Niels Laurens

Protein sliding and DNA denaturation are essential for DNA organization by human mitochondrial transcription factor A

Mitochondria organize their genome in protein-DNA complexes called nucleoids. The mitochondrial transcription factor A (TFAM), a protein that regulates mitochondrial transcription, is abundant in these nucleoids. TFAM is believed to be essential for mitochondrial DNA compaction, yet the exact mechanism has not been resolved. Here we use a combination of single-molecule manipulation and fluorescence microscopy to show the nonspecific DNA-binding dynamics and compaction by TFAM. We observe that single TFAM proteins diffuse extensively over DNA (sliding) and, by collisions, form patches on DNA in a cooperative manner. Moreover, we demonstrate that TFAM induces compaction by changing the flexibility of the DNA, which can be explained by local denaturation of the DNA (melting). Both sliding of TFAM and DNA melting are also necessary characteristics for effective, specific transcription regulation by TFAM. This apparent connection between transcription and DNA organization clarifies how TFAM can accomplish two complementary roles in the mitochondrial nucleoid at the same time.

Alba shapes the archaeal genome using a delicate balance of bridging and stiffening the DNA

Architectural proteins have an important role in shaping the genome and act as global regulators of gene expression. How these proteins jointly modulate genome plasticity is largely unknown. In archaea, one of the most abundant proteins, Alba, is considered to have a key role in organizing the genome. Here we characterize the multimodal architectural properties and interplay of the Alba1 and Alba2 proteins using single-molecule imaging and manipulation techniques. We demonstrate that the two paralogues can bridge and rigidify DNA and that the interplay between the two proteins influences the balance between these effects. Our data yield a structural model that explains the multimodal behaviour of Alba proteins and its impact on genome folding.

Effect of temperature on the intrinsic flexibility of DNA and its interaction with architectural proteins.

The helical structure of double-stranded DNA is destabilized by increasing temperature. Above a critical temperature (the melting temperature), the two strands in duplex DNA become fully separated. Below this temperature, the structural effects are localized. Using tethered particle motion in a temperature-controlled sample chamber, we systematically investigated the effect of increasing temperature on DNA structure and the interplay between this effect and protein binding. Our measurements revealed that (1) increasing temperature enhances DNA flexibility, effectively leading to more compact folding of the double-stranded DNA chain, and (2) temperature differentially affects different types of DNA-bending chromatin proteins from mesophilic and thermophilic organisms. Thus, our findings aid in understanding genome organization in organisms thriving at moderate as well as extreme temperatures. Moreover, our results underscore the importance of carefully controlling and measuring temperature in single-molecule DNA (micromanipulation) experiments.

Photothermal detection of individual gold nanoparticles: perspectives for high-throughput screening.

We use photothermal microscopy to detect and image individual gold nanoparticles that are either embedded in a polymer film or immobilized in an aqueous environment. Reducing the numerical aperture of the detection optics allows us to achieve a 200-fold-enlarged detection volume while still retaining sufficient detectivity. We characterize the capabilities of this approach for the detection of gold colloids with a diameter of 20 nm, with emphasis on practical aspects that are important for high-throughput-screening applications. The extended detection volume in combination with the stability of the photothermal signal are major advantages compared to fluorescence-based approaches, which are limited by photoblinking and photobleaching. Careful consideration is given to the trade-off between the maximum increase in local temperature that can be tolerated by a biological specimen and the minimum integration time needed to reliably determine whether a given volume contains a target species. We find that our approach has the potential to increase the detection-limited flow rate (i.e. the limit given by the detection volume divided by the minimum detection time) by two to three orders of magnitude.

Direct quantitative detection of Doc2b-induced hemifusion in optically trapped membranes.

Ca2+-sensor proteins control the secretion of many neuroendocrine substances. Calcium-secretion coupling may involve several mechanisms. First, Ca2+-dependent association of their tandem C2 domains with phosphatidylserine may induce membrane curvature and thereby enhance fusion. Second, their association with SNARE complexes may inhibit membrane fusion in the absence of a Ca2+ trigger. Here we present a method using two optically trapped beads coated with SNARE-free synthetic membranes to elucidate the direct role of the C2AB domain of the soluble Ca2+-sensor Doc2b. Contacting membranes are often coupled by a Doc2b-coated membrane stalk that resists forces up to 600 pN upon bead separation. Stalk formation depends strictly on Ca2+ and phosphatidylserine. Real-time fluorescence imaging shows phospholipid but not content mixing, indicating membrane hemifusion. Thus, Doc2b acts directly on membranes and stabilizes the hemifusion intermediate in this cell-free system. In living cells, this mechanism may co-occur with progressive SNARE complex assembly, together defining Ca2+-secretion coupling.

Diverse architectural properties of Sso10a proteins: Evidence for a role in chromatin compaction and organization.

Sso10a proteins are small DNA-binding proteins expressed by the crenarchaeal model organism Sulfolobus solfataricus. Based on the structure of Sso10a1, which contains a winged helix-turn-helix motif, it is believed that Sso10a proteins function as sequence-specific transcription factors. Here we show that Sso10a1 and Sso10a2 exhibit different distinct DNA-binding modes. While the ability to bend DNA is shared between the two proteins, DNA bridging is observed only for Sso10a1 and only Sso10a2 exhibits filament formation along DNA. The architectural properties of Sso10a proteins suggest that these proteins fulfil generic roles in chromatin organization and compaction. As these proteins exhibit different binding behaviour depending on their DNA binding stoichiometry, altered levels of expression in the cell can be exploited to drive changes in local genome folding, which may operate to modulate transcription.

Optical Pushing: A Tool for Parallelized Biomolecule Manipulation

The ability to measure and manipulate single molecules has greatly advanced the field of biophysics. Yet, the addition of more single-molecule tools that enable one to measure in a parallel fashion is important to diversify the questions that can be addressed. Here we present optical pushing (OP), a single-molecule technique that is used to exert forces on many individual biomolecules tethered to microspheres using a single collimated laser beam. Forces ranging from a few femtoNewtons to several picoNewtons can be applied with a submillisecond response time. To determine forces exerted on the tethered particles by the laser, we analyzed their measured Brownian motion using, to our knowledge, a newly derived analytical model and numerical simulations. In the model, Brownian rotation of the microspheres is taken into account, which proved to be a critical component to correctly determine the applied forces. We used our OP technique to map the energy landscape of the protein-induced looping dynamics of DNA. OP can be used to apply loading rates in the range of 10(-4)-10(6) pN/s to many molecules at the same time, which makes it a tool suitable for dynamic force spectroscopy.

Tethered Particle Motion Analysis of the DNA Binding Properties of Architectural Proteins

Architectural DNA binding proteins are key to the organization and compaction of genomic DNA inside cells. Tethered Particle Motion (TPM) permits analysis of DNA conformation and detection of changes in conformation induced by such proteins at the single molecule level in vitro. As many individual protein-DNA complexes can be investigated in parallel, these experiments have high throughput. TPM is therefore well suited for characterization of the effects of protein-DNA stoichiometry and changes in physicochemical conditions (pH, osmolarity, and temperature). Here, we describe in detail how to perform Tethered Particle Motion experiments on complexes between DNA and architectural proteins to determine their structural and biochemical characteristics.

Versatile Quadruple-Trap Optical Tweezers for Dual DNA Experiments

Optical manipulation techniques provide researchers the powerful ability to directly move, probe and interrogate molecular complexes. Quadruple optical trapping is an emerging method for optical manipulation and force spectroscopy that has found its primary use in studying dual DNA interactions, but is certainly not limited to DNA investigations. The key benefit of quadruple optical trapping is that two molecular strands can be manipulated independently and simultaneously. The molecular geometries of the strands can thus be controlled and their interactions can be quantified by force measurements. Accurate control of molecular geometry is of critical importance for the analysis of, for example, protein-mediated DNA-bridging, which plays an important role in DNA compaction. Here, we describe the design of a dedicated and robust quadruple optical trapping-instrument. This instrument can be switched straightforwardly to a high-resolution dual trap and it is integrated with microfluidics and single-molecule fluorescence microscopy, making it a highly versatile tool for correlative single-molecule analysis of a wide range of biomolecular systems.

Optical Pushing: A Tool for Parallelized Biomolecule Manipulation (vol 110, pg 44, 2016)

The analytical model. To determine the exerted force on tethered molecules it is important to understand the motion of the system. Apart from translational Brownian motion the microspheres also experiences rotational Brownian motion causing them to swivel around the attachment point as shown schematically for one dimension in Supplementary Fig. 2a,b. Existing force calibration methods for tethered microspheres, which are used for instance for MT, are not directly applicable to our system. Microsphere rotation is neglected in these models because of the fixed alignment of the paramagnetic microsphere with the external magnetic field. Neglected microsphere rotation leads to an underestimation of the applied force (Supplementary Fig. 3). We therefore derived a power spectrum for the microspheres motion parallel to the surface using the Langevin equation for both the translation as the rotation: m dt = ?⃗?brown(t) − ?⃗? ∙ ?⃗?(t) + ?⃗?ext(t) I dω ���⃗ dt = T�⃗brown(t) − β ∙ ω�⃗ (t) + T�⃗ ext(t) , (5)