, women account for more than half of all new cases and most of these women acquire the infection through heterosexual contact. The endocervix is believed to be a susceptible site for HIV entry. Cervical mucus, which coats the endocervix, should play a protective role against the viruses. Thus, we developed a methodology to apply time-resolved confocal microscopy to examine the motion of HIV viruses that were added to samples of untreated cervical mucus. From the images, we identified the viruses, tracked them over time, and calculated changes of the statistical mean-squared displacement (MSD) of each virus. Approximately half of tracked viruses appear constrained while the others show mobility with MSDs that are proportional to τα+ν2τ2, over time range τ, depicting a combination of anomalous diffusion (0<α<0.4) and flow-like behavior. The MSD data also reveal plateaus attributable to possible stalling of the viruses. Although a more extensive study is warranted, these results support the assumption of mucus being a barrier against the motion of these viruses.