High mobility group (HMG) proteins of the HMGB family are chromatin-associated proteins that as architectural factors are involved in the regulation of transcription and other DNA-dependent processes. HMGB proteins are generally considered nuclear proteins, although mammalian HMGB1 can also be detected in the cytoplasm and outside of cells. Plant HMGB proteins studied so far were found exclusively in the cell nucleus. Using immunofluorescence and fluorescence microscopy of HMGB proteins fused to the green fluorescent protein, we have examined the subcellular localization of the Arabidopsis (Arabidopsis thaliana) HMGB2/3 and HMGB4 proteins, revealing that, in addition to a prominent nuclear localization, they can be detected also in the cytoplasm. The nucleocytoplasmic distribution appears to depend on the cell type. By time-lapse fluorescence microscopy, it was observed that the HMGB2 and HMGB4 proteins tagged with photoactivatable green fluorescent protein can shuttle between the nucleus and the cytoplasm, while HMGB1 remains nuclear. The balance between the basic amino-terminal and the acidic carboxyl-terminal domains flanking the central HMG box DNA-binding domain critically influences the nucleocytoplasmic distribution of the HMGB proteins. Moreover, protein kinase CK2-mediated phosphorylation of the acidic tail modulates the intranuclear distribution of HMGB2. Collectively, our results show that, in contrast to other Arabidopsis HMGB proteins such as HMGB1 and HMGB5, the HMGB2/3 and HMGB4 proteins occur preferentially in the cell nucleus, but to various extents also in the cytoplasm.Within the cell nucleus, the genomic DNA is organized with histones and other proteins into a nucleoprotein complex termed chromatin. This packaging of the DNA has crucial consequences for DNA-dependent processes, including the transcription of genes. The chromatin structure is highly dynamic and is modulated by a variety of chromatin-associated proteins. Among these proteins are the high mobility group (HMG) proteins that represent a heterogeneous class of proteins that, after the histones, are the second most abundant family of chromosomal proteins

T he intermediate filament (IF) protein desmin is encodedby the gene DES and contributes to the mechanical stabilization of the sarcomeres and cell contacts within the cardiac intercalated disk (ID). Desmin is the predominant IF protein of striated muscles. It belongs to the type III IF proteins characterized by a uniform assembly mechanism. In the first step of the in vitro assembly, 2 coiled-coil dimers form an antiparallel tetramer.1 These tetramers are the essential building blocks of the IF. Eight tetramers anneal in lateral orientation into unit length filaments. In the longitudinal elongation step, these unit length filaments are assembled and radially compacted into IF.2 Since the first reports on DES mutations, [3][4][5] it became obvious that DES mutations cause skeletal myopathies and different forms of cardiomyopathies. 6,7 Clinical Perspective on p 623In the meantime, >60 different DES mutations distributed over the whole sequence are known, which lead to filament formation defects with deposition of cytoplasmic desmin aggregates in the majority of cases. 8,9 However, the pathomechanisms of desmin aggregation leading to skeletal or cardiac myopathies are mechanistically not understood in detail. Moreover, 10-15 ARVC is an inherited cardiomyopathy clinically characterized by arrhythmias and predominately right ventricular dilatation leading to cardiac syncope, heart failure, or even sudden cardiac death (SCD). 16 It is well established that mutations in the genes coding for desmosomal plaque proteins cause ARVC [17][18][19] and rare forms of dilated cardiomyopathy. 20 In the cardiac muscle, desmin is found in costamers, the z-disk, and connected via plaque proteins to the cardiac desmosome within the ID. The molecular processes contributing to the destabilization of the ID through desmin filaments are fragmentarily understood. Especially, it is not known, how and which of the desmin mutations impair the connection of the IF system to the cardiac desmosome. Background-TheIn this study, we report a novel pathogenic DES mutation (c.359C>A, p.A120D), which seems to interfere particularly with the connection of desmin IF to the ID. Furthermore, we investigate whether the DES variants p.A120D and p.H326R (c.977A>G) affect the IF formation using ectopic expression cell culture systems and atomic force microscopy (AFM). These data reveal that desmin-p.A120D but not desmin-p. H326R inhibits the longitudinal assembly step, confirming its pathogenic potential. Materials and Methods Clinical Description of the PatientsIn family A, the 34-year-old female index patient (III:24) presented with atrial flutter, variable atrioventricular conduction ( Figure I in the online-only Data Supplement), and dilated atria. The average ventricular frequency was 64 bpm, and the atrial frequency was 120 bpm. In the ECG, some polymorphic premature ventricular contractions (PVCs) with a frequency of 45 to 111 bpm were detected ( Figure I in the online-only Data Supplement). The cardiological evaluation including 2-dimensional, M-m...

We introduce a versatile and high precision three-dimensional optical tweezers setup with minimal optical interference to measure small forces and manipulate single molecules in the vicinity of a weak reflective surface. Our tweezers system integrates an inverted optical microscope with a single IR-laser beam that is spatially filtered in an appropriate way to allow force measurements in three dimensions with remarkably high precision when operated in backscattered light detection mode. The setup was tested by overstretching a lambda-DNA in x and z directions (perpendicular and along the optical axis), and by manipulating individual lambda-DNA molecules in the vicinity of a nanopore that allowed quantitative single molecule threading experiments with minimal optical interference.

The binding kinetics of the intercalative binding of Triostin A to lambda-DNA was investigated by measuring the force extension response of the DNA-ligand complexes with an optical tweezers system. These force response curves, containing the information about different binding properties, were analyzed based on a recent method (put forth by another research group) for monointercalators that was extended to bisintercalators. Our binding analysis reveals an exponential dependence of the association constant on the applied external force as well as a decreasing binding site size. In general, our results are in agreement with those for the monointercalator ethidium. However, to explain the high-force binding site size, a new model for bisintercalation of Triostin A at high forces is proposed.

Fluorescent DNA dyes are broadly used in many biotechnological applications for detecting and imaging DNA in cells and gels. Their binding alters the structural and nanomechanical properties of DNA and affects the biological processes that are associated with it. Although interaction modes like intercalation and minor groove binding already have been identified, associated mechanic effects like local elongation, unwinding, and softening of the DNA often remain in question. We used magnetic tweezers to quantitatively investigate the impact of three DNA-binding dyes (YOYO-1, DAPI, and DRAQ5) in a concentration-dependent manner. By extending and overwinding individual, torsionally constrained, nickfree dsDNA molecules, we measured the contour lengths and molecular forces that allow estimation of thermodynamic and nanomechanical binding parameters. Whereas for YOYO-1 and DAPI the binding mechanisms could be assigned to bis-intercalation and minor groove binding, respectively, DRAQ5 exhibited both binding modes in a concentration-dependent manner.