CENP-C/H/I/K/M/T/W/N/L and hMis12 but not CENP-S/X participate in complex formation in the nucleoplasm of living human interphase cells outside centromeres

<div><p>Kinetochore proteins assemble onto centromeric chromatin and regulate DNA segregation during cell division. The inner kinetochore proteins bind centromeres while most outer kinetochore proteins assemble at centromeres during mitosis, connecting the complex to microtubules. Here, we measured the co-migration between protein pairs of the constitutive centromere associated network (CCAN) and hMis12 complexes by fluorescence cross-correlation spectroscopy (FCCS) in the nucleoplasm outside centromeres in living human interphase cells. FCCS is a method that can tell if in living cells two differently fluorescently labelled molecules migrate independently, or co-migrate and thus are part of one and the same soluble complex. We also determined the apparent dissociation constants (K_d) of the hetero-dimers CENP-T/W and CENP-S/X. We measured co-migration between CENP-K and CENP-T as well as between CENP-M and CENP-T but not between CENP-T/W and CENP-S/X. Furthermore, CENP-C co-migrated with CENP-H, and CENP-K with CENP-N as well as with CENP-L. Thus, in the nucleoplasm outside centromeres, a large fraction of the CENP-H/I/K/M proteins interact with CENP-C, CENP-N/L and CENP-T/W but not with CENP-S/X. Our FCCS analysis of the Mis12 complex showed that hMis12, Nsl1, Dsn1 and Nnf1 also form a complex outside centromeres of which at least hMis12 associated with the CENP-C/H/I/K/M/T/W/N/L complex.</p></div

SW-FCCS analysis of CENP-T and CENP-W.

<p>(a): ACF curves of EGFP-(s)-CENP-W (green) and mCherry-(s)-CENP-T (red), and CCF curves (blue, purple) in the nucleoplasm of interphase HEK293 cells. The data show cross-correlation between CENP-T and CENP-W, indicating interaction. (b) and (c): <i>K</i>_<i>d</i> determination using Scatter plot (b) and a histogram (c) of multiple SW-FCCS measurements to determine an effective <i>K</i>_<i>d</i> of the interaction. (d): ACF curves of EGFP-(s)-CENP-T^∆N (green) and mCherry-(s)-CENP-W (red), and CCF curves (blue, purple) in the nucleoplasm of interphase HEK293 cells. The data show reduced cross-correlation between CENP-T^∆N and CENP-W. (e) and (f): <i>K</i>_<i>d</i> determination using Scatter plot (e) and a histogram (f) of multiple SW-FCCS measurements to determine the effective <i>K</i>_<i>d</i> of this interaction between the histone-fold domain of CENP-T (CENP-T^∆N) and CENP-W. A defined interaction is detected by both, the linear fit of the scatter plot as well as the log-normal fit of the histogram.</p

DC-FCCS of EGFP-(s)-CENP-T and mCherry-(s)-CENP-S.

<p>A) Displayed are G versus lag time. Red: FCS- or autocorrelation-curve G (τ) for mCherry, green: FCS- or autocorrelation-curve G (τ) for EGFP, black: cross-correlation-curve G (τ), AC = autocorrelation. CC = cross-correlation, A(AC) = amplitude of autocorrelation curve, A(CC) = amplitude of cross-correlation curve. The cross-correlation analyses are amplified in inserts a. Count rates are displayed over 1 sec (inserts b; green = EGFP and red = mCherry). For the pair EGFP-(s)-CENP-T and mCherry-(s)-CENP-S no indication for complex formation in the nucleoplasm was detected (A(CC)/A(AC_mCherry) = 0%). The cross-correlation analysis (with a magnified scale of G (τ); insert a) resulted in a correlation of 1.001, whereas the autocorrelations yielded 1.322 for EGFP-(s)-CENP-T and 1.106 for mCherry-(s)-CENP-S. This ratio indicates that no nucleoplasmic CENP-T and -S are part of a common complex. B) Localization of cotransfected EGFP-(s)-CENP-T (EGFP) and mCherry-(s)-CENP-S (mCherry) in living human HEp-2 cells which were used for FCCS analysis. White bar = 10 μm. A cell nucleus is displayed showing co-localisation at centromeres (merge) and weak fluorescence in the nucleoplasm. Two locations of the same size and shape, a centromere (spot 1) and the centromere-free position of an FCCS measurement, as shown in Fig 4A (spot 2), in the nucleoplasm were selected for fluorescence intensity analysis. For the analyzed centromere in spot 1 the ratios of nucleoplasmic to centromeric fluorescence intensities was 1:43 for EGFP-(s)-CENP-T and 1:33 for mCherry-(s)-CENP-S. The concentrations of nucleoplasmic proteins, estimated by FCCS, was 6 nM for EGFP-(s)-CENP-T and 14 nM for mCherry-(s)-CENP-S.</p

Cross-correlation by DC-FCCS.

<p>Cross-correlation by DC-FCCS.</p

Protein-protein co-migration of CCAN and Mis12 proteins.

<p>The degree of co-migration (corrected) in the nucleoplasm outside centromeres of human interphase cells is color-coded (red dashed arrows: no or hardly detectable co-migration, green arrow: 5–30%, blue arrows: 30–60%, black arrows: above 60% co-migration). Please note: these green, blue or black arrows do not necessarily indicate direct protein-protein interaction; the labeled proteins co-migrate in a complex: their interaction might be either direct or mediated by a third (or more) protein.</p

Cross-correlation by SW-FCCS.

<p>Cross-correlation by SW-FCCS.</p

Accuracy and Precision in Camera-Based Fluorescence Correlation Spectroscopy Measurements

Imaging fluorescence correlation spectroscopy (FCS) performed using array detectors has been successfully used to quantify the number, mobility, and organization of biomolecules in cells and organisms. However, there have not been any systematic studies on the errors in these estimates that are introduced due to instrumental and experimental factors. State-of-the-art array detectors are still restricted in the number of frames that can be recorded per unit time, sensitivity and noise characteristics, and the total number of frames that can be realistically recorded. These limitations place constraints on the time resolution, the signal-to-noise ratio, and the total measurement time, respectively. This work addresses these problems by using a combination of simulations and experiments on lipid bilayers to provide characteristic performance parameters and guidelines that govern accuracy and precision of diffusion coefficient and concentration measurements in camera-based FCS. We then proceed to demonstrate the effects of these parameters on the capability of camera-based FCS to determine membrane heterogeneity via the FCS diffusion laws, showing that there is a lower length scale limit beyond which membrane organization cannot be detected and which can be overcome by choosing suitable experimental parameters. On the basis of these results, we provide guidelines for an efficient experimental design for camera-based FCS to extract information on mobility, concentration, and heterogeneity

Bayesian Approach to the Analysis of Fluorescence Correlation Spectroscopy Data II: Application to Simulated and In Vitro Data

Fluorescence correlation spectroscopy (FCS) is a powerful approach to characterizing the binding and transport dynamics of macromolecules. The unbiased interpretation of FCS data relies on the evaluation of multiple competing hypotheses to describe an underlying physical process under study, which is typically unknown a priori. Bayesian inference provides a convenient framework for this evaluation based on the temporal autocorrelation function (TACF), as previously shown theoretically using model TACF curves (He, J.; Guo, S.; Bathe, M. Anal. Chem. 2012, 84). Here, we apply this procedure to simulated and experimentally measured photon-count traces analyzed using a multitau correlator, which results in complex noise properties in TACF curves that cannot be modeled easily. As a critical component of our technique, we develop two means of estimating the noise in TACF curves based either on multiple independent TACF curves themselves or a single raw underlying intensity trace, including a general procedure to ensure that independent, uncorrelated samples are used in the latter approach. Using these noise definitions, we demonstrate that the Bayesian approach selects the simplest hypothesis that describes the FCS data based on sampling and signal limitations, naturally avoiding overfitting. Further, we show that model probabilities computed using the Bayesian approach provide a reliability test for the downstream interpretation of model parameter values estimated from FCS data. Our procedure is generally applicable to FCS and image correlation spectroscopy and therefore provides an important advance in the application of these methods to the quantitative biophysical investigation of complex analytical and biological systems

Mitochondrial Routing of Glucose and Sucrose Polymers after Pinocytotic Uptake: Avenues for Drug Delivery

Mitochondria are key organelles organizing cellular metabolic flux. Therefore, a targeted drug delivery to mitochondria promises the advancement of medicine in fields that are associated with mitochondrial dysfunction. However, successful mitochondrial drug delivery is limited by complex transport steps across organelle membranes and fast drug efflux in cases of multidrug resistance. Strategies to deliver small-molecular-weight drugs to mitochondria are very limited, while the use of complex polymeric carriers is limited by a lack of clinical feasibility. We show here that clinically established macromolecules such as a sucrose copolymer (Ficoll 70/400 kDa) and polyglucose (dextran 70/500 kDa) are micropinocytosed swiftly by mesenchymal stem cells and subsequently routed to mitochondria. The intracellular level of Ficoll appears to decrease over time, suggesting that it does not persist within cells. After coupling to polysucrose, the low-molecular-weight photodynamic drug Rose Bengal reached mitochondria and thus exhibited an increased destructive potential after laser excitation. These findings support new opportunities to deliver already clinically approved drugs to mitochondria

A Funneled Conformational Landscape Governs Flavivirus Fusion Peptide Interaction with Lipid Membranes

During host cell infection by flaviviruses such as dengue and Zika, acidic pH within the endosome triggers a conformational change in the envelope protein on the outer surface of the virion. This results in exposure of the ∼15 residue fusion peptide (FP) region, freeing it to induce fusion between the viral and endosomal membranes. A better understanding of the conformational dynamics of the FP in the presence of membranes, and the basis for its selectivity for anionic lipid species present within the endosome, would facilitate its therapeutic targeting with antiviral drugs and antibodies. In this work, multiscale modeling, simulations, and free energy calculations (including a total of ∼75 μs of atomic-resolution sampling), combined with imaging total internal reflection fluorescence correlation spectroscopy experiments, were employed to investigate the mechanisms of interaction of FP variants with lipid bilayers. Wild-type FPs (in the presence or absence of a fluorescein isothiocyanate tag) were shown to possess a funneled conformational landscape governing their exit from solvent and penetration into the lipid phase and to exhibit an electrostatically favored >2-fold affinity for membranes containing anionic species over purely zwitterionic ones. Conversely, the landscape was abolished in a nonfunctional point mutant, leading to a 2-fold drop in host membrane affinity. Collectively, our data reveal how the highly conserved flavivirus FP has evolved to funnel its conformational space toward a maximally fusogenic state anchored within the endosomal membrane. Therapeutically targeting the accessible ensemble of FP conformations may represent a new, rational strategy for blocking viral infection