Dissecting the binding mechanism of the linker histone in live cells: an integrated FRAP analysis

The linker histone H1 has a fundamental role in DNA compaction. Although models for H1 binding generally involve the H1 C‐terminal tail and sites S1 and S2 within the H1 globular domain, there is debate about the importance of these binding regions and almost nothing is known about how they work together. Using a novel fluorescence recovery after photobleaching (FRAP) procedure, we have measured the affinities of these regions individually, in pairs, and in the full molecule to demonstrate for the first time that binding among several combinations is cooperative in live cells. Our analysis reveals two preferred H1 binding pathways and we find evidence for a novel conformational change required by both. These results paint a complex, highly dynamic picture of H1–chromatin binding, with a significant fraction of H1 molecules only partially bound in metastable states that can be readily competed against. We anticipate the methods we have developed here will be broadly applicable, particularly for deciphering the binding kinetics of other nuclear proteins that, similar to H1, interact with and modify chromatin.     

Messages Do Diffuse Faster than Messengers: Reconciling Disparate Estimates of the Morphogen Bicoid Diffusion Coefficient

The gradient of Bicoid (Bcd) is key for the establishment of the anterior-posterior axis in Drosophila embryos. The gradient properties are compatible with the SDD model in which Bcd is synthesized at the anterior pole and then diffuses into the embryo and is degraded with a characteristic time. Within this model, the Bcd diffusion coefficient is critical to set the timescale of gradient formation. This coefficient has been measured using two optical techniques, Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Correlation Spectroscopy (FCS), obtaining estimates in which the FCS value is an order of magnitude larger than the FRAP one. This discrepancy raises the following questions: which estimate is "correct''''; what is the reason for the disparity; and can the SDD model explain Bcd gradient formation within the experimentally observed times? In this paper, we use a simple biophysical model in which Bcd diffuses and interacts with binding sites to show that both the FRAP and the FCS estimates may be correct and compatible with the observed timescale of gradient formation. The discrepancy arises from the fact that FCS and FRAP report on different effective (concentration dependent) diffusion coefficients, one of which describes the spreading rate of the individual Bcd molecules (the messengers) and the other one that of their concentration (the message). The latter is the one that is more relevant for the gradient establishment and is compatible with its formation within the experimentally observed times.     

Fluorescence Correlation Spectroscopy Measurements of the Membrane Protein TetA in Escherichia coli Suggest Rapid Diffusion at Short Length Scales

Structural inhomogeneities in biomembranes can lead to complex diffusive behavior of membrane proteins that depend on the length or time scales that are probed. This effect is well studied in eukaryotic cells, but has been explored only recently in bacteria. Here we used fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) to study diffusion of the membrane protein TetA-YFP in E. coli. We find that the diffusion constant determined from FRAP is comparable to other reports of inner membrane protein diffusion constants in E. coli. However, FCS, which probes diffusion on shorter length scales, gives a value that is almost two orders of magnitude higher and is comparable to lipid diffusion constants. These results suggest there is a population of TetA-YFP molecules in the membrane that move rapidly over short length scales (∼ 400 nm) but move significantly more slowly over the longer length scales probed by FRAP.     

Measuring Mobility in Chromatin by Intensity-Sorted FCS

The architectural organization of chromatin can play an important role in genome regulation by affecting the mobility of molecules within its surroundings via binding interactions and molecular crowding. The diffusion of molecules at specific locations in the nucleus can be studied by fluorescence correlation spectroscopy (FCS), a well-established technique based on the analysis of fluorescence intensity fluctuations detected in a confocal observation volume. However, detecting subtle variations of mobility between different chromatin regions remains challenging with currently available FCS methods. Here, we introduce a method that samples multiple positions by slowly scanning the FCS observation volume across the nucleus. Analyzing the data in short time segments, we preserve the high temporal resolution of single-point FCS while probing different nuclear regions in the same cell. Using the intensity level of the probe (or a DNA marker) as a reference, we efficiently sort the FCS segments into different populations and obtain average correlation functions that are associated to different chromatin regions. This sorting and averaging strategy renders the method statistically robust while preserving the observation of intranuclear variations of mobility. Using this approach, we quantified diffusion of monomeric GFP in high versus low chromatin density regions. We found that GFP mobility was reduced in heterochromatin, especially within perinucleolar heterochromatin. Moreover, we found that modulation of chromatin compaction by ATP depletion, or treatment with solutions of different osmolarity, differentially affected the ratio of diffusion in both regions. Then, we used the approach to probe the mobility of estrogen receptor-α in the vicinity of an integrated multicopy prolactin gene array. Finally, we discussed the coupling of this method with stimulated emission depletion FCS for performing FCS at subdiffraction spatial scales.     

The nucleolar SUMO-specific protease SMT3IP1/SENP3 attenuates Mdm2-mediated p53 ubiquitination and degradation

SUMO (small ubiquitin-like modifier) modification plays multiple roles in several cellular processes. Sumoylation is reversibly regulated by SUMO-specific proteases. SUMO-specific proteases have recently been implicated in cell proliferation and early embryogenesis, but the underlying mechanisms remain unknown. Here, we show that a nucleolar SUMO-specific protease, SMT3IP1/SENP3, controls the p53–Mdm2 pathway. We found that SMT3IP1 interacts with p53 and Mdm2, and desumoylates both proteins. Overexpression of SMT3IP1 in cells resulted in the accumulation of Mdm2 in the nucleolus and increased stability of the p53 protein. In addition, SMT3IP1 bound to the acidic domain of Mdm2, which also mediates the p53 interaction, and competed with p53 for binding. Increasing expression of SMT3IP1 suppressed Mdm2-mediated p53 ubiquitination and subsequent proteasomal degradation. Interestingly, the desumoylation activity of SMT3IP1 was not necessary for p53 stabilization. These results suggest that SMT3IP1 is a new regulator of the p53–Mdm2 pathway.     

On the structure and dynamics of the complex of the nucleosome and the linker histone

Several different models of the linker histone (LH)–nucleosome complex have been proposed, but none of them has unambiguously revealed the position and binding sites of the LH on the nucleosome. Using Brownian dynamics-based docking together with normal mode analysis of the nucleosome to account for the flexibility of two flanking 10 bp long linker DNAs (L-DNA), we identified binding modes of the H5-LH globular domain (GH5) to the nucleosome. For a wide range of nucleosomal conformations with the L-DNA ends less than 65 Å apart, one dominant binding mode was identified for GH5 and found to be consistent with fluorescence recovery after photobleaching (FRAP) experiments. GH5 binds asymmetrically with respect to the nucleosomal dyad axis, fitting between the nucleosomal DNA and one of the L-DNAs. For greater distances between L-DNA ends, docking of GH5 to the L-DNA that is more restrained and less open becomes favored. These results suggest a selection mechanism by which GH5 preferentially binds one of the L-DNAs and thereby affects DNA dynamics and accessibility and contributes to formation of a particular chromatin fiber structure. The two binding modes identified would, respectively, favor a tight zigzag chromatin structure or a loose solenoid chromatin fiber.     

Methods for measuring rates of protein binding to insoluble scaffolds in living cells: histone H1-chromatin interactions

Understanding of cell regulation is limited by our inability to measure molecular binding rates for proteins within the structural context of living cells, and many systems biology models are hindered because they use values obtained with molecules binding in solution. Here, we present a kinetic analysis of GFP-histone H1 binding to chromatin within nuclei of living cells that allows both the binding rate constant k(ON) and dissociation rate constant k(OFF) to be determined based on data obtained from fluorescence recovery after photobleaching (FRAP) analysis. This is accomplished by measuring the ratio of bound to free concentration of protein at steady state, and identifying the rate-determining step during FRAP recovery experimentally, combined with mathematical modeling. We report k(OFF) = 0.0131/s and k(ON) = 0.14/s for histone H1.1 binding to chromatin. This work brings clarity to the interpretation of FRAP experiments and provides a way to determine binding kinetics for nuclear proteins and other cellular molecules that interact with insoluble scaffolds within living cells.     

Measuring antibody affinity and performing immunoassay at the single molecule level

Fluorescence correlation spectroscopy (FCS) enables direct observation of the translational diffusion of single fluorescent molecules in solution. When fluorescent hapten binds to antibody, analysis of FCS data yields the fractional amounts of free and bound hapten, allowing determination of the equilibrium binding constant. Equilibrium dissociation constants of anti-digoxin antibodies and corresponding fluorescein-labeled digoxigenin obtained by FCS and fluorescence polarization measurements are identical. It is also possible to follow a competitive displacement of the tracer from the antibody by unlabeled hapten using FCS in an immunoassay format. The fluorescence polarization immunoassay for vancomycin detection was used to test the FCS approach. Fitting of the FCS data for the molar fractions of free and bound fluorescein-labeled vancomycin yielded a calibration curve which could serve for determination of the vancomycin concentration in biological samples.     

Early identification of false positives in high-throughput screening for activators of p53-DNA interaction

Naturally occurring mutant forms of p53 are deficient for specific DNA binding. However, their specific DNA binding can be reactivated. The search for small molecules that reactivate latent p53 is considered to be a cornerstone in cancer therapy. The authors describe a new homogeneous fluorescent assay approach for the characterization of binding affinities of human wild-type latent and activated p53 using DNA(*)spec(26), with and without the addition of the antibody PAb421, respectively, and fluorescence correlation spectroscopy (FCS)/2-dimensional fluorescence-intensity distribution analysis anisotropy as the detection methods. FCS was compared with 2D-FIDA anisotropy, and a very good correlation of the results with both readouts was observed (K(D)s for nonspecific DNA binding of 24.4+/-3.5 nM with 2D-FIDA anisotropy and of 29.5+/-5.5 nM with FCS). The presence of poly(dI-dC) led to a 10-fold increase of binding affinity (K(D) of 3.3+/-0.5 nM in the presence of PAb421). 2D-FIDA anisotropy was demonstrated to be the most accurate readout; hence, this detection technology was selected for a 25,000 compound member high-throughput screening (HTS) campaign. The hits obtained were qualified using a novel data evaluation algorithm that identifies false positives and moreover assesses the validity of true hits in the presence of the deteriorating artifact. This process step is of utmost importance for decreasing the attrition in fluorescence-based HTS.     

An automated fluorescence videomicroscopy assay for the detection of mitotic catastrophe

Mitotic catastrophe can be defined as a cell death mode that occurs during or shortly after a prolonged/aberrant mitosis, and can show apoptotic or necrotic features. However, conventional procedures for the detection of apoptosis or necrosis, including biochemical bulk assays and cytofluorometric techniques, cannot discriminate among pre-mitotic, mitotic and post-mitotic death, and hence are inappropriate to monitor mitotic catastrophe. To address this issue, we generated isogenic human colon carcinoma cell lines that differ in ploidy and p53 status, yet express similar amounts of fluorescent biosensors that allow for the visualization of chromatin (histone H2B coupled to green fluorescent protein (GFP)) and centrosomes (centrin coupled to the Discosoma striata red fluorescent protein (DsRed)). By combining high-resolution fluorescence videomicroscopy and automated image analysis, we established protocols and settings for the simultaneous assessment of ploidy, mitosis, centrosome number and cell death (which in our model system occurs mainly by apoptosis). Time-lapse videomicroscopy showed that this approach can be used for the high-throughput detection of mitotic catastrophe induced by three mechanistically distinct anti-mitotic agents (dimethylenastron (DIMEN), nocodazole (NDZ) and paclitaxel (PTX)), and – in this context – revealed an important role of p53 in the control of centrosome number.