The voices of Ukrainian and Russian scientists.

The brutal attack on Ukraine by the Russian Federation has shocked the world. While the world works to end the violence and help refugees, as a scientific journal, our thoughts are also with those in the scientific community who are directly or indirectly impacted by the war. We have been inspired by and applaud the labs around the world that have opened their doors to displaced scientists and remain committed to supporting scientists, whoever and wherever they are. Because science requires collaboration and trust, we urge the scientific community to continue efforts like this and to remain united, especially in times as difficult as these. In this Voices piece, we feature short comments from scientists from Ukraine and scientists from Russia. This small sampling is far from exhaustive, but our sincere thanks go to those scientists who were willing to share their thoughts on this volatile and emotionally charged situation; the views expressed are those of the contributors alone. We join the world in hoping for a swift resolution to the conflict, for the good of humanity.

Regulation of microtubule dynamics, mechanics and function through the growing tip.

Microtubule dynamics and their control are essential for the normal function and division of all eukaryotic cells. This plethora of functions is, in large part, supported by dynamic microtubule tips, which can bind to various intracellular targets, generate mechanical forces and couple with actin microfilaments. Here, we review progress in the understanding of microtubule assembly and dynamics, focusing on new information about the structure of microtubule tips. First, we discuss evidence for the widely accepted GTP cap model of microtubule dynamics. Next, we address microtubule dynamic instability in the context of structural information about assembly intermediates at microtubule tips. Three currently discussed models of microtubule assembly and dynamics are reviewed. These are considered in the context of established facts and recent data, which suggest that some long-held views must be re-evaluated. Finally, we review structural observations about the tips of microtubules in cells and describe their implications for understanding the mechanisms of microtubule regulation by associated proteins, by mechanical forces and by microtubule-targeting drugs, prominently including cancer chemotherapeutics.

Theory of tip structure-dependent microtubule catastrophes and damage-induced microtubule rescues.

Microtubules are essential cytoskeletal polymers that exhibit stochastic switches between tubulin assembly and disassembly. Here, we examine possible mechanisms for these switches, called catastrophes and rescues. We formulate a four-state Monte Carlo model, explicitly considering two biochemical and two conformational states of tubulin, based on a recently conceived view of microtubule assembly with flared ends. The model predicts that high activation energy barriers for lateral tubulin interactions can cause lagging of curled protofilaments, leading to a ragged appearance of the growing tip. Changes in the extent of tip raggedness explain some important but poorly understood features of microtubule catastrophe: weak dependence on tubulin concentration and an increase in its probability over time, known as aging. The model predicts a vanishingly rare frequency of spontaneous rescue unless patches of guanosine triphosphate tubulin are artificially embedded into microtubule lattice. To test our model, we used in vitro reconstitution, designed to minimize artifacts induced by microtubule interaction with nearby surfaces. Microtubules were assembled from seeds overhanging from microfabricated pedestals and thus well separated from the coverslip. This geometry reduced the rescue frequency and the incorporation of tubulins into the microtubule shaft compared with the conventional assay, producing data consistent with the model. Moreover, the rescue positions of microtubules nucleated from coverslip-immobilized seeds displayed a nonexponential distribution, confirming that coverslips can affect microtubule dynamics. Overall, our study establishes a unified theory accounting for microtubule assembly with flared ends, a tip structure-dependent catastrophe frequency, and a microtubule rescue frequency dependent on lattice damage and repair.

Expanding Stereoelectronic Limits of endo-tet Cyclizations: Synthesis of Benz[b]azepines from Donor-Acceptor Cyclopropanes.

The importance of intramolecular constraints in cyclic transition-state geometries is especially pronounced in n-endo-tet cyclizations, where the usual backside approach of a nucleophile to the breaking bond is impossible for the rings containing less than eight atoms. Herein, we expand the limits of endo-tet cyclizations and show that donor-acceptor cyclopropanes can provide a seven-membered ring via a genuine 6-endo-tet process. Substrates containing a N-alkyl-N-arylcarbamoyl moiety as an acceptor group undergo Lewis acid-induced cyclization to form tetrahydrobenz[b]azepin-2-ones in high yields. The reaction proceeds with the inversion of the configuration at the electrophilic carbon. In this process, a formally six-membered transition state yields a seven-membered ring as the pre-existing cycle is merged into the forming ring. The stereochemistry of the products can be controlled by the reaction time and by the nature of Lewis acid, opening access to both diastereomers by tuning of the reaction conditions.

Mechanical properties of tubulin intra- and inter-dimer interfaces and their implications for microtubule dynamic instability.

Thirteen tubulin protofilaments, made of αß-tubulin heterodimers, interact laterally to produce cytoskeletal microtubules. Microtubules exhibit the striking property of dynamic instability, manifested in their intermittent growth and shrinkage at both ends. This behavior is key to many cellular processes, such as cell division, migration, maintenance of cell shape, etc. Although assembly and disassembly of microtubules is known to be linked to hydrolysis of a guanosine triphosphate molecule in the pocket of ß-tubulin, detailed mechanistic understanding of corresponding conformational changes is still lacking. Here we take advantage of the recent generation of in-microtubule structures of tubulin to examine the properties of protofilaments, which serve as important microtubule assembly and disassembly intermediates. We find that initially straight tubulin protofilaments, relax to similar non-radially curved and slightly twisted conformations. Our analysis further suggests that guanosine triphosphate hydrolysis primarily affects the flexibility and conformation of the inter-dimer interface, without a strong impact on the shape or flexibility of αß-heterodimer. Inter-dimer interfaces are significantly more flexible compared to intra-dimer interfaces. We argue that such a difference in flexibility could be key for distinct stability of the plus and minus microtubule ends. The higher flexibility of the inter-dimer interface may have implications for development of pulling force by curving tubulin protofilaments during microtubule disassembly, a process of major importance for chromosome motions in mitosis.

Probing Mitotic CENP-E Kinesin with the Tethered Cargo Motion Assay and Laser Tweezers.

Coiled-coil stalks of various kinesins differ significantly in predicted length and structure; this is an adaption that helps these motors carry out their specialized functions. However, little is known about the dynamic stalk configuration in moving motors. To gain insight into the conformational properties of the transporting motors, we developed a theoretical model to predict Brownian motion of a microbead tethered to the tail of a single, freely walking molecule. This approach, which we call the tethered cargo motion (TCM) assay, provides an accurate measure of the mechanical properties of motor-cargo tethering, verified using kinesin-1 conjugated to a microbead via DNA links in vitro. Applying the TCM assay to the mitotic kinesin CENP-E unexpectedly revealed that when walking along a microtubule track, this highly elongated molecule with a contour length of 230 nm formed a 20-nm-long tether. The stalk of a walking CENP-E could not be extended fully by application of sideways force with optical tweezers (up to 4 pN), implying that CENP-E carries its cargo in a compact configuration. Assisting force applied along the microtubule track accelerates CENP-E walking, but this increase does not depend on the presence of the CENP-E stalk. Our results suggest that the unusually large stalk of CENP-E has little role in regulating its function as a transporter. The adjustable stalk configuration may represent a regulatory mechanism for controlling the physical reach between kinetochore-bound CENP-E and spindle microtubules, or it may assist localizing various kinetochore regulators in the immediate vicinity of the kinetochore-embedded microtubule ends. The TCM assay and underlying theoretical framework will provide a general guide for determining the dynamic configurations of various molecular motors moving along their tracks, freely or under force.

Long tethers provide high-force coupling of the Dam1 ring to shortening microtubules.

Microtubule kinetochore attachments are essential for accurate mitosis, but how these force-generating connections move chromosomes remains poorly understood. Processive motion at shortening microtubule ends can be reconstituted in vitro using microbeads conjugated to the budding yeast kinetochore protein Dam1, which forms microtubule-encircling rings. Here, we report that, when Dam1 is linked to a bead cargo by elongated protein tethers, the maximum force transmitted from a disassembling microtubule increases sixfold compared with a short tether. We interpret this significant improvement with a theory that considers the geometry and mechanics of the microtubule-ring-bead system. Our results show the importance of fibrillar links in tethering microtubule ends to cargo: fibrils enable the cargo to align coaxially with the microtubule, thereby increasing the stability of attachment and the mechanical work that it can do. The force-transducing characteristics of fibril-tethered Dam1 are similar to the analogous properties of purified yeast kinetochores, suggesting that a tethered Dam1 ring comprises the main force-bearing unit of the native attachment.

Molecular and Mechanical Causes of Microtubule Catastrophe and Aging.

Tubulin polymers, microtubules, can switch abruptly from the assembly to shortening. These infrequent transitions, termed "catastrophes", affect numerous cellular processes but the underlying mechanisms are elusive. We approached this complex stochastic system using advanced coarse-grained molecular dynamics modeling of tubulin-tubulin interactions. Unlike in previous simplified models of dynamic microtubules, the catastrophes in this model arise owing to fluctuations in the composition and conformation of a growing microtubule tip, most notably in the number of protofilament curls. In our model, dynamic evolution of the stochastic microtubule tip configurations over a long timescale, known as the system's "aging", gives rise to the nonexponential distribution of microtubule lifetimes, consistent with experiment. We show that aging takes place in the absence of visible changes in the microtubule wall or tip, as this complex molecular-mechanical system evolves slowly and asymptotically toward the steady-state level of the catastrophe-promoting configurations. This new, to our knowledge, theoretical basis will assist detailed mechanistic investigations of the mechanisms of action of different microtubule-binding proteins and drugs, thereby enabling accurate control over the microtubule dynamics to treat various pathologies.

Synthesis and evaluation of tetrahydropyrrolo[1,2-a]quinolin-1(2H)-ones as new tubulin polymerization inhibitors.

Here we explored new 1,5-disubstituted pyrrolidin-2-ones 1, 2 and 5-aryl-3,3a,4,5-tetrahydropyrrolo[1,2-a]quinoline-1(2H)-ones 3 as inhibitors of tubulin polymerization. We evaluated their effects on microtubule dynamics in vitro and on the proliferation of A549 cells, using flow cytometry-based cell cycle analysis. The results were verified with phase-contrast microscopy in three cancer cell lines: A549, HeLa and MCF-7. Guided by molecular modeling of the interactions between tubulin and the most active of the identified compounds, we designed, synthesized, and tested the 3-hydroxyphenyl-substituted compound 3c. This compound was further shown to bind to the colchicine site of tubulin and reduce microtubule growth rates in vitro. Moreover, compound 3c arrested division of the A549 cells in the low micromolar range (IC50 = 5.9 µM) and exhibited cytotoxicity against four different cell lines in the MTT assay for cell proliferation. Our findings demonstrate that 5-aryltetrahydropyrrolo[1,2-a]quinoline-1(2H)-one is a promising scaffold for the development of novel tubulin polymerization inhibitors.

Measuring and modeling forces generated by microtubules.

Tubulins are essential proteins, which are conserved across all eukaryotic species. They polymerize to form microtubules, cytoskeletal components of paramount importance for cellular mechanics. The microtubules combine an extraordinarily high flexural rigidity and a non-equilibrium behavior, manifested in their intermittent assembly and disassembly. These chemically fueled dynamics allow microtubules to generate significant pushing and pulling forces at their ends to reposition intracellular organelles, remodel membranes, bear compressive forces, and transport chromosomes during cell division. In this article, we review classical and recent studies, which have allowed the quantification of microtubule-generated forces. The measurements, to which we owe most of the quantitative information about microtubule forces, were carried out in biochemically reconstituted systems in vitro. We also discuss how mathematical and computational modeling has contributed to the interpretations of these results and shaped our understanding of the mechanisms of force production by tubulin polymerization and depolymerization.

Microtubule rescue control by drugs and MAPs examined with in vitro pedestal assay.

Microtubules are essential cytoskeletal polymers, which exhibit stochastic transitions between assembly and disassembly, known as catastrophes and rescues. Understanding of catastrophes, rescues, and their control by drugs and microtubule associated proteins (MAPs) has been informed by in vitro reconstitutions of microtubule dynamics. In such experiments microtubules are typically observed on a flat surface of the coverslip. In contrast, we have recently proposed a modified setup in which microtubules assemble from stabilized seeds, overhanging from microfabricated pedestals, so that their dynamic extensions are fully isolated from contact with the coverslip. This assay allows to eliminate potential artifacts, which may substantially affect the frequency of microtubule rescues in vitro. Here we use the pedestal assay to study the sensitivity of microtubules to paclitaxel, one of the best-known inhibitors of microtubule dynamics. By comparing observations in the conventional and the pedestal assays, we find that microtubule dynamics are substantially more sensitive to paclitaxel when the polymers can contact the coverslip. We interpret this as a consequence of the coverslip-induced microtubule assembly perturbation, leading to formation of lattice with defects, and thereby enhancing the efficiency of paclitaxel binding to microtubules in the conventional assay. To test this idea, we use vinblastine, another small-molecule inhibitor, which had been previously shown to cause microtubule growth perturbations. We find that in the pedestal assay vinblastine sensitizes microtubules to paclitaxel to the level, observed in the conventional assay. Interestingly, a minimal fragment of MAP called CLASP2, a previously characterized rescue factor, has a strong effect on microtubule rescues, regardless of the type of assay. Overall, our study underscores the role of microtubule damage in promoting rescues and highlights the utility of the in vitro pedestal assay to study microtubule dynamics modulation by tubulin inhibitors and MAPs.

VII Congress of Russian Biophysicists-2023, Krasnodar, Russia.

This special issue of Biophysical Reviews contains the materials presented at the VII Congress of Biophysicists of Russia, held from 17 to 23 April in Krasnodar. We believe that we have managed to prepare a selection of articles that well reflects the current state of biophysical science in Russia and its place in the world science. The VII Russian Congress on Biophysics was held in Krasnodar in April 2023, continuing the tradition of the series of biophysics conferences held every 4 years. The congress discussed physical principles and mechanisms of biological processes occurring at different life levels-from molecular to cellular and population levels. The results of fundamental and applied research in molecular biophysics, cell biophysics, and biophysics of complex systems were presented at plenary, sectional, and poster sessions. The works in the field of medical biophysics and neurobiology were especially widely presented. The structure and dynamics of biopolymers and fundamental mechanisms underlying the effects of physicochemical factors on biological systems, membrane, and transport processes were actively discussed. Much attention was paid to new experimental methods of biophysical research, methods of bioinformatics, computer, and mathematical modeling as necessary tools of the research at all levels of living systems. Along with fundamental problems of studying biophysical mechanisms of regulation of processes at the molecular, subcellular, and cellular levels, much attention was paid to applied research in the field of biotechnology and environmental monitoring. The Congress has formed the National Committee of Russian biophysicists.

An open call for contributions to a special issue of Biophysical Reviews highlighting the research themes of VII Congress of Russian Biophysicists 2023.

We announce a call for contributions to a Special Issue of Biophysical Reviews associated with the VII Congress of Russian Biophysicists (to be held in Krasnodar, Russia, 17-23 April 2023). The Congress is the main biophysical meeting held within Russia and is organized every four years. The Congress will focus on both the physical principles and mechanisms of biological processes occurring at different levels of structural organization, from molecular to cellular to organism and to population levels. The Special Issue will accept reviews on topics from molecular biophysics, structure and dynamics of biopolymers, biophysics of the cell, energy transformation mechanisms, biophotonics, ecological biophysics, and medical biophysics, following the sections of the Congress. The VII Congress of Russian Biophysicists is supported by International Union of Pure and Applied Biophysics (IUPAB). Here we describe main topics and sections of the coming event, the paper types for the journal issue, and the key deadline dates.

The Dam1 ring binds microtubules strongly enough to be a processive as well as energy-efficient coupler for chromosome motion.

Accurate chromosome segregation during mitotic division of budding yeast depends on the multiprotein kinetochore complex, Dam1 (also known as DASH). Purified Dam1 heterodecamers encircle microtubules (MTs) to form rings that can function as "couplers," molecular devices that transduce energy from MT disassembly into the motion of a cargo. Here we show that MT depolymerization develops a force against a Dam1 ring that is sixfold larger than the force exerted on a coupler that binds only one side of an MT. Wild-type rings slow depolymerization fourfold, but rings that include a mutant Dam1p with truncated C terminus slow depolymerization less, consistent with the idea that this tail is part of a strong bond between rings and MTs. A molecular-mechanical model for Dam1-MT interaction predicts that binding between this flexible tail and the MT wall should cause a Dam1 ring to wobble, and Fourier analysis of moving, ring-attached beads corroborates this prediction. Comparison of the forces generated against wild-type and mutant complexes confirms the importance of tight Dam1-MT association for processive cargo movement under load.

Correction: Brownian dynamics simulation of protofilament relaxation during rapid freezing.

[This corrects the article DOI: 10.1371/journal.pone.0247022.].

Brownian dynamics simulation of protofilament relaxation during rapid freezing.

Electron cryo-microscopy (Cryo-EM) is a powerful method for visualizing biological objects with up to near-angstrom resolution. Instead of chemical fixation, the method relies on very rapid freezing to immobilize the sample. Under these conditions, crystalline ice does not have time to form and distort structure. For many practical applications, the rate of cooling is fast enough to consider sample immobilization instantaneous, but in some cases, a more rigorous analysis of structure relaxation during freezing could be essential. This difficult yet important problem has been significantly under-reported in the literature, despite spectacular recent developments in Cryo-EM. Here we use Brownian dynamics modeling to examine theoretically the possible effects of cryo-immobilization on the apparent shapes of biological polymers. The main focus of our study is on tubulin protofilaments. These structures are integral parts of microtubules, which in turn are key elements of the cellular skeleton, essential for intracellular transport, maintenance of cell shape, cell division and migration. We theoretically examine the extent of protofilament relaxation within the freezing time as a function of the cooling rate, the filament's flexural rigidity, and the effect of cooling on water's viscosity. Our modeling suggests that practically achievable cooling rates are not rapid enough to capture tubulin protofilaments in conformations that are incompletely relaxed, suggesting that structures seen by cryo-EM are good approximations to physiological shapes. This prediction is confirmed by our analysis of curvatures of tubulin protofilaments, using samples, prepared and visualized with a variety of methods. We find, however, that cryofixation may capture incompletely relaxed shapes of more flexible polymers, and it may affect Cryo-EM-based measurements of their persistence lengths. This analysis will be valuable for understanding of structures of different types of biopolymers, observed with Cryo-EM.

α-tubulin tail modifications regulate microtubule stability through selective effector recruitment, not changes in intrinsic polymer dynamics.

Microtubules are non-covalent polymers of αß-tubulin dimers. Posttranslational processing of the intrinsically disordered C-terminal α-tubulin tail produces detyrosinated and Δ2-tubulin. Although these are widely employed as proxies for stable cellular microtubules, their effect (and of the α-tail) on microtubule dynamics remains uncharacterized. Using recombinant, engineered human tubulins, we now find that neither detyrosinated nor Δ2-tubulin affect microtubule dynamics, while the α-tubulin tail is an inhibitor of microtubule growth. Consistent with the latter, molecular dynamics simulations show the α-tubulin tail transiently occluding the longitudinal microtubule polymerization interface. The marked differential in vivo stabilities of the modified microtubule subpopulations, therefore, must result exclusively from selective effector recruitment. We find that tyrosination quantitatively tunes CLIP-170 density at the growing plus end and that CLIP170 and EB1 synergize to selectively upregulate the dynamicity of tyrosinated microtubules. Modification-dependent recruitment of regulators thereby results in microtubule subpopulations with distinct dynamics, a tenet of the tubulin code hypothesis.

Mechanisms of microtubule dynamics and force generation examined with computational modeling and electron cryotomography.

Microtubules are dynamic tubulin polymers responsible for many cellular processes, including the capture and segregation of chromosomes during mitosis. In contrast to textbook models of tubulin self-assembly, we have recently demonstrated that microtubules elongate by addition of bent guanosine triphosphate tubulin to the tips of curving protofilaments. Here we explore this mechanism of microtubule growth using Brownian dynamics modeling and electron cryotomography. The previously described flaring shapes of growing microtubule tips are remarkably consistent under various assembly conditions, including different tubulin concentrations, the presence or absence of a polymerization catalyst or tubulin-binding drugs. Simulations indicate that development of substantial forces during microtubule growth and shortening requires a high activation energy barrier in lateral tubulin-tubulin interactions. Modeling offers a mechanism to explain kinetochore coupling to growing microtubule tips under assisting force, and it predicts a load-dependent acceleration of microtubule assembly, providing a role for the flared morphology of growing microtubule ends.

Microtubules grow by the addition of bent guanosine triphosphate tubulin to the tips of curved protofilaments.

We used electron tomography to examine microtubules (MTs) growing from pure tubulin in vitro as well as two classes of MTs growing in cells from six species. The tips of all these growing MTs display bent protofilaments (PFs) that curve away from the MT axis, in contrast with previously reported MTs growing in vitro whose tips are either blunt or sheetlike. Neither high pressure nor freezing is responsible for the PF curvatures we see. The curvatures of PFs on growing and shortening MTs are similar; all are most curved at their tips, suggesting that guanosine triphosphate-tubulin in solution is bent and must straighten to be incorporated into the MT wall. Variations in curvature suggest that PFs are flexible in their plane of bending but rigid to bending out of that plane. Modeling by Brownian dynamics suggests that PF straightening for MT growth can be achieved by thermal motions, providing a simple mechanism with which to understand tubulin polymerization.