Biology subjects

Speckner, K.

Publications and source records attributed to Speckner, K..

3 recordsLinked to original sources

bioRxiv2023

Live-cell imaging reveals the trade-off between target search flexibility and efficiency for Cas9 and Cas12a

CRISPR-Cas systems have widely been adopted as genome editing tools, with two frequently employed Cas nucleases being SpyCas9 and LbCas12a. Although both nucleases use RNA guides to find and cleave target DNA sites, the two enzymes differ in terms of protospacer-adjacent motif (PAM) requirements, guide architecture and cleavage mechanism. In the last years, rational engineering led to the creation of PAM-relaxed variants SpRYCas9 and impLbCas12a to broaden the targetable DNA space. By employing their catalytically inactive variants (dCas9/dCas12a), we quantified how the protein-specific characteristics impact the target search process. To allow quantification, we fused these nucleases to the photoactivatable fluorescent protein PAmCherry2.1 and performed single-particle tracking in cells of Escherichia coli. From our tracking analysis, we derived kinetic parameters for each nuclease with a non-targeting RNA guide, strongly suggesting that interrogation of DNA by LbdCas12a variants proceeds faster than that of SpydCas9. In the presence of a targeting RNA guide, both simulations and imaging of cells confirmed that LbdCas12a variants are faster and more efficient in finding a specific target site. Our work demonstrates the trade-off of relaxing PAM requirements in SpydCas9 and LbdCas12a using a powerful framework, which can be applied to other nucleases to quantify their DNA target search.

biophysics↗

bioRxiv2023

Intermittent subdiffusion of short nuclear actin rods due to interactions with chromatin

The interior of cellular nuclei, the nucleoplasm, is a crowded fluid that is pervaded by protein-decorated DNA polymers, the chromatin. Due to the complex architecture of chromatin and a multitude of associated non-equilbrium processes, e.g. DNA repair, the nucleoplasm can be expected to feature non-trivial material properties and hence anomalous transport phenomena. Here, we have used single-particle tracking on nuclear actin rods, which are important players in DNA repair, to probe such transport phenomena. Our analysis reveals that short actin rods in the nucleus show an intermittent, anti-persistent subdiffusion with clear signatures of fractional Brownian motion. Moreover, the diffusive motion is heterogeneous with clear signatures of an intermittent switching of trajectories between at least two different mobilities, most likely due to transient associations with chromatin. In line with this interpretation, hyperosmotic stress is seen to stall the motion of nuclear actin rods, whereas hypoosmotic conditions yield a reptation-like motion. Our data highlight the local heterogeneity of the nucleoplasm, e.g. distinct biochemical microenvironments and chromosome territories, that need to be taken into account for an understanding of nucleoplasmic transport and the mechanobiology of nuclei.

biophysics↗

bioRxiv2018

Diffusion of exit sites on the endoplasmic reticulum - a random walk on a shivering backbone

Major parts of the endoplasmic reticulum (ER) in eukaryotic cells are organized as a dynamic network of membrane tubules connected by three-way junctions. On this network, self-assembled membrane domains, called ER exit sites (ERES), provide platforms at which nascent cargo proteins are packaged into vesicular carriers for subsequent transport along the secretory pathway. While ERES appear stationary and spatially confined on long time scales, we show here via single-particle tracking that they exhibit a microtubule-dependent anomalous diffusion behavior on short and intermediate time scales. By quantifying key parameters of their random walk, we show that the subdiffusive motion of ERES is distinct from that of ER junctions, i.e. ERES are not tied to junctions but rather are mobile on ER tubules. We complement and corroborate our experimental findings with model simulations that also indicate that ERES are not actively moved by microtubules. Altogether, our study shows that ERES perform a random walk on the shivering ER backbone, indirectly powered by microtubular activity. Similar phenomena can be expected for other domains on subcellular structures, setting a caveat for the interpretation of domain tracking data.

biophysics↗