Irena Bronshtein

Loss of lamin A function increases chromatin dynamics in the nuclear interior

Chromatin is organized in a highly ordered yet dynamic manner in the cell nucleus, but the principles governing this organization remain unclear. Similarly, it is unknown whether, and how, various proteins regulate chromatin motion and as a result influence nuclear organization. Here by studying the dynamics of different genomic regions in the nucleus of live cells, we show that the genome has highly constrained dynamics. Interestingly, depletion of lamin A strikingly alters genome dynamics, inducing a dramatic transition from slow anomalous diffusion to fast and normal diffusion. In contrast, depletion of LAP2α, a protein that interacts with lamin A and chromatin, has no such effect on genome dynamics. We speculate that chromosomal inter-chain interactions formed by lamin A throughout the nucleus contribute to chromatin dynamics, and suggest that the molecular regulation of chromatin diffusion by lamin A in the nuclear interior is critical for the maintenance of genome organization.

Universal Algorithm for Identification of Fractional Brownian Motion. A Case of Telomere Subdiffusion

We present a systematic statistical analysis of the recently measured individual trajectories of fluorescently labeled telomeres in the nucleus of living human cells. The experiments were performed in the U2OS cancer cell line. We propose an algorithm for identification of the telomere motion. By expanding the previously published data set, we are able to explore the dynamics in six time orders, a task not possible earlier. As a result, we establish a rigorous mathematical characterization of the stochastic process and identify the basic mathematical mechanisms behind the telomere motion. We find that the increments of the motion are stationary, Gaussian, ergodic, and even more chaotic--mixing. Moreover, the obtained memory parameter estimates, as well as the ensemble average mean square displacement reveal subdiffusive behavior at all time spans. All these findings statistically prove a fractional Brownian motion for the telomere trajectories, which is confirmed by a generalized p-variation test. Taking into account the biophysical nature of telomeres as monomers in the chromatin chain, we suggest polymer dynamics as a sufficient framework for their motion with no influence of other models. In addition, these results shed light on other studies of telomere motion and the alternative telomere lengthening mechanism. We hope that identification of these mechanisms will allow the development of a proper physical and biological model for telomere subdynamics. This array of tests can be easily implemented to other data sets to enable quick and accurate analysis of their statistical characteristics.

On the Correlation Between Hydrophobicity, Liposome Binding and Cellular Uptake of Porphyrin Sensitizers

Abstract A crucial factor in choosing a porphyrin or analogous photosensitizer for photodynamic therapy (PDT) is its ability to incorporate into the cells. For hydrophobic compounds that partition passively into the cytoplasmic membrane, a partition coefficient between an organic solvent and water, P, is one factor that could be used to predict the molecules ability to diffuse into biomembranes. We synthesized several porphyrins, modified with two, three or four meso-substituents and studied their spectroscopic and photophysical properties. The octanol–water partitioning coefficients, log P, were calculated as a parameter for hydrophobicity. We found these porphyrins to be very hydrophobic, with log P values in the range of 8.9–11.8. These were correlated with the binding constants of these porphyrins into liposomes, Kb, as well as to their uptake by cells. The correlation between the estimated log P and Kb is nearly linear but negative, indicating, apparently, that there is lesser binding to liposomes with increased hydrophobicity. On the other hand, all of the studied porphyrins are taken up by cells, but there is no clear correlation between cellular uptake and the log P or Kb. Lipinskis pharmacological “rule of 5” predicts poor permeation of drugs into cells when log P is greater than five. This may be relevant for diffusional binding to liposomes, where aqueous aggregation can interfere strongly with cellular uptake. In such extreme conditions, neither liposome binding nor other rules seem to predict porphyrin behavior in vitro.

Improved estimation of anomalous diffusion exponents in single-particle tracking experiments

The mean square displacement is a central tool in the analysis of single-particle tracking experiments, shedding light on various biophysical phenomena. Frequently, parameters are extracted by performing time averages on single-particle trajectories followed by ensemble averaging. This procedure, however, suffers from two systematic errors when applied to particles that perform anomalous diffusion. The first is significant at short-time lags and is induced by measurement errors. The second arises from the natural heterogeneity in biophysical systems. We show how to estimate and correct these two errors and improve the estimation of the anomalous parameters for the whole particle distribution. As a consequence, we manage to characterize ensembles of heterogeneous particles even for rather short and noisy measurements where regular time-averaged mean square displacement analysis fails. We apply this method to both simulations and in vivo measurements of telomere diffusion in 3T3 mouse embryonic fibroblast cells. The motion of telomeres is found to be subdiffusive with an average exponent constant in time. Individual telomere exponents are normally distributed around the average exponent. The proposed methodology has the potential to improve experimental accuracy while maintaining lower experimental costs and complexity.

In vitro and in vivo photosensitization by protoporphyrins possessing different lipophilicities and vertical localization in the membrane

Abstract Photodynamic therapy (PDT) is being evaluated in clinical trials for treatment of various oncologic and ophthalmic diseases. The main cause for cell inactivation and retardation of tumor growth after photoactivation of sensitizers is very short-lived singlet oxygen molecules that are produced and have limited diffusion distances. In this paper we show that the extent of biological damage can be modulated by using protoporphyrin, which was modified to increase its lipophilicity, and which also places the tetrapyrrole core deeper within the membrane by the carboxylate groups being anchored at the lipid:water interface. The uptake of the parent molecule (PPIX) and its diheptanoic acid analogue (PPIXC6) by WiDR and CT26 cells was investigated by fluorescence microscopy and by fluorescence intensity from the cells. The uptake of PPIXC6 increased almost linearly with incubation length for over 24 h, whereas for PPIX only 1 h was needed to reach maximal intracellular concentration. Fluorescence microscopy of both cell lines indicated that both drugs were distributed diffusely in the plasma membrane and cytoplasm, but remained outside the nucleus. The efficiency of in vitro inactivation of WiDr and CT26 cells increased with the length of the alkylcarboxylic chain. Tumors in mice that were treated with PPIX-PDT grew more slowly than control tumors. However, tumors that were given PPIXC6 followed by light exposure showed a significant delay in their growth.

Exploring chromatin organization mechanisms through its dynamic properties

Abstract The organization of the genome in the nucleus is believed to be crucial for different cellular functions. It is known that chromosomes fold into distinct territories, but little is known about the mechanisms that maintain these territories. To explore these mechanisms, we used various live-cell imaging methods, including single particle tracking to characterize the diffusion properties of different genomic regions in live cells. Chromatin diffusion is found to be slow and anomalous; in vast contrast, depletion of lamin A protein significantly increases chromatin motion, and the diffusion pattern of chromatin transforms from slow anomalous to fast normal. More than this, depletion of lamin A protein also affects the dynamics of nuclear bodies. Our findings indicate that chromatin motion is mediated by lamin A and we suggest that constrained chromatin mobility allows to maintain chromosome territories. Thus, the discovery of this function of nucleoplasmic lamin A proteins sheds light on the maintenance mechanism of chromosome territories in the interphase nucleus, which ensures the proper function of the genome.

The binding of analogs of porphyrins and chlorins with elongated side chains to albumin

In previous studies, we demonstrated that elongation of side chains of several sensitizers endowed them with higher affinity for artificial and natural membranes and caused their deeper localization in membranes. In the present study, we employed eight hematoporphyrin and protoporphyrin analogs and four groups containing three chlorin analogs each, all synthesized with variable numbers of methylenes in their alkyl carboxylic chains. We show that these tetrapyrroles’ affinity for bovine serum albumin (BSA) and their localization in the binding site are also modulated by chain lengths. The binding constants of the hematoporphyrins and protoporphyrins to BSA increased as the number of methylenes was increased. The binding of the chlorins depended on the substitution at the meso position opposite to the chains. The quenching of the sensitizers’ florescence by external iodide ions decreased as the side chains became longer, indicating to deeper insertion of the molecules into the BSA binding pocket. To corroborate this conclusion, we studied the efficiency of photodamage caused to tryptophan in BSA upon illumination of the bound sensitizers. The efficiency was found to depend on the side-chain lengths of the photosensitizer. We conclude that the protein site that hosts these sensitizers accommodates different analogs at positions that differ slightly from each other. These differences are manifested in the ease of access of iodide from the external aqueous phase, and in the proximity of the photosensitizers to the tryptophan. In the course of this study, we developed the kinetic equations that have to be employed when the sensitizer itself is being destroyed.

Genome organization in the nucleus: From dynamic measurements to a functional model

A biological system is by definition a dynamic environment encompassing kinetic processes that occur at different length scales and time ranges. To explore this type of system, spatial information needs to be acquired at different time scales. This means overcoming significant hurdles, including the need for stable and precise labeling of the required probes and the use of state of the art optical methods. However, to interpret the acquired data, biophysical models that can account for these biological mechanisms need to be developed. The structure and function of a biological system are closely related to its dynamic properties, thus further emphasizing the importance of identifying the rules governing the dynamics that cannot be directly deduced from information on the structure itself. In eukaryotic cells, tens of thousands of genes are packed in the small volume of the nucleus. The genome itself is organized in chromosomes that occupy specific volumes referred to as chromosome territories. This organization is preserved throughout the cell cycle, even though there are no sub-compartments in the nucleus itself. This organization, which is still not fully understood, is crucial for a large number of cellular functions such as gene regulation, DNA breakage repair and error-free cell division. Various techniques are in use today, including imaging, live cell imaging and molecular methods such as chromosome conformation capture (3C) methods to better understand these mechanisms. Live cell imaging methods are becoming well established. These include methods such as Single Particle Tracking (SPT), Continuous Photobleaching (CP), Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Correlation Spectroscopy (FCS) that are currently used for studying proteins, RNA, DNA, gene loci and nuclear bodies. They provide crucial information on its mobility, reorganization, interactions and binding properties. Here we describe how these dynamic methods can be used to gather information on genome organization, its stabilization mechanisms and the proteins that take part in it.

Studies of Single Molecules in their Natural Form

Single molecule studies make possible the characterization of molecu- lar processes and the identification of biophysical sub-populations that are not acces - sible through ensemble studies. We describe tethered particle motion, a method that allows one to study single molecules in their natural form without having to apply any external forces. The method combines darkfield microscopy with a metal nano- bead. It permits the study of the biophysical properties of the tethered particles, as well as protein-DNA interactions. The method is not suitable for in vivo studies, and we therefore describe two other methods that are appropriate for live-cell imaging.

Study of Nuclear Organization through the Dynamic Properties of Chromatin

Chromosomes occupy specific nuclear volumes called chromosome territories and their motion is highly constrained. Little is known about which proteins and structures organize chromosome territories. A major object of our research is to understand the biophysical mechanisms that maintain this organization. We turned to study the diffusion properties of genome in order to shed light on this maintenance mechanism. The diffusion character of species depends on its properties and on the environment, thereby providing an excellent method for studying the nuclear maintenance mechanism. We examined genome mobility by focusing on three different genomic elements: telomeres, centromeres and specific gene loci. We developed method that allows measuring the diffusion in time-range of 10−2 - 104 sec. Such broad time range allowed us to identify the transient anomalous diffusion of different genomic regions that could not be identified by other techniques. Anomalous diffusion usually depends on environmental constrains, such as temporal binding. Therefore, we propose a model for chromatin organization maintenance in the nucleus that is based on temporal binding of chromatin to itself, or to other nuclear entities. In order to prove this hypothesis, we decided to focus on identifying the possible molecular source of the suggested binding. We conduct our research on measuring the effect of loss of Lamin A on chromatins diffusion properties. We found that telomeres and centromes motion in cells without Lamin A is ∼8 times less constrained compared to normal cells. It also shows normal diffusion, while in normal cells diffusion was found anomalous. Based on our results we can conclude that lack of Lamin A leads to looser chromatin. Finding other proteins that are responsible for such binding is a great challenge that we are now pursuing.