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.

Single-Particle Tracking Reveals Anti-Persistent Subdiffusion in Cell Extracts.

Single-particle tracking (SPT) has become a powerful tool to quantify transport phenomena in complex media with unprecedented detail. Based on the reconstruction of individual trajectories, a wealth of informative measures become available for each particle, allowing for a detailed comparison with theoretical predictions. While SPT has been used frequently to explore diffusive transport in artificial fluids and inside living cells, intermediate systems, i.e., biochemically active cell extracts, have been studied only sparsely. Extracts derived from the eggs of the clawfrog Xenopus laevis, for example, are known for their ability to support and mimic vital processes of cells, emphasizing the need to explore also the transport phenomena of nano-sized particles in such extracts. Here, we have performed extensive SPT on beads with 20 nm radius in native and chemically treated Xenopus extracts. By analyzing a variety of distinct measures, we show that these beads feature an anti-persistent subdiffusion that is consistent with fractional Brownian motion. Chemical treatments did not grossly alter this finding, suggesting that the high degree of macromolecular crowding in Xenopus extracts equips the fluid with a viscoelastic modulus, hence enforcing particles to perform random walks with a significant anti-persistent memory kernel.

Unscrambling exit site patterns on the endoplasmic reticulum as a quenched demixing process.

The endoplasmic reticulum (ER) is a vital organelle in mammalian cells with a complex morphology. Consisting of sheet-like cisternae in the cell center, the peripheral ER forms a vast tubular network on which a dispersed pattern of a few hundred specialized domains (ER exit sites (ERESs)) is maintained. Molecular details of cargo sorting and vesicle formation at individual ERESs, fueling the early secretory pathway, have been studied in some detail. The emergence of spatially extended ERES patterns, however, has remained poorly understood. Here, we show that these patterns are determined by the underlying ER morphology, suggesting ERESs to emerge from a demixing process that is quenched by the ER network topology. In particular, we observed fewer but larger ERESs when transforming the ER network to more sheet-like morphologies. In contrast, little to no changes with respect to native ERES patterns were observed when fragmenting the ER, indicating that hampering the diffusion-mediated coarse graining of domains is key for native ERES patterns. Model simulations support the notion of effective diffusion barriers impeding the coarse graining and maturation of ERES patterns. We speculate that tuning a simple demixing mechanism by the ER topology allows for a robust but flexible adaption of ERES patterns, ensuring a properly working early secretory pathway in a variety of conditions.

Microtubule polyglutamylation is important for regulating cytoskeletal architecture and motility in Trypanosoma brucei.

The shape of kinetoplastids, such as Trypanosoma brucei, is precisely defined during the stages of the life cycle and governed by a stable subpellicular microtubule cytoskeleton. During the cell cycle and transitions between life cycle stages, this stability has to transiently give way to a dynamic behaviour to enable cell division and morphological rearrangements. How these opposing requirements of the cytoskeleton are regulated is poorly understood. Two possible levels of regulation are activities of cytoskeleton-associated proteins and microtubule post-translational modifications (PTMs). Here, we investigate the functions of two putative tubulin polyglutamylases in T. brucei, TTLL6A and TTLL12B. Depletion of both proteins leads to a reduction in tubulin polyglutamylation in situ and is associated with disintegration of the posterior cell pole, loss of the microtubule plus-end-binding protein EB1 and alterations of microtubule dynamics. We also observe a reduced polyglutamylation of the flagellar axoneme. Quantitative motility analysis reveals that the PTM imbalance correlates with a transition from directional to diffusive cell movement. These data show that microtubule polyglutamylation has an important role in regulating cytoskeletal architecture and motility in the parasite T. bruceiThis article has an associated First Person interview with the first author of the paper.

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. Although ERES appear stationary and spatially confined on long timescales, we show here via single-particle tracking that they exhibit a microtubule-dependent and heterogeneous anomalous diffusion behavior on short and intermediate timescales. 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.

Anomalous dynamics of the endoplasmic reticulum network.

Large portions of the endoplasmic reticulum (ER) in eukaryotic cells are organized as dynamic networks whose segments are connected by three-way junctions. Here we show that ER junctions move subdiffusively with signatures of fractional Brownian motion and a strong dependence on the cytoskeleton's integrity: The time-averaged mean square displacement scales as 〈r^{2}(τ)〉_{t}∼τ^{α} with α≈0.5 in untreated cells and α≈0.3 when disrupting microtubules, with successive steps being anticorrelated in both cases. We explain our observations by considering ER junctions to move like monomers in (semi)flexible polymer segments immersed in a viscoelastic environment. We also report that ER networks have a nontrivial fractal dimension d_{f}≈1.6 on mesoscopic scales and we provide evidence that the organelle's dynamics is governed by fractons.