Erik Schäffer

During land colonization, plants evolved new microtubule structures that have no functional analogues in opisthokonts, namely animals and fungi. The appearance of these unique structures, such as cortical microtubule arrays, the pre-prophase band, and the phragmoplast, coincided with the expansion of the family of kinesin motors. While most plant kinesins are classified into the same families as their opisthokont orthologues, many plant kinesins not only have increased functional redundancy but have acquired additional or completely new functionality in various cellular processes. Although many opisthokont kinesins have been scrutinized down to the mechanics of a single motor, much less is known about the molecular details of plant kinesins. Insights from opisthokont kinesins are often used to infer how their corresponding plant kinesin counterparts might work in vivo, with little molecular verification. In this review, we summarize current advances in in vitro characterization of plant kinesins and highlight examples of how Arabidopsis thaliana kinesins have diverged functionally. These examples illustrate that insights from opisthokont kinesins cannot be easily transferred to plant kinesins. Furthermore, they motivate the in vitro characterization of each individual plant kinesin, a challenge we hope to overcome by promoting the use of reconstituted plant kinesin assays.

Near-resonance optical trapping of upconversion nanoparticles can double the trap stiffness demonstrated by using dual-wavelength optical tweezers. Yet, nanoparticle clustering can lead to an overestimation of trapping efficiency.

Tissue development and function rely on correct cell patterning. In plants, patterns are determined by the new cell wall formed during mitosis. During preprophase in embryophytes, ring-like positional cues already mark the future division plane on the plasma membrane. These cues include the kinesin-12 motors PHRAGMOPLAST ORIENTING KINESIN 2 (POK2, also known as KIN12D) and its paralogue POK1 (KIN12C). They are essential for correctly aligning the phragmoplast – a microtubule scaffold for cell wall formation. Although we have a basic understanding of how these motors align the phragmoplast, we currently lack information on how they are targeted to and maintained at the plasma membrane. Here, we reconstituted recombinant POK2 tail fragments on microtubules and vesicles in vitro. We found that the POK2 tail interacted with microtubules, the microtubule-associated protein MAP65-3, as well as with certain anionic lipids. We identified a short element in the POK2 tail responsible for all three interactions. Our data suggest a sequential and cooperative mechanism that targets POK2 specifically to the future division site. There, it is robustly maintained through interactions with the plasma membrane to establish the cell division plane.

Precise force measurements are crucial for understanding fundamental physics or nanoscale interactions, such as those of molecular machines in biology. Optical tweezers are versatile force transducers for such measurements, enabling meticulous manipulation of small particles. However, achieving high-resolution, subfemtonewton force measurements under physiological conditions remains challenging due to thermal fluctuations and instrument noise. Here, we employed an ultrastable optical tweezers setup in an isolated environment with precise temperature control, which minimized instrumental noise and enabled prolonged, low-force measurements. We utilized water-suspended, high-refractive index silicon nanospheres for improved resolution and trapping stability. Our system achieved a force resolution of ≈60 aN, with a sensitivity of 2.7 fN Hz^-0.5, allowing us to measure forces as low as 0.30 ± 0.06 fN. Our drag force measurements demonstrate the importance of optimized experimental conditions for low-force measurements, providing a robust framework for scientific investigations that require high-precision force characterization.

Genome maintenance and stability rely on the repair of DNA double-strand breaks. Breaks can be repaired via the single-strand-annealing pathway mediated by the protein RAD52. RAD52 oligomerizes to rings that are thought to promote annealing. However, rings have only been observed at micromolar concentrations at which annealing activity is impaired. Thus, it is unclear which oligomeric form is responsible for annealing. We combined single-molecule mass photometry with biochemical assays to determine the in vitro oligomeric states of human RAD52. We found that RAD52 was mostly monomeric at lower nanomolar concentrations. With increasing concentration, RAD52 oligomerized and formed rings with a variable stoichiometry from heptamers to tridecamers consistent with an oligomerization model of noncooperative assembly coupled with preferential cyclization. Under conditions where hardly any rings were present, RAD52 already promoted single-strand annealing in vitro. Our findings indicate that in vitro single-strand annealing can be mediated by monomers and short oligomers of RAD52. The oligomerization model suggests that ring formation is similar to a phase transition whereby rings are a reservoir to replenish the monomer and short oligomer pool. This pool has a nearly constant concentration which may be optimal for annealing and would be independent, for example, of the amount of DNA damage, protein upregulation, or the cell cycle.

In cells, mitochondria undergo constant fusion and fission. An essential factor for fission is the mammalian dynamin-related protein 1 (Drp1). Dysregulation of Drp1 is associated with neurodegenerative diseases including Parkinson’s, cardiovascular diseases and cancer, making Drp1 a pivotal biomarker for monitoring mitochondrial status and potential pathophysiological conditions. Here, we developed nanobodies (Nbs) as versatile binding molecules for proteomics, advanced microscopy and live cell imaging of Drp1. To specifically enrich endogenous Drp1 with interacting proteins for proteomics, we functionalized high-affinity Nbs into advanced capture matrices. Furthermore, we detected Drp1 by bivalent Nbs combined with site-directed fluorophore labelling in super-resolution STORM microscopy. For real-time imaging of Drp1, we intracellularly expressed fluorescently labelled Nbs, so-called chromobodies (Cbs). To improve the signal-to-noise ratio, we further converted Cbs into a “turnover-accelerated” format. With these imaging probes, we visualized the dynamics of endogenous Drp1 upon compound-induced mitochondrial fission in living cells. Considering the wide range of research applications, the presented Nb toolset will open up new possibilities for advanced functional studies of Drp1 in disease-relevant models.

Genome maintenance and stability rely on the repair of DNA double-strand breaks. The break repair can be mediated by the single-strand annealing protein RAD52. RAD52 forms rings that are thought to promote annealing. However, RAD52’s annealing activity decreases with increasing concentrations that favor ring formation. Thus, which oligomeric form and how RAD52 anneals DNA strands and detects sequence homology is unclear. We combine mass photometry with biochemical assays to quantify oligomeric states of human RAD52 with and without DNA and put forward an alternative mechanism illustrating the critical role of short oligomers for single-stranded DNA annealing. We found that while truncated RAD52 formed undecameric rings at nanomolar concentrations, full-length RAD52 was mostly monomeric at lower nanomolar, physiological concentrations. At higher concentrations, it formed rings with a variable stoichiometry from heptamers to tridecamers. At low concentrations, with hardly any rings present, RAD52 already promoted single-strand annealing. Rings and short oligomers could bind at least two single DNA strands, but if complementary strands were both bound to rings annealing was inhibited. Our findings suggest that single-strand annealing and homology detection is mediated by short oligomers of RAD52 instead of rings.

Single‐molecule assays often require functionalized surfaces. One approach for microtubule assays renders surfaces hydrophobic and uses amphiphilic blocking agents. However, the optimal hydrophobicity is unclear, protocols take long, produce toxic waste, and are susceptible to failure. Our method uses plasma activation with hydrocarbons for hexamethyldisilazane (HMDS) silanization in the gas phase. We measured the surface hydrophobicity, its effect on how well microtubule filaments were bound to the surface, and the number of nonspecific interactions with kinesin motor proteins. Additionally, we tested and discuss the use of different silanes and activation methods. We found that even weakly hydrophobic surfaces were optimal. Our environmentally friendly method significanty reduced the overall preparation effort and resulted in reproducible, high‐quality surfaces with low variability. We expect the method to be applicable to a wide range of other single‐molecule assays.