Moshe Lindner

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.

HU Protein Induces Incoherent DNA Persistence Length

HU is a highly conserved protein that is believed to play an important role in the architecture and dynamic compaction of bacterial DNA. Its ability to control DNA bending is crucial for functions such as transcription and replication. The effects of HU on the DNA structure have been studied so far mainly by single molecule methods that require us to apply stretching forces on the DNA and therefore may perturb the DNA-protein interaction. To overcome this hurdle, we study the effect of HU on the DNA structure without applying external forces by using an improved tethered particle motion method. By combining the results with DNA curvature analysis from atomic force microscopy measurements we find that the DNA consists of two different curvature distributions and the measured persistence length is determined by their interplay. As a result, the effective persistence length adopts a bimodal property that depends primarily on the HU concentration. The results can be explained according to a recently suggested model that distinguishes single protein binding from cooperative protein binding.

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.

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.

Protein-DNA Interactions Studies with Single Tethered Molecule Techniques

The last decade has seen a leap forward in the understanding of molecular and cellular mechanisms with the development of advanced techniques for observing, manipulating and imaging single molecules. In contrast to conventional biochemical techniques which yield information derived from population averages, single molecule techniques give access to the dynamics and properties of individual biomolecules in situ.

Plasmonic Scattering as an Efficient Tool for a Force-Free Technique to Follow Single DNA Molecules

One of the promising methods for single molecule studies is Tethered Particle Motion (TPM). The technique layout that was developed a decade ago (1), is based on anchoring one end of a DNA molecule (or any other polymer of interest) to a surface, and labeling the opposite end with an optical marker. In solution, the marker moves randomly in a volume that is governed by the restrictions set by the DNA molecule. Tracking and analyzing its position distribution provides an essential tool to follow the dynamics of the DNA conformations (2, 3). Most of the TPM systems use a CCD camera to detect the projected position of the bead on a two-dimensional plane, and the information on the bead height above the surface is lost. Here we show how TPM can be exploited for three-dimensional particle tracking using Total Internal Reflection (TIR) (4) illumination system. We also report of the deviations between the lateral distribution and the axial distribution.

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.