Biology subjects

Diez, S.

Publications and source records attributed to Diez, S..

33 records · Page 2Linked to original sources

bioRxiv2023

Self-assembly and contraction of micron-scale DNA rings

Contractile rings formed from cytoskeletal filaments mediate the division of cells. The reverse-engineering of synthetic contractile rings could shed light on fundamental physical principles of the ring self-assembly and dynamics independent of the natural protein-based compounds. Here, we engineer DNA nanotubes and crosslink them with a synthetic peptide-functionalized star-PEG construct. The star-PEG construct induces the formation of DNA nanotube bundles composed of several tens of individual DNA nanotubes. Importantly, the DNA nanotube bundles curve into closed micron-scale DNA rings in a high-yield one-pot self-assembly process resulting in several thousand rings per microliter. The crosslinked DNA rings can undergo contraction to less than half of their initial diameter by two distinct mechanisms, triggered by increasing molecular crowding or temperature. DNA-based contractile rings expand the toolbox of DNA nanotechnology and could be a future element of an artificial division machinery in synthetic cells.

biophysics↗

bioRxiv2022

Vesicles driven by dynein and kinesin exhibit directional reversals without external regulators

Intracellular transport along cytoskeletal filaments propelled by molecular motors ensures the targeted delivery of cargoes to their destinations. Such transport is rarely unidirectional but rather bidirectional, including intermittent pauses and directional reversals owing to the simultaneous presence of opposite-polarity motors. It has been unclear whether such a complex motility pattern results from the sole mechanical interplay between opposite-polarity motors or requires external regulators. Here, we addressed this outstanding question by reconstituting cargo motility along microtubules in vitro by attaching purified Dynein-Dynactin-BICD2 (DDB) and kinesin-3 (KIF16B) to large unilamellar vesicles. Strikingly, we found that this minimal system is sufficient to recapitulate runs, pauses and reversals similar to in vivo cargo motility. In our experiments, reversals were always preceded by vesicle pausing and the transport directionality could be tuned by the relative numbers of opposite-polarity motors on the vesicles. Unexpectedly, during all runs the vesicle velocity was not influenced by the presence of the opposing motors. To gain mechanistic insight into bidirectional transport, we developed a mathematical model which predicts that low numbers of engaged motors are critical to transition between runs and pauses. Taken together, our results suggest that motors diffusively anchored to membranous cargo transiently engage in a tug-of-war during pauses where stochastic motor attachment and detachment events can lead to directional reversals without the necessity of external regulators.

biophysics↗

bioRxiv2021

Crosstalk between guanosine nucleotides regulates cellular heterogeneity in protein synthesis during nutrient limitation

Phenotypic heterogeneity of microbial populations can facilitate survival in dynamic environments by generating sub-populations of cells that may have differential fitness in a future environment. Bacillus subtilis cultures experiencing nutrient limitation contain distinct sub-populations of cells exhibiting either comparatively high or low protein synthesis activity. This heterogeneity requires the production of phosphorylated guanosine nucleotides (pp)ppGpp by three synthases: SasA, SasB, and RelA. Here we show that these enzymes differentially affect this bimodality: RelA and SasB are necessary to generate the sub-population of cells exhibiting low protein synthesis whereas SasA is necessary to generate cells exhibiting comparatively higher protein synthesis. The RelA product (pppGpp) allosterically activates SasB and we find, in contrast, that the SasA product (pGpp) competitively inhibits this activation. Finally, we provide in vivo evidence that this antagonistic interaction mediates the observed heterogeneity in protein synthesis. This work therefore identifies the mechanism underlying phenotypic heterogeneity in the central physiological process of protein synthesis. Author SummaryUpon encountering conditions that are unfavorable to growth, such as nutrient limitation, bacteria enter into a quiescent phenotype that is mediated by group of guanosine nucleotides collectively known as (pp)pGpp. These nucleotides direct the down-regulation of energy intensive processes and are essential for a striking heterogeneity in protein synthesis observed during exit from rapid growth. Here, we show that a network of (pp)pGpp synthases is responsible for this heterogeneity and describe a mechanism that allows for the integration of multiple signals into the decision to down regulate the most energy intensive process in a cell.

microbiology↗

bioRxiv2021

Intraflagellar transport trains can turn around without the ciliary tip complex

Cilia and flagella are microtubule doublet based organelles found across the eukaryotic tree of life. Their very high aspect ratio and crowded interior are unfavourable to diffusive transport for their assembly and maintenance. Instead, a highly dynamic system of intraflagellar transport (IFT) trains moves rapidly up and down the cilium. However, the mechanism of how these trains turn around upon reaching the ciliary tip has remained elusive. It has been hypothesized that there exists a dedicated calcium-dependent protein-based machinery at the ciliary tip to mediate this conversion. In this work, we use physical and chemical methods to manipulate IFT in the cilia of the unicellular green alga Chlamydomonas reinhardtii to show that no such stationary tip-machinery is required for IFT turnaround. Instead, we demonstrate that the conversion from anterograde to retrograde IFT trains is a calcium independent intrinsic ability of the IFT system.

cell biology↗

bioRxiv2020

Highly-parallel, microfluidics-based force spectroscopy on single motor proteins

Cytoskeletal motors transform chemical energy into mechanical work to drive essential cellular functions. Optical trapping experiments have provided crucial insights into the operation of these molecular machines under load. However, the throughput of such force spectroscopy experiments is typically limited to one measurement at a time. Here, we introduce a highly-parallel, microfluidics-based method that allows for rapid collection of force-dependent motility parameters of cytoskeletal motors with two orders of magnitude improvement in throughput compared to currently available methods. We apply tunable hydrodynamic forces to stepping kinesin-1 motors via DNA-tethered beads and utilize a large field of view to simultaneously track the velocities, run lengths and interaction times of hundreds of individual kinesin-1 molecules under varying resisting and assisting loads. Importantly, the 16-m long DNA tethers between the motors and the beads significantly reduces the vertical component of the applied force pulling the motors away from the microtubule. Our approach is readily applicable to other molecular systems and constitutes a new methodology for parallelized single-molecule force studies on cytoskeletal motors.

biophysics↗

bioRxiv2020

Stable tug-of-war between kinesin-1 and cytoplasmic dynein upon different ATP and roadblock concentrations

The maintenance of intracellular processes like organelle transport and cell division depend on bidirectional movement along microtubules. These processes typically require kinesin and dynein motor proteins which move with opposite directionality. Because both types of motors are often simultaneously bound to the cargo, regulatory mechanisms are required to ensure controlled directional transport. Recently, it has been shown that parameters like mechanical motor activation, ATP concentration and roadblocks on the microtubule surface differentially influence the activity of kinesin and dynein motors in distinct manners. However, how these parameters affect bidirectional transport systems has not been studied. Here, we investigate the regulatory influence of these three parameter using in vitro gliding motility assays and stochastic simulations. We find that the number of active kinesin and dynein motors determines the transport direction and velocity, but that variations in ATP concentration and roadblock density have no significant effect. Thus, factors influencing the force balance between opposite motors appear to be important, whereas the detailed stepping kinetics and bypassing capabilities of the motors have only little effect.

biophysics↗

bioRxiv2020

Intrinsically disordered domain of kinesin-3 Kif14 enables unique functional diversity

In addition to their force-generating motor domains, kinesin motor proteins feature various accessory domains enabling them to fulfil a variety of functions in the cell. Human kinesin-3, Kif14, localizes to the midbody of the mitotic spindle and is involved in the progression of cytokinesis. The specific motor properties enabling Kif14s cellular functions, however, remain unknown. Here, we show in vitro that it is the intrinsically disordered N-terminal domain of Kif14 that enables unique functional diversity of the motor. Using single molecule TIRF microscopy we observed that the presence of the disordered domain i) increased the Kif14 run-length by an order of magnitude, rendering the motor super-processive and enabling the motor to pass through highly crowded microtubule areas shielded by cohesive layers of microtubule-associated protein tau, which blocks less processive motors ii) enabled robust, autonomous Kif14 tracking of growing microtubule tips, independent of microtubule end-binding (EB) proteins and iii) enabled Kif14 to crosslink parallel microtubules and to drive the relative sliding of antiparallel ones. We explain these features of Kif14 by the observed increased affinity of the disordered domain for GTP-like tubulin and the observed diffusible interaction of the disordered domain with the microtubule lattice. We hypothesize that the disordered domain tethers the motor domain to the microtubule forming a diffusible foothold. We suggest that the intrinsically disordered N-terminal anchoring domain of Kif14 is a regulatory hub supporting the various cellular functions of Kif14 by tuning the motors interaction with microtubules.

biophysics↗

bioRxiv2020

Anillin propels myosin-independent constriction of actin rings

Constriction of the actin cytokinetic ring is an essential step of cell division. In a generally accepted view, the constriction is driven by relative sliding of actin filaments propelled by myosin motors. However, in multiple organisms, the ring constriction is myosin independent. How actin rings constrict in the absence of motor activity remains unclear. Here, we demonstrate that actin contractility can be propelled by anillin, a diffusible non-motor actin crosslinker, localising to the cytokinetic ring. We in vitro observed the formation and constriction of rings comprising multiple actin filaments bundled by anillin. Rings constricted due to anillin-generated forces maximising the overlap lengths between the filaments. Actin disassembly promoted constriction. We propose that actin crosslinkers, generating forces complementary to molecular motors, contribute to the contractility of diverse actin structures, including the cytokinetic ring.

cell biology↗

bioRxiv2020

Mitochondria-adaptor TRAK1 promotes kinesin-1 driven transport in crowded environments

Intracellular trafficking of organelles, driven by kinesin-1 stepping along microtubules, underpins essential processes including neuronal activity. In absence of other proteins on the microtubule surface, kinesin-1 performs micron-long runs. Under protein crowding conditions, however, kinesin-1 motility is drastically impeded. It is thus unclear how kinesin-1 acts as an efficient transporter in crowded intracellular environments. Here, we demonstrate that TRAK1 (Milton), an adaptor protein essential for mitochondrial trafficking, activates kinesin-1 and increases its robustness of stepping in protein crowding conditions. Interaction with TRAK1 i) facilitated kinesin-1 navigation around obstacles, ii) increased the probability of kinesin-1 passing through cohesive envelopes of tau and iii) increased the run length of kinesin-1 in cell lysate. We explain the enhanced motility by the observed direct interaction of TRAK1 with microtubules, providing an additional anchor for the kinesin-1-TRAK1 complex. We propose adaptor-mediated tethering as a mechanism regulating kinesin-1 motility in various cellular environments.

cell biology↗

bioRxiv2019

(p)ppGpp directly regulates translation initiation during entry into quiescence

Many bacteria exist in a state of metabolic quiescence where they must minimize energy consumption so as to maximize available resources over a potentially extended period of time. As protein synthesis is the most energy intensive metabolic process in a bacterial cell, it would be an appropriate target for downregulation during the transition from growth to quiescence. We find that when Bacillus subtilis exits growth, a subpopulation of cells emerges with very low levels of protein synthesis dependent on synthesis of the nucleotides (p)ppGpp. We show that (p)ppGpp inhibits protein synthesis in vivo and in vitro by preventing the allosteric activation of the essential GTPase Initiation Factor 2 (IF2) during translation initiation. Finally, we demonstrate that IF2 is an authentic in vivo target of (p)ppGpp during the entry into quiescence, thus providing a mechanistic basis for the observed attenuation of protein synthesis.

microbiology↗

bioRxiv2019

The kinesin-14, Ncd, drives a right-handed, helical motion of antiparallel microtubules around each other

Within the mitotic spindle, several kinesin motors crosslink and slide microtubules. While some of them (e.g. kinesin-5, kinesin-8 and kinesin-14) have been shown to exhibit sideways components in their step cycles, the impact of the resulting off-axis power strokes on motility and force generation in the spindle has not been investigated so far. Here, we develop and utilize a novel three-dimensional in vitro motility assay to explore the kinesin-14, Ncd, driven sliding of crosslinked, fluorescently-labeled microtubules. We find that free microtubules, sliding in an antiparallel orientation on microtubules suspended between nanofabricated ridges, not only rotate around their own axis but also move around the suspended microtubules with right-handed helical trajectories. In contrast, microtubules crosslinked in parallel orientation are static with neither longitudinal nor helical motion. Further, our technique allows us to measure the in situ spatial extension of the motors between the crosslinked microtubules to be about 20 nm. We argue that the capability of microtubule-crosslinking kinesins to cause helical motion of microtubules around each other allows for flexible filament organization, roadblock circumvention and torque generation in the mitotic spindle.

biophysics↗

bioRxiv2019

A Brownian ratchet model explains the biased sidestepping movement of single-headed kinesin-3 KIF1A

The kinesin-3 motor KIF1A is involved in long-ranged axonal transport in neurons. In order to ensure vesicular delivery, motors need to navigate the microtubule lattice and overcome possible roadblocks along the way. The single-headed form of KIF1A is a highly diffusive motor that has been shown to be a prototype of Brownian motor by virtue of a weakly-bound diffusive state to the microtubule. Recently, groups of single-headed KIF1A motors were found to be able to sidestep along the microtubule lattice, creating left-handed helical membrane tubes when pulling on giant unilamellar vesicles in vitro. A possible hypothesis is that the diffusive state enables the motor to explore the microtubule lattice and switch protofilaments, leading to a left-handed helical motion. Here we study microtubule rotation driven by single-headed KIF1A motors using fluorescene-interference contrast (FLIC) microscopy. We find an average rotational pitch of [~=] 1.4 m which is remarkably robust to changes in the gliding velocity, ATP concentration and motor density. Our experimental results are compared to stochastic simulations of Brownian motors moving on a two-dimensional continuum ratchet potential, which quantitatively agree with the FLIC experiments. We find that single-headed KIF1A sidestepping can be explained as a consequence of the intrinsic handedness and polarity of the microtubule lattice in combination with the diffusive mechanochemical cycle of the motor.

biophysics↗

bioRxiv2018

Kinetically distinct phases of tau on microtubules regulate kinesin motors and severing enzymes

Tau is an intrinsically disordered protein, which diffuses on microtubules. In neurodegenerative diseases collectively termed tauopathies, tau malfunction and its detachment from axonal microtubules is correlated with microtubule degradation. It is known that tau can protect microtubules from microtubule-degrading enzymes, such as katanin. However, how tau can fulfill such regulative function is still unclear. Using in vitro reconstitution, we here show that tau molecules on microtubules cooperatively form islands of an ordered layer with regulatory qualities distinct from a comparably dense layer of diffusible tau. These islands shield the microtubules from katanin and kinesin-1 but are penetrable by kinesin-8 which causes the islands to disassemble. Our results indicate a new phase of tau, constituting an adjustable protective sheath around microtubules.

biophysics↗

bioRxiv2017

Challenges in estimating the motility parameters of single processive motor proteins

Cytoskeletal motor proteins are essential to the function of a wide range of intracellular mechanosystems. The biophysical characterization of their movement along their filamentous tracks is therefore of large importance. Towards this end, single-molecule, in vitro stepping-motility assays are commonly used to determine motor velocity and run length. However, comparing results from such experiments has proved difficult due to influences from variations in the experimental conditions and the data analysis methods. Here, we investigate the movement of fluorescently-labeled, processive, dimeric motor proteins and propose a unified algorithm to correct the measurements for finite filament length as well as photobleaching. Particular emphasis is put on estimating the statistical errors associated with the proposed evaluation method as knowledge of these values is crucial when comparing measurements from different experiments. Testing our approach with simulated and experimental data from GFP-labeled kinesin-1 motors stepping along immobilized microtubules, we show (i) that velocity distributions should be fitted by a t location-scale probability density function rather than by a norm*al distribution, (ii) that the impossibility to measure events shorter than the image acquisition time needs to be accounted for, (iii) that the interaction time and run length of the motors can be estimated independent of the filament length distribution, and (iv) that the dimeric nature of the motors needs to be considered when correcting for photobleaching. Moreover, our analysis reveals that controlling the temperature during the experiments with a precision below 1 K is of importance. We believe, our method will not only improve the evaluation of experimental data, but will also allow for better statistical comparisons between different populations of motor proteins (e.g. with distinct mutations or linked to different cargos) and filaments (e.g. in distinct nucleotide states or with different posttranslational modifications).

biophysics↗

bioRxiv2017

Local Nucleation Of Microtubule Bundles Through Tubulin Concentration Into A Condensed Tau Phase

Non-centrosomal microtubule bundles play important roles in cellular organization and function. Although many diverse proteins are known that can bundle microtubules, biochemical mechanisms by which cells could locally control the nucleation and formation of microtubule bundles are understudied. Here, we demonstrate that concentration of tubulin into a condensed, liquid-like compartment composed of the unstructured neuronal protein tau is sufficient to nucleate microtubule bundles. We show that under conditions of macro-molecular crowding, tau forms liquid drops. Tubulin partitions into these drops, efficiently increasing tubulin concentration and driving the nucleation of microtubules. These growing microtubules form bundles enclosed in a liquid sheath of tau. Our data suggest that condensed compartments of microtubule bundling proteins could promote the local formation of microtubule bundles in neurons by acting as non-centrosomal microtubule nucleation centers, and that liquid-like tau encapsulation could provide both stability and plasticity to long axonal microtubule bundles.

cell biology↗