Biology subjects

Pyrpassopoulos, S.

Publications and source records attributed to Pyrpassopoulos, S..

6 recordsLinked to original sources

bioRxiv2026

Curved microtubule regions mark sites of lattice compaction in cells and neurons

Angstrom-scale changes in microtubule (MT) lattice spacing regulate the selective recruitment of MT-associated proteins, yet how these structural states operate in cells remains poorly understood. Here, we show that MT lattice expansion, induced by protein-based expanders or microtubule-stabilizing agents such as Taxol and epothilone D, drives the relocalization of compact lattice-binding proteins, including tau, doublecortin (DCX), and the C1 domain-containing signaling protein GEF-H1, into highly curved MT-associated domains, whereas the compaction-inducing agent laulimalide suppresses this response. In contrast, the tumor suppressor RASSF1A preferentially associates with expanded lattice states, revealing differential lattice sensitivity among closely related C1 domain-containing proteins. These short, curved assemblies are enriched at MT intersections and discrete MT segments, revealing spatially heterogeneous lattice states within individual microtubules. At substoichiometric levels, compact lattice-binding proteins behave as both MT compactors and curvature sensors. Changes in osmotic pressure selectively promote dissociation of compact lattice-binding proteins, whereas expanded lattice-binding proteins remain largely unaffected. Using curved filament formation as an in-cellulo readout of compact lattice regions, we identify widespread lattice-state sensitivity across diverse MT-associated and signaling proteins. Finally, we show that these principles extend to neurons, where somatic, but not axonal, tau exhibits sensitivity to lattice expansion despite the expanded lattice architecture of distal axonal microtubules, suggesting additional neuron-specific regulation of lattice accessibility. Together, our findings identify the MT lattice as a dynamic mechanochemical platform whose nanoscale structural states spatially organize protein recruitment and signaling in cells and neurons.

cell biology↗

bioRxiv2026

Mechanical tension expands the microtubule lattice stepwise and modulates kinesin-1 binding in an isoform-dependent manner

Recent work has shown that the microtubule lattice possesses remarkable structural plasticity, with its conformation modulated by microtubule-associated proteins and motor proteins. However, how this plasticity responds to mechanical forces remains poorly understood. Here, we developed optical tweezers and fluorescence microscopy assays to measure the effect of tensile forces on single microtubules. Quantum dot decoration enabled nanometre-precision measurement of lattice distortions of [~]0.33% under a change of mean tensile force of [<]{Delta}F[>] = 10.6 pN, within the range Fmin = 1.29 pN to Fmax = 22.3 pN -- comparable to forces from one to three kinesin-1 motors. Within this force range, the binding rate of kinesin-1 isoform KIF5B decreased reversibly within seconds by [~]20% and the dissociation rate increased by [~]10%, reducing mean run length, that in extreme cases decreased by up to 46%. Substantial heterogeneity was also observed along individual microtubules, where distinct lattice regions responded differently to applied force, implying that lattice expansion is not always uniform. Consistent heterogeneity was observed in cells, where MAPs with competing conformational preferences assembled in non-overlapping patches along the same microtubule. A cooperatively-switching lattice Ising model based on tubulin conformational bistability, supported by dynamics simulations, quantitatively reproduces these observations with a critical switching force Fc = 8.5 pN, similar to established mechanosensory proteins such as talin and E-catenin. Strikingly, no significant effects were observed for KIF5C, revealing a kinesin isoform-dependent mechanoresponse. Together, these findings establish microtubules as mechanochemical signal transducers, converting mechanical forces into biochemical signals with the speed, spatial precision and sensitivity required for rapid cellular responses. Significance StatementMicrotubules have been implicated as mechanotransducers in both mammalian and plant cells, yet a physical characterization of how mechanical forces are sensed and transduced into biochemical signals has been lacking. The present study demonstrates that modest tensile forces of less than 20 pN are sufficient to expand cooperatively the microtubule lattice by [~]0.3%, which in turn modulates its biochemical interactions with kinesin-1 in an isoform-dependent manner, selectively affecting KIF5B motor activity but not KIF5C. Strikingly, this mechanotransduction occurs on a timescale of seconds, implying that microtubules are highly efficient conduits for propagating mechanical information across the cell body. These findings establish microtubules as bona fide mechanochemical signal transducers with the speed and sensitivity required for rapid cellular responses.

biophysics↗

bioRxiv2023

An endocytic myosin essential for plasma membrane invagination powers motility against resistance

Myosins are required for clathrin-mediated endocytosis, but their precise molecular roles in this process are not known. This is, in part, because the biophysical properties of the relevant motors have not been investigated. Myosins have diverse mechanochemical activities, ranging from powerful contractility against mechanical loads to force-sensitive anchoring. To better understand the essential molecular contribution of myosin to endocytosis, we studied the in vitro force-dependent kinetics of the Saccharomyces cerevisiae endocytic type I myosin called Myo5, a motor whose role in clathrin-mediated endocytosis has been meticulously studied in vivo. We report that Myo5 is a low-duty-ratio motor that is activated [~]10-fold by phosphorylation, and that its working stroke and actin-detachment kinetics are relatively force-insensitive. Strikingly, the in vitro mechanochemistry of Myo5 is more like that of cardiac myosin than like that of slow anchoring myosin-1s found on endosomal membranes. We therefore propose that Myo5 generates power to augment actin assembly-based forces during endocytosis in cells. SummaryPedersen, Snoberger et al. measure the force-sensitivity of the yeast endocytic the myosin-1 called Myo5 and find that it is more likely to generate power than to serve as a force-sensitive anchor in cells. Implications for Myo5s role in clathrin-mediated endocytosis are discussed.

cell biology↗

bioRxiv2022

KIF1A is kinetically tuned to be a super-engaging motor under hindering loads

KIF1A is a highly processive vesicle transport motor in the kinesin-3 family. Mutations in KIF1A lead to neurodegenerative diseases including hereditary spastic paraplegia. We applied optical tweezers to study the ability of KIF1A to generate and sustain force against hindering loads. We used both the three-bead assay, where force is oriented parallel to the microtubule, and the traditional single-bead assay, where force is directed along the radius of the bead, resulting in a vertical force component. The average force and attachment duration of KIF1A in the three-bead assay were substantially greater than those observed in the single-bead assay. Thus, vertical forces accelerate termination of force ramps of KIF1A. Average KIF1A termination forces were slightly lower than the kinesin-1 KIF5B, and the median attachment duration of KIF1A was >10-fold shorter than KIF5B under hindering loads. KIF1A rapidly reengages with microtubules after detachment, as observed previously. Strikingly, quantification enabled by the three-bead assay shows that reengagement largely occurs within 2 ms of detachment, indicating that KIF1A has a nearly tenfold faster reengagement rate than KIF5B. We found that rapid microtubule reengagement is not due to KIF1As positively charged loop-12; however, removal of charge from this loop diminished the unloaded run length at near physiological ionic strength. Both loop-12 and the microtubule nucleotide state have modulatory effects on reengagement under load, suggesting a role for the microtubule lattice in KIF1A reengagement. Our results reveal adaptations of KIF1A that lead to a novel model of super-engaging transport under load.

biophysics↗

bioRxiv2019

Strain-Dependent Kinetic Properties of KIF3A and KIF3C Tune the Mechanochemistry of the KIF3AC Heterodimer

KIF3AC is a mammalian neuron-specific kinesin-2 implicated in intracellular cargo transport. It is a heterodimer of KIF3A and KIF3C motor polypeptides which have distinct biochemical and motile properties as engineered homodimers. Single-molecule motility assays show that KIF3AC moves processively along microtubules at a rate faster than expected given the motility rates of the KIF3AA and much slower KIF3CC homodimers. To resolve the stepping kinetics of KIF3A and KIF3C motors in homo-and heterodimeric constructs, and to determine their transport potential under mechanical load, we assayed motor activity using interferometric scattering (iSCAT) microscopy and optical trapping. The distribution of stepping durations of KIF3AC molecules is described by a rate (k1 = 11 s-1) without apparent kinetic asymmetry in stepping. Asymmetry was also not apparent under hindering or assisting mechanical loads of 1 pN in the optical trap. KIF3AC shows increased force sensitivity relative to KIF3AA, yet is more capable of stepping against mechanical load than KIF3CC. Microtubule gliding assays containing 1:1 mixtures of KIF3AA and KIF3CC result in speeds similar to KIF3AC, indicating the homodimers mechanically impact each others motility to reproduce the behavior of the heterodimer. We conclude that the stepping of KIF3C can be activated by KIF3A in a strain-dependent manner which is similar to application of an assisting load, and the behavior of KIF3C mirrors prior studies of kinesins with increased interhead compliance. These results suggest that KIF3AC-based cargo transport likely requires multiple motors, and its mechanochemical properties arise due to the strain-dependences of KIF3A and KIF3C.\n\nSignificance StatementKinesins are important long-range intracellular transporters in neurons required by the extended length of the axon and dendrites and selective cargo transport to each. The mammalian kinesin-2, KIF3AC, is a neuronal heterodimer of fast and slow motor polypeptides. Our results show that KIF3AC has a single observed stepping rate in the presence and absence of load and detaches from the microtubule rapidly under load. Interestingly, both KIF3A and assisting loads accelerate the kinetics of KIF3C. These results suggest that KIF3AC is an unconventional cargo transporter and its motile properties do not represent a combination of alternating fast and slow step kinetics. We demonstrate that the motile properties of KIF3AC represent a mechanochemistry that is specific to KIF3AC and may provide functional advantages in neurons.

biophysics↗

bioRxiv2019

Modulation of kinesin’s load-bearing capacity by force geometry and the microtubule track

Kinesin motors and their associated microtubule tracks are essential for long-distance transport of cellular cargos. Intracellular activity and proper recruitment of kinesins is regulated by biochemical signaling, cargo adaptors, microtubule associated proteins and mechanical forces. In this study, we found that the effect of opposing forces on the kinesin-microtubule attachment duration depends strongly on experimental assay geometry. Using optical tweezers and the conventional single-bead assay we show that detachment of kinesin from the microtubule is likely accelerated by forces vertical to the long-axis of the microtubule due to contact of the single bead with the underlying surface. We used the three-bead assay to minimize the vertical force component and found that when the opposing forces are mainly parallel to the microtubule the median attachment duration between kinesin and microtubules can be up to 10-fold longer than observed using the single-bead assay. Using the three-bead assay, we also found that not all microtubule protofilaments are equivalent interacting substrates for kinesin and that the median attachment duration (median-{Delta}t) of kinesin varies by more than 10-fold, depending on the relative angular position of the forces along the circumference of the microtubule. Thus, depending on the geometry of forces across the microtubule, kinesin can switch from a fast detaching motor (median-{Delta}t < 0.2 s) to a persistent motor that sustains attachment (median-{Delta}t > 3 s) at high forces (5 pN). Our data show that the load-bearing capacity of the kinesin motor is highly variable and can be dramatically affected by off-axis forces and forces across the microtubule lattice which has implications for a range of cellular activities including cell division and organelle transport.\n\nSignificance StatementKinesins are cytoskeletal motors responsible for the transport of cargoes along microtubules. It is well known that opposing forces decrease kinesins speed and run length. In this study, we found that when the pair of opposing forces applied on the kinesin-microtubule complex are parallel to the microtubule, the ability of kinesin to remain attached to the microtubule can vary by more than an order of magnitude depending on the relative azimuthal position of the pair of forces along the periphery of the microtubule. These results reveal a previously unknown versatility of kinesins load bearing capacity and as such have implications for the potential physiological roles of kinesin in a wide range of cell activities, including organelle transport and cell division.

biophysics↗